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有助于许多疾病的发展。例如，导致转录因子突变的染色体易位与几种癌症的发生有关。Ewing肉瘤是一种儿童和青少年癌症，由易位引起，将Friend白血病病毒整合1 （FLI1）转录因子的DNA结合域与EWS蛋白的反激活域融合，形成融合的EWS/FLI癌蛋白。Ewing肉瘤细胞对EWS/FLI癌蛋白有严格的依赖性，通过实验调节EWS/FLI活性（如RNAi敲除、反义RNA）可导致肿瘤细胞凋亡、细胞周期阻滞和致瘤性丧失。EWS/FLI在尤文氏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的药物发现方法是难治的，通常被认为是“不可药物的”。在本提案中，我们合作了几种创新方法来鉴定和随后验证EWS/FLI的小分子抑制剂。这些方法规避了许多阻碍特异性靶向转录因子的疗法鉴定的障碍。在第一个

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.