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

描述(申请人提供)：B-catenin癌基因的激活突变在广泛的常见人类癌症中被发现，包括结直肠癌、肝细胞癌、子宫内膜癌、甲状腺癌等。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，因为它们代表在癌细胞中发现的所需的基因功能获得，而在患者的正常组织中不存在。与这一想法一致，我们以前已经实验证明，靶向致癌的B-连环蛋白可能是一种有效的抗癌策略(Mol can Ther 1：1355,2002)。在这项应用中，我们建议实施一种基于细胞的筛选，以鉴定对携带致癌基因B-catenin等位基因的人结肠癌细胞具有细胞毒性/细胞抑制作用的小分子和天然产物先导化合物。为了做到这一点，我们将使用一组以基因为靶标的人类癌细胞配对，这些细胞仅在存在或不存在突变(致癌)B-连环蛋白的内源性等位基因时有所不同。由于细胞在其他方面是同源的，因此，具有特异性杀伤或抑制亲本细胞生长的化合物(含有致癌的B-连环蛋白)，而不是通过基因靶向删除致癌的B-连环蛋白的衍生物，是很好的B-连环蛋白靶向先导化合物。这样的点击量将在各种次级屏幕上进一步验证，以证明可重复性和通用性。一旦通过筛选大约4000种化合物证明了可行性，我们将申请将筛选转移到NIH路线图筛选中心进行高通量筛选工作。

DESCRIPTION (provided by applicant): 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. Activitated 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.