Mechanisms of Resistance to Cell Cycle Checkpoint Kinase Inhibitors

描述（由申请人提供）：DNA损伤抗癌剂在整个细胞周期的各个检查点诱导阻滞。这种保护机制使细胞有时间修复损伤。UCN-01（7-羟基星孢素）被鉴定为DNA损伤诱导的S和G2阻滞的有效抑制剂，从而引起细胞杀伤的显着增强。p53肿瘤抑制蛋白可阻止ucn -01介导的正常细胞凋亡。因此，UCN-01可能选择性地增强肿瘤的化疗，同时保留正常组织。然而，一些p53缺陷型肿瘤对UCN-01也有耐药性，而一些p53野生型肿瘤对UCN-01敏感。这就引出了本提案中的主要问题：什么是对检查点抑制剂反应的决定因素？Aim 1将专注于p53缺陷的肿瘤细胞系，并研究Chk1（被UCN-01抑制）和其他检查点激酶在阻止细胞周期进展中的作用。各种细胞系对不同检查点抑制剂的反应将被评估。将分析三种耐药肿瘤细胞模型的替代激酶，解释它们对Chk1和Chk2抑制剂的耐药性；候选激酶包括hSAD1、PLK3、MAPKAPK2和JNK。为了确认每个激酶在检查点调节中的作用，将产生被siRNA阻止激酶表达的细胞系。Aim 2将解决为什么一些p53野生型肿瘤保留对Chk1抑制剂的敏感性，尽管事实上非致瘤性系具有耐药性。最近的研究结果表明，p53通过基因激活（p21waf1）和抑制（cyclin B）来调节检查点，并且在ucn -01敏感的p53野生型肿瘤中，这两种蛋白的调节都是有缺陷的；p21在S期阻滞期间不能被诱导，而cyclin B在G2期阻滞期间不能被抑制。这两种蛋白的转录和翻译后调控将被研究，并对其差异调控的贡献者进行评估。实验方法将包括通过启动子分析和染色质免疫沉淀分析来解剖通路。随着新的检查点抑制剂进入临床试验，这些研究的结果将为患者分层提供基础，从而提高开发成功治疗方案的可能性。对于那些没有反应的肿瘤，这些实验可能会确定药物发现的替代靶点。

DESCRIPTION (provided by applicant): DNA damaging anticancer agents induce arrest at various checkpoints throughout the cell cycle. This protective mechanism allows cells time to repair damage before progressing. UCN-01 (7-hydroxy-staurosporine) was identified as a potent inhibitor of the DNA damage-induced S and G2 arrest, thereby causing a marked enhancement in cell killing. UCN-01-mediated abrogation of normal cells is prevented by the p53 tumor suppressor protein. Thus, UCN-01 may selectively enhance chemotherapy in the tumor while sparing normal tissue. However, some p53-defective tumors are also resistant to UCN-01, while some p53-wildtype tumors are sensitive. This leads to the major question in this proposal: what are the determinants of response to checkpoint inhibitors? Aim 1 will focus on p53-defective tumor cell lines and investigate the role of Chk1 (inhibited by UCN-01) and other checkpoint kinases in arresting cell cycle progression. The response of various cell lines to different checkpoint inhibitors will be assessed. Three resistant tumor cell models will be analyzed for alternate kinases that explain their resistance to Chk1 and Chk2 inhibitors; candidate kinases include hSAD1, PLK3, MAPKAPK2 and JNK. To confirm the role of each kinase in checkpoint regulation, cell lines will be generated in which the kinase expression is prevented by siRNA. Aim 2 will address the question as to why some p53-wildtype tumors retain sensitivity to Chk1 inhibitors despite the fact that non-tumorigenic lines are resistant. Recent results demonstrate that p53 regulates the checkpoint through both gene activation (p21waf1) and repression (cyclin B) and that regulation of both of these proteins is defective in UCN-01-sensitive p53 wildtype tumors; p21 fails to be induced during S phase arrest, while cyclin B fails to be repressed during G2 arrest. The transcriptional and post-translational regulation of these two proteins will be studied and contributors to their differential regulation assessed. Experimental approaches will include dissection of the pathways through promoter analysis and chromatin immunoprecipitation assays. As novel checkpoint inhibitors enter clinical trial, the results of these studies will provide a basis upon which to stratify patients and thereby enhance the probability of developing a successful therapeutic regimen. For those tumors in which a response is not indicated, these experiments will likely identify alternate targets for drug discovery.