Targeting the ETS Transcription Factor Rearrangement EWS/FLI in Ewing's Sarcoma

靶向尤文肉瘤中的 ETS 转录因子重排 EWS/FLI

• 批准号: 8327461
• 负责人: ANDREW L KUNG
• 金额: $ 4.38万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8327461
• 关键词: Antisense RNA; Apoptosis; Binding; Biological Assay; Cell Cycle; Cell Cycle Arrest; Cell Line; Cells; Chemical Structure; Chemicals; Chemistry; Childhood; Chromosomal translocation; Cytomegalovirus; DNA; DNA Binding Domain; DNA Sequence Rearrangement; Dependency; Development; Disease; Dose; Drug Delivery Systems; EZH2 gene; Engineering; Ewings sarcoma; FLI1 Transcription Factor; Family; Firefly Luciferases; Functional disorder; Gene Expression; Gene Targeting; Genes; Genetic Transcription; HCT116 Cells; Hela Cells; ID2 gene; Immunoblotting; Inhibitory Concentration 50; Lead; Luciferases; Malignant Neoplasms; Mediating; Morphologic artifacts; NR0B1 gene; Oncogene Proteins; Outcome; Patients; Phase; Physiology; Property; Proteins; RNA Interference; RNA-Binding Protein EWS; Refractory; Renilla Luciferases; Reporter; Repression; Reproducibility; Role; Screening procedure; Specificity; Structure-Activity Relationship; Transactivation; Transcription Factor Oncogene; Translations; Tumorigenicity; Ubiquitin C; Work; base; cancer type; drug development; drug discovery; high throughput screening; improved; inhibitor/antagonist; innovation; internal control; knock-down; mutant; neoplastic cell; novel; novel therapeutic intervention; promoter; response; scaffold; small molecule; success; transcription factor; tumorigenesis; young adult

DESCRIPTION (provided by applicant): Perturbations in the normal function of transcription factors contribute to the development of a number of diseases. For example, chromosomal translocations that result in mutant transcription factors are associated with the development of several types of cancers. Ewing sarcoma is a pediatric and young adult cancer that results from a translocation that fuses the DNA binding domain of the Friend leukemia virus integration 1 (FLI1) transcription factor to the transactivation domain of the EWS protein, forming a fusion EWS/FLI oncoprotein. Ewing sarcoma cells have a strict dependency on the EWS/FLI oncoprotein, demonstrated by the fact that experimental modulation of EWS/FLI activity (e.g., RNAi knock-down, antisense RNA) results in tumor cell apoptosis, cell cycle arrest, and loss of tumorigenicity. The central role of EWS/FLI in Ewing sarcoma tumorigenesis makes it an attractive drug target; however transcription factor oncoproteins have largely been refractory to conventional drug discovery approaches and are generally considered "undruggable." In this proposal, we partner several innovative approaches to identify and subsequently validate small-molecule inhibitors of EWS/FLI. These approaches circumvent many of the hurdles that have hindered the identification of therapies specifically targeting transcription factors. In the first Specific Aim, we will use a well-validated reporter of EWS/FLI activity to screen for small molecules that inhibit EWS/FLI reporter activity, but not a constitutive internal control. Because reporter assays are subject to frequent false-positives, in the second Specific Aim we will use a novel bead-based assay to assess the expression of 123 genes to identify compounds that modulate endogenous EWS/FLI transcriptional activity in Ewing sarcoma cells. Finally, positive hits will be evaluated in the third Specific Aim with a variety of validated tertiary assays to identify potential mechanisms of action. We will work to define and refine the chemical structure of promising lead compounds to identify probes with properties that can be used to develop new therapeutic approaches to selectively target Ewing sarcoma cells. The assays that constitute this discovery pipeline have been optimized for high-throughput screening and are immediately ready for implementation with the MLPCN. Success in these studies will not only identify mature leads for development of Ewing sarcoma targeted therapies, but will also establish a paradigm that can be generalized for identification of inhibitory molecules targeting a variety of transcription factor oncogenes.

描述（由申请人提供）：转录因子正常功能的扰动有助于许多疾病的发展。例如，导致突变转录因子的染色体易位与几种类型的癌症的发展有关。尤文肉瘤是一种儿科和年轻成人癌症，由Friend白血病病毒整合1（FLI 1）转录因子的DNA结合结构域融合到EWS蛋白的反式激活结构域，形成融合EWS/FLI癌蛋白的易位引起。尤文肉瘤细胞对EWS/FLI癌蛋白具有严格的依赖性，这通过EWS/FLI活性的实验调节（例如，RNAi敲低、反义RNA）导致肿瘤细胞凋亡、细胞周期停滞和致瘤性丧失。EWS/FLI在尤因肉瘤肿瘤发生中的核心作用使其成为一个有吸引力的药物靶点;然而，转录因子癌蛋白在很大程度上对传统药物发现方法很难治疗，并且通常被认为是“不可治疗的”。“在这项提案中，我们合作了几种创新方法来鉴定并随后验证EWS/FLI的小分子抑制剂。这些方法规避了许多阻碍鉴定特异性靶向转录因子的疗法的障碍。上 具体目的，我们将使用经充分验证的EWS/FLI活性报告基因筛选抑制EWS/FLI报告基因活性的小分子，但不是组成型内部对照。由于报告基因检测经常出现假阳性，在第二个特定目的中，我们将使用一种新型的基于珠的检测来评估123个基因的表达，以鉴定调节尤文肉瘤细胞中内源性EWS/FLI转录活性的化合物。最后，将在第三个特定目标中使用各种经验证的三级试验评价阳性命中，以确定潜在的作用机制。我们将努力定义和完善有前途的先导化合物的化学结构，以确定具有可用于开发新的治疗方法以选择性靶向尤文肉瘤细胞的特性的探针。构成这一发现管道的检测试剂盒已针对高通量筛选进行了优化，并立即准备好与MLPCN一起实施。这些研究的成功不仅将为尤文肉瘤靶向治疗的发展确定成熟的线索，而且还将建立一个范式，可以推广到识别靶向各种转录因子癌基因的抑制分子。