Isogenic Cell-Based Screen:Cancer-Targeting Catenin(RMI)

B-连环蛋白癌基因的激活突变在一系列常见的人类癌症中被发现， 包括结直肠癌，肝细胞癌，子宫内膜癌，甲状腺癌，以及 其他。激活的癌基因，如B-连环蛋白，是一个吸引人的抗癌药物靶点，因为它们代表着一种 在癌细胞中发现了必需的基因功能获得，但在患者的正常组织中没有发现。与一致 这个想法，我们之前已经实验证明，靶向致癌的B-连环蛋白很可能是 有效的抗癌策略(Mol Can Ther 1：1355,2002)。 在这个应用中，我们建议实现一个基于细胞的筛选来识别小分子和天然的 产品先导化合物，对携带阿司匹林的人结肠癌细胞具有特异性的细胞毒性/细胞抑制作用 致癌B-连环蛋白的等位基因。为了做到这一点，我们将使用一对基因靶向的人类癌细胞 唯一不同的是有没有突变的(致癌的)B-连环蛋白的内源性等位基因。 由于这些细胞在其他方面是同源的，所以专门杀死或抑制亲本细胞生长的化合物 (含有致癌的B-连环蛋白)，但不是其中致癌的B-连环蛋白已被删除的衍生物 基因靶向是B-连环蛋白靶向先导化合物的良好候选者。这样的打击将进一步 在各种次级筛选中进行验证，以证明可重复性和可推广性。一次 通过筛选约4000种化合物证明了可行性，我们将申请转移 该筛查到NIH路线图筛查中心进行高通量筛查的努力。

Activating mutations in the B-catenin oncogene are found in a wide range of common human cancers, including colorectal carcinoma, hepatocellular carcinoma, endometrial carcinoma, thyroid carcinoma, and others. Activated oncogenes such as B-catenin are an appealing cancer drug target, since they represent a required genetic gain-of-function found in cancer cells but not in the patient's normal tissues. Consistent with this idea, we have previously experimentally demonstrated that targeting oncogenic B-catenin is likely to be an effective anticancer strategy (Mol Can Ther 1:1355, 2002). In this application we propose to implement a cell-based screen to identify small molecule and natural product lead compounds that are specifically cytotoxic/cytostatic to human colon cancer cells harboring an allele of oncogenic B-catenin. To do this we will employ a paired set of gene-targeted human cancer cells that differ only in the presence or absence of their endogenous allele of mutant (oncogenic) B-catenin. Since the cells are otherwise isogenic, compounds that specifically kill or inhibit the growth of parental cells (harboring oncogenic B-catenin) but not the derivatives in which oncogenic B-catenin has been deleted by gene targeting are good candidates as B-catenin targeted lead compounds. Such hits will be further validated in a variety of secondary screens to demonstrate reproducibility and generalizability. Once feasibility has been demonstrated by screening approximately 4000 compounds, we will apply to transfer the screen to an NIH Roadmap screening center for high throughput screening efforts.