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

描述(由申请人提供)：转录因子正常功能的扰动导致了许多疾病的发生。例如，导致转录因子突变的染色体易位与几种类型癌症的发生有关。尤文肉瘤是一种儿童和青年癌症，其原因是易位将Friend白血病病毒整合1(FLI1)转录因子的DNA结合域与EWS蛋白的反式激活域融合，形成融合的EWS/FLI癌蛋白。尤文肉瘤细胞对EWS/FLI癌蛋白具有严格的依赖性，这一事实证明，实验上调节EWS/FLI活性(例如，RNAi下调、反义RNA)会导致肿瘤细胞凋亡、细胞周期停滞和致瘤性丧失。EWS/FLI在尤文肉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的药物发现方法无效，通常被认为是“不可用药”。在这项提案中，我们合作了几种创新的方法来识别和随后验证EWS/FLI的小分子抑制剂。这些方法绕过了许多障碍，这些障碍阻碍了专门针对转录因子的治疗的确定。在第一个 为了明确目标，我们将使用经过良好验证的EWS/FLI活性报告程序来筛选抑制EWS/FLI报告程序活性的小分子，但不是结构性内部对照。由于报告分析经常受到假阳性的影响，在第二个特定目标中，我们将使用一种新的基于珠子的分析来评估123个基因的表达，以确定调节尤文肉瘤细胞内源性EWS/FLI转录活性的化合物。最后，在第三个特定目标中，将通过各种有效的第三级检测来评估阳性命中，以确定潜在的作用机制。我们将致力于定义和提炼有前景的先导化合物的化学结构，以确定具有可用于开发选择性靶向尤文肉瘤细胞的新治疗方法的探针。构成这一发现管道的分析已经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开发确定成熟的线索，而且还将建立一个可推广用于识别针对各种转录因子癌基因的抑制分子的范例。

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.