Mechanisms of Resistance to Cell Cycle Checkpoint Kinase Inhibitors

细胞周期检查点激酶抑制剂的耐药机制

基本信息

批准号：
7257577
负责人：
ALAN R EASTMAN
金额：
$ 30.38万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-10 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7257577
关键词：
7-Hydroxystaurosporine Address Antineoplastic Agents Binding Biological Assay CDKN1A gene Caffeine Cell Cycle Cell Cycle Arrest Cell Cycle Checkpoint Cell Cycle Progression Cell Line Cell model Cells Chemotherapy-Oncologic Procedure Cisplatin Clinical Trials Condition Cyclin B DNA Damage Defect Disease Dissection Drug Combinations G2 Phase G2 Phase Arrest Gene Activation Goals HCT116 Cells Human Incubated Individual Lead Literature MAPK8 gene MAPKAPK2 gene MCF7 cell Mediating Mitosis Normal Cell Normal tissue morphology Outcome PLK3 gene Pathway interactions Patients Pharmaceutical Preparations Phase Phase I Clinical Trials Phenotype Phosphotransferases Plasma Proteins Post-Translational Regulation Probability Proliferating Property Protein p53 Proteins Range Regulation Regulatory Pathway Reporting Repression Research Resistance Role Serum Small Interfering RNA Staurosporine Structure Structure-Activity Relationship T47D TP53 gene Therapeutic Time Treatment Efficacy Treatment Protocols Tumor Cell Line Tumor Suppressor Proteins analog base cell killing chemotherapy chromatin immunoprecipitation drug discovery human CHEK1 protein immortalized cell improved in vivo inhibitor/antagonist kinase inhibitor mutant neoplastic cell novel oncoprotein p21 prevent programs promoter protective effect repaired research study resistance mechanism response tumor tumorigenic

项目摘要

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.

描述（由申请人提供）：在整个细胞周期中，DNA破坏抗癌药在各种检查点引起停滞。这种保护机制使细胞在进行之前有时间修复损坏。 UCN-01（7-羟基星孢菌素）被确定为DNA损伤诱导的S和G2停滞的有效抑制剂，从而导致细胞杀伤的明显增强。 p53肿瘤抑制蛋白可以预防UCN-01介导的正常细胞废除。因此，UCN-01可以选择性地增强肿瘤中的化学疗法，同时保留正常组织。但是，一些p53缺陷的肿瘤也对UCN-01具有抗性，而某些p53-WildType肿瘤对此很敏感。这导致了本提案中的主要问题：对检查点抑制剂的回答的决定因素是什么？ AIM 1将重点放在P53缺陷型肿瘤细胞系上，并研究CHK1（UCN-01抑制）和其他检查点激酶在阻止细胞周期进程中的作用。将评估各种细胞系对不同检查点抑制剂的响应。将分析三个抗性肿瘤细胞模型的替代激酶，以解释其对CHK1和CHK2抑制剂的抗性。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，将产生细胞系，其中siRNA预防激酶表达。 AIM 2将解决一个问题，说明为什么某些p53-wildtype肿瘤仍然对CHK1抑制剂保持敏感性，尽管非肿瘤线具有抗性。最近的结果表明，p53通过基因激活（P21WAF1）和抑制（Cyclin b）调节检查点，并且这两种蛋白质的调节在UCN-01敏感的p53 WildType肿瘤中都是有缺陷的。 p21在S阶段逮捕期间未能诱导，而在G2逮捕期间，Cyclin B未能被抑制。将研究这两种蛋白质的转录和翻译后调节，并评估其差异调节。实验方法将包括通过启动子分析和染色质免疫沉淀测定法解剖途径。随着新型检查点抑制剂进入临床试验，这些研究的结果将提供分层患者的基础，从而提高了开发成功治疗方案的可能性。对于未表明反应的肿瘤，这些实验可能会识别出药物发现的替代靶标。