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

• 批准号: 7864084
• 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
• 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/7864084
• 关键词: 7-Hydroxystaurosporine; Address; Antineoplastic Agents; Binding; Biological Assay; Caffeine; Cell Cycle; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Line; Cell model; Cells; Chemotherapy-Oncologic Procedure; Cisplatin; Clinical Trials; 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; Regimen; Regulation; Regulatory Pathway; Reporting; Repression; Research; Resistance; Role; Serum; Small Interfering RNA; Staurosporine; Structure; Structure-Activity Relationship; T47D; TP53 gene; Therapeutic; Time; Treatment Efficacy; Tumor Cell Line; Tumor Suppressor Proteins; analog; base; cell killing; chemotherapy; chromatin immunoprecipitation; drug discovery; effective therapy; human CHEK1 protein; immortalized cell; improved; in vivo; inhibitor/antagonist; kinase inhibitor; mutant; neoplastic cell; novel; prevent; programs; promoter; protective effect; repaired; research study; resistance mechanism; response; tumor; tumorigenic

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（由申请方提供）：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在进展之前修复损伤。UCN-01（7-羟基-星形孢菌素）被鉴定为DNA损伤诱导的S和G2停滞的有效抑制剂，从而引起细胞杀伤的显著增强。UCN-01介导的正常细胞的废除被p53肿瘤抑制蛋白阻止。因此，UCN-01可以选择性地增强肿瘤中的化疗，同时保留正常组织。然而，一些p53缺陷型肿瘤也对UCN-01具有抗性，而一些p53野生型肿瘤则敏感。这就引出了这个提议中的主要问题：对检查点抑制剂反应的决定因素是什么？目的1将关注p53缺陷的肿瘤细胞系，并研究Chk 1（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。将评估各种细胞系对不同检查点抑制剂的反应。将分析三种耐药肿瘤细胞模型中解释其对Chk 1和Chk 2抑制剂耐药的替代激酶;候选激酶包括hSAD 1、PLK 3、MAPKAPK 2和JNK。为了证实每种激酶在检查点调节中的作用，将产生其中激酶表达被siRNA阻止的细胞系。目的2将解决的问题，为什么一些p53-野生型肿瘤保持敏感性Chk 1抑制剂，尽管事实上，非致瘤株耐药。最近的研究结果表明，p53通过基因激活（p21 waf 1）和抑制（细胞周期蛋白B）调节检查点，这两种蛋白的调节在UCN-01敏感的p53野生型肿瘤中是有缺陷的; p21在S期阻滞期间不能被诱导，而细胞周期蛋白B在G2期阻滞期间不能被抑制。这两种蛋白质的转录和翻译后调控将进行研究，并评估其差异调节的贡献者。实验方法将包括通过启动子分析和染色质免疫沉淀分析的途径解剖。随着新型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。