Identifying CDK4 and CDK6 substrates in cancers and cancer therapy

 描述(由申请人提供)：本申请寻求确定在癌症发生和基于细胞周期的癌症治疗中具有关键作用的细胞周期蛋白D依赖的激酶的底物。控制细胞周期的基因突变是癌细胞最常见的基因变化之一。虽然许多细胞周期蛋白与癌症有关，但细胞周期蛋白D依赖的激酶CDK4和CDK6从根本上与癌症的发生有关。CDK4/6活性缺失的突变在肿瘤中很常见，而CDK4/6活性对肿瘤细胞的维持至关重要。CDK4/6在肿瘤发生中的重要作用刺激了药物CDK4/6抑制剂的发展，最终可能实现基于细胞周期的癌症治疗的前景。然而，与其他使许多底物磷酸化的CDK不同，人们所知的CDK4/6底物很少。事实上，几乎所有以前的工作都集中在视网膜母细胞瘤蛋白家族上，而其他已知底物的缺乏限制了人们对CDK4/6相关癌症的机制和针对这些激酶的治疗的深入了解。尽管需要识别与肿瘤发生和治疗相关的CDK4/6底物，但许多技术挑战阻碍了识别这些蛋白质的努力。我们开发了利用ATP类似物敏感(AS)CDK和底物硫代磷酸化的蛋白质组学方法，使我们能够有效地识别CDK底物，并实施了这种称为原位底物检测的方法，以便更接近地模拟CDK底物磷酸化的生理环境。我们现在将应用这些方法来鉴定癌症中的CDK4/6底物，我们的初步研究已经证明了它们能够检测内源性和新的CDK6底物。这项应用的目标是应用这些创新的蛋白质组学方法来识别有助于肿瘤发生的新的CDK4/6底物，以及评估CDK4/6抑制剂的疗效。如果成功，这项研究将确定CDK4/6可能导致癌症发生的新活性，将使未来对CDK4/6相关癌症的机制研究和药理CDK4/6抑制剂成为可能，并可能导致新的治疗靶点。

 DESCRIPTION (provided by applicant): This application seeks to identify substrates of cyclin D-dependent kinases with critical roles in carcinogenesis and cell cycle-based cancer therapy. Mutations in the genes that control the cell cycle are among the most common genetic changes in cancer cells. Although many cell cycle proteins are implicated in cancers, the cyclin D-dependent kinases, CDK4 and CDK6, are fundamentally linked to carcinogenesis. Mutations that deregulate CDK4/6 activity are common in tumors, and CDK4/6 activity is critical for tumor cell maintenance. The essential roles of CDK4/6 in tumorigenesis stimulated the development of pharmacologic CDK4/6 inhibitors that may finally realize the promise of cell cycle-based cancer therapy. However, unlike other CDKs, which phosphorylate many substrates, very few CDK4/6 substrates are known. Indeed, almost all previous work has focused on the Retinoblastoma protein family, and the paucity of other known substrates has limited insights into the mechanisms of CDK4/6- associated cancer and therapy targeting these kinases. Despite the need to identify the CDK4/6 substrates relevant for tumorigenesis and therapy, numerous technical challenges have impeded efforts to identify these proteins. We have developed proteomic methods utilizing ATP analog-sensitive (AS) CDKs and substrate thiophosphorylation that enable us to efficiently identify CDK substrates, and have implemented this approach, termed in situ substrate detection, so as to closely mimic the physiologic environment of CDK substrate phosphorylation. We will now apply these methods to identify CDK4/6 substrates in cancers, and our preliminary studies have demonstrated their ability to detect endogenous and novel CDK6 substrates. This application's goal is to apply these innovative proteomic methods to identify novel CDK4/6 substrates that contribute to tumorigenesis, as well as to evaluate the efficacy of CDK4/6 inhibitors. If successful, this research will define new activities of CDK4/6 that may drive carcinogenesis, will enable future mechanistic studies of CDK4/6-associated cancer and pharmacologic CDK4/6 inhibitors, and may lead to new therapeutic targets.