DNA Repair, Cell Cycle Checkpoints and Apoptosis as Targets for Anticancer Drugs

DNA 修复、细胞周期检查点和细胞凋亡作为抗癌药物的靶点

• 批准号: 8348897
• 负责人: YVES POMMIER
• 金额: $ 115.79万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348897
• 关键词: Alkaloids; Alkylating Agents; Antineoplastic Agents; Apoptosis; Apoptotic; Area; Arsenic Trioxide; Ataxia Telangiectasia; Base Excision Repairs; Binding; Bioinformatics; Biological Markers; Bleomycin; CCR; Camptothecin; Cancer cell line; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Line; Cells; Cellular Assay; Chemicals; Chromatin; Chromosome Condensation; Chromosomes; Clinical; Clinical Trials; Code; Collaborations; Communities; Complex; Cyclin-Dependent Kinase Inhibitor; Cyclins; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA-dependent protein kinase; Data; Databases; Defect; Development; Developmental Therapeutics Program; Drug Combinations; Drug Delivery Systems; Drug usage; Etoposide; Event; Exons; FDA approved; Family; Gene Expression; Genes; Genome Stability; Genomics; Goals; Histones; Laboratories; Lesion; Malignant Neoplasms; Manuscripts; Marines; Measures; Mediating; Microtubules; Mitochondria; Mitochondrial DNA; Molecular; Molecular Target; Monitor; Mutation; Normal Cell; Nuclear; Oligonucleotides; Oncogenes; Paclitaxel; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phase; Phosphorylation; Physical condensation; Platinum; Play; Poly(ADP-ribose) Polymerases; Preclinical Drug Evaluation; Proteins; Proteomics; Publications; Published Comment; Reactive Oxygen Species; Recombinants; Reporting; Resolution; Role; Stimulus; Systems Biology; TDP-1; TNFSF10 gene; Therapeutic; Time; Topoisomerase; Topoisomerase Inhibitors; Type I DNA Topoisomerases; United States National Institutes of Health; Variant; Vinblastine; Work; XRCC1 gene; Xeroderma Pigmentosum; analog; austin; cancer cell; cell type; chemotherapeutic agent; chromatin modification; cofactor; drug discovery; exome; high throughput screening; human TOP1 protein; inhibitor/antagonist; interest; lasonolide A; mRNA Expression; marine natural product; molecular marker; mutant; neoplastic cell; novel; overexpression; pre-clinical; premature; programs; repair enzyme; repaired; response; therapeutic development; tool; tumor; tyrosyl-DNA phosphodiesterase; vector; web site

Because most cancers have alterations in DNA repair pathways, cell cycle checkpoint pathways (p53, pRb, Chk2) and cell cycle machinery (BLM, cyclins, cyclin-dependent kinase inhibitors, such as p16), we are dissecting the alterations that are most relevant for DNA targeted anticancer agents, especially topoisomerase inhibitors, and developing inhibitors of DNA repair and cell cycle checkpoints as novel anticancer agents. DNA repair defects not only predispose to cancers (for instance BLM, Mre11, Xeroderma Pigmentosum and ataxia Telangiectasia), but also play an important role in the response of cancer cells to treatments that target DNA and chromatin. We have set up high-throughput screens for inhibitors of tyrosyl-DNA phosphodiesterase 1 (Tdp1), an enzyme that repairs topoisomerase I-mediated DNA damage. We are identifying Tdp1 inhibitors with the goal of finding new drugs with therapeutic potential. High throughput screens have been set up with the NIH National Chemical Genomic Center (NCGC; Dr. Christopher Austin) and the CCR Molecular Therapeutics Drug Discovery Program (MTDP; Dr. Barry O Keefe). We have also shown that Tdp1 inhibitors should be synergistic in combination with Top1-targeted agents and a range of anticancer drugs including bleomycin, etoposide and alkylating agents. We have also shown that Tdp1 is regulated in response to DNA damage. Tdp1 phosphorylation by ATM and DNA-PK stabilizes Tdp1 and promotes the binding of Tdp1 to the DNA repair base excision factor, XRCC1. In a more recent study, we have also shown that Tdp1 enters mitochondria and is critical for the repair of mitochondrial DNA. This is especially important because mitochondria contain their own topoisomerase (Top1mt), which was discovered in our laboratory, and because mitochondria produce oxygen radicals and produce mitochondrial DNA damage, which is repaired by Tdp1. We are investigating the role and cancer-specific alterations of Chk2 in cell cycle checkpoint response and genomic stability. We just finished a study (available online at Oncogene) showing for the first time the differential status of Chk2 (determined by proteomic, phosphoproteomic, gene expression and exome sequencing) in the 60 cell lines of the NCI-DTP screen. Those data demonstrate that cancer cells belong to one of at least 2 groups depending on their Chk2 status. One group consists of cells with Chk2 inactivation. Those cells include all the p53 wild-type cells. The other group consists of cells with endogenous Chk2 activation/phosphorylation by ATM/ATR/DNA-PK. Those cells are all p53 mutants and probably require Chk2 to survive. We have also set up a high throughput screen to discover Chk2 inhibitors (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI, and colleagues at Provid Pharma) and discovered a novel family of Chk2 inhibitors, the bis-guanidylhydrazones. These new drugs act as competitive ATP inhibitors against Chk2. Analogs have been synthesized and co-crystallized with Chk2 in collaboration with David Waugh (CCR). Cellular assays have been developed to measure Chk2 inhibition in cells and to determine whether Chk2 inhibitors can be used to synergize with Top1 inhibitors and other currently available chemotherapeutic agents (microtubule inhibitors). We have proposed that Chk2 inhibitors could be selectively active against tumor cells overexpressing activated Chk2. To approach and study the pathways involved in cancer from a global system biology viewpoint, we are investigating and charactering the 60 cancer cell lines (The NCI-60) from NCI Developmental Therapeutics Program (DTP) in collaboration with our colleagues at DTP and the Meltzer group in CCR. This database is unique in the world because it includes the activity patterns of more than 16,000 drugs including the FDA-approved anticancer drugs. We just finished the full sequencing of all the coding sequences of all genes (exome) for the NCI-60. Our databases will be made available at the LMP Genomics & Bioinformatics Group (GBG) web site: http://discover.nci.nih.gov following publication of our first manuscript. This project takes advantage of the unique databases for the 60 cancer cell lines that constitute the DTP Drug Screen. These databases include several gene expression platforms (Affymetrix and Agilent) for all the genes and all the exons. They also include high resolution SNIPs, array CGH, SKY and chromosome parameters. This year, we implemented two novel databases: a z-score tool that encompasses in one parameter the entire mRNA expression data for any given gene, and a high resolution array CGH (NimbleGen platform). This project is a collaboration between CCR and DCTD. Its uniqueness resides in the high quality genomic databases in the LMP-GBG and the drug responses generated by DCTD. Crossing these various databases (vectors) enables the comparison between gene expression, genomic copy number variants (CNV), mutations (exome sequencing) and drug response. This provide unique ways to correlate drug response with specific genes and genes to genes. We are studying several new drugs in preclinical and early clinical development including agents from the NCI-Developmental Therapeutics Program (DTP). We are focusing on drugs that alter chromatin and cell cycle progression. We are currently looking for the molecular target of lasonolide A, a marine natural product that produces chromatin condensation. We have also characterized the molecular pharmacology of the novel poly(ADPribose) polymerase (PARP) inhibitor, Veliparib (ABT-888) and elucidated the close relationship between PARP and Tdp1 in the repair of topoisomerase-induced DNA damage. Our studies on apoptosis are focused on chromatin modifications. We were the first to demonstrate that one of the early events in apoptosis is the induction of apoptotic topoisomerase I-DNA complexes. We, and others have found that the apoptotic Top1-DNA complexes are induced by a variety of apoptotic stimuli: arsenic trioxide, etoposide, camptothecin, platinum derivatives, taxol, and vinblastine. Our working hypothesis that these apoptotic Top1-DNA complexes are produced by oxidative lesion of genomic DNA, which trap Top1 bound to chromatin. Apoptotic Top1-DNA complexes in turn activate additional apoptotic responses/pathways and might represent an irreversible apoptotic activation loop. To further elucidate the molecular events induced by the apoptotic program, we have focused our recent studies on nuclear alterations produced by TRAIL, which is in clinical trials. We were the first to report the induction of a novel chromatin alteration in early apoptosis: the apoptotic ring. We have demonstrated that the apoptotic ring contains a subset of the DNA damage response (DDR) proteins. This could have two implications. From a basic standpoint, the apoptotic ring may be used to better understand the chromatin changes that take place during programmed cell death. From a translational standpoint, the apoptotic ring could be used to score tumors that respond to TRAIL and other agents that act against cancer cells by inducing apoptosis.

由于大多数癌症在 DNA 修复途径、细胞周期检查点途径（p53、pRb、Chk2）和细胞周期机制（BLM、细胞周期蛋白、细胞周期蛋白依赖性激酶抑制剂，如 p16）方面发生改变，因此我们正在剖析与 DNA 靶向抗癌药物（尤其是拓扑异构酶抑制剂）最相关的改变，并开发 DNA 修复和细胞周期抑制剂 检查点作为新型抗癌药物。 DNA 修复缺陷不仅容易患癌症（例如 BLM、Mre11、色素性干皮病和毛细血管扩张性共济失调），而且在癌细胞对针对 DNA 和染色质的治疗的反应中发挥着重要作用。我们已经建立了酪氨酰 DNA 磷酸二酯酶 1 (Tdp1) 抑制剂的高通量筛选，Tdp1 是一种修复拓扑异构酶 I 介导的 DNA 损伤的酶。我们正在鉴定 Tdp1 抑制剂，目标是寻找具有治疗潜力的新药。 NIH 国家化学基因组中心 (NCGC；Christopher Austin 博士) 和 CCR 分子治疗药物发现计划 (MTDP；Barry O Keefe 博士) 已建立高通量筛选。我们还表明，Tdp1 抑制剂应与 Top1 靶向药物和一系列抗癌药物（包括博莱霉素、依托泊苷和烷化剂）联合使用时具有协同作用。我们还表明 Tdp1 在 DNA 损伤反应中受到调节。 ATM 和 DNA-PK 使 Tdp1 磷酸化，稳定 Tdp1 并促进 Tdp1 与 DNA 修复碱基切除因子 XRCC1 结合。在最近的一项研究中，我们还表明 Tdp1 进入线粒体并且对于线粒体 DNA 的修复至关重要。这一点尤其重要，因为线粒体含有自己的拓扑异构酶（Top1mt），这是我们实验室发现的，而且线粒体会产生氧自由基并产生线粒体DNA损伤，而线粒体DNA损伤是由Tdp1修复的。 我们正在研究 Chk2 在细胞周期检查点反应和基因组稳定性中的作用和癌症特异性改变。我们刚刚完成了一项研究（可在 Oncogene 在线获取），首次显示了 NCI-DTP 筛选的 60 个细胞系中 Chk2 的差异状态（通过蛋白质组、磷酸蛋白质组、基因表达和外显子组测序确定）。这些数据表明，癌细胞根据其 Chk2 状态属于至少 2 个组中的一组。一组由 Chk2 失活的细胞组成。这些细胞包括所有 p53 野生型细胞。另一组由通过 ATM/ATR/DNA-PK 进行内源 Chk2 激活/磷酸化的细胞组成。这些细胞都是 p53 突变体，可能需要 Chk2 才能生存。我们还建立了高通量筛选来发现 Chk2 抑制剂（与 Shoemaker 博士和 Scudiero 博士、DTP、NCI 以及 Provid Pharma 的同事合作），并发现了 Chk2 抑制剂的新家族，即双胍基腙。这些新药可作为 Chk2 的竞争性 ATP 抑制剂。与 David Waugh (CCR) 合作，合成了类似物并与 Chk2 共结晶。细胞测定已被开发用于测量细胞中的 Chk2 抑制，并确定 Chk2 抑制剂是否可用于与 Top1 抑制剂和其他现有化疗药物（微管抑制剂）协同作用。我们提出 Chk2 抑制剂可以选择性地对抗过度表达激活 Chk2 的肿瘤细胞。为了从全球系统生物学的角度探讨和研究涉及癌症的通路，我们正在与 DTP 的同事和 CCR 的 Meltzer 小组合作，对来自 NCI 发展治疗计划 (DTP) 的 60 种癌细胞系 (NCI-60) 进行研究和表征。该数据库在世界上是独一无二的，因为它包含超过 16,000 种药物的活性模式，其中包括 FDA 批准的抗癌药物。我们刚刚完成了 NCI-60 所有基因（外显子组）所有编码序列的完整测序。在我们的第一篇手稿发表后，我们的数据库将在 LMP 基因组学和生物信息学组 (GBG) 网站上提供：http://discover.nci.nih.gov。该项目利用构成 DTP 药物筛选的 60 种癌细胞系的独特数据库。这些数据库包括针对所有基因和所有外显子的多个基因表达平台（Affymetrix 和 Agilent）。它们还包括高分辨率 SNIP、阵列 CGH、SKY 和染色体参数。今年，我们实施了两个新颖的数据库：一个 z 评分工具，在一个参数中包含任何给定基因的整个 mRNA 表达数据，以及一个高分辨率阵列 CGH（NimbleGen 平台）。该项目是 CCR 和 DCTD 之间的合作。其独特之处在于 LMP-GBG 中的高质量基因组数据库以及 DCTD 生成的药物反应。交叉这些不同的数据库（载体）可以比较基因表达、基因组拷贝数变异（CNV）、突变（外显子组测序）和药物反应。这提供了将药物反应与特定基因以及基因与基因相关联的独特方法。我们正在研究几种处于临床前和早期临床开发阶段的新药，包括来自 NCI 开发治疗计划 (DTP) 的药物。我们专注于改变染色质和细胞周期进程的药物。我们目前正在寻找 Lasonolide A 的分子靶标，Lasonolide A 是一种产生染色质缩合的海洋天然产物。我们还表征了新型聚（ADP核糖）聚合酶（PARP）抑制剂 Veliparib (ABT-888) 的分子药理学，并阐明了 PARP 和 Tdp1 在修复拓扑异构酶诱导的 DNA 损伤中的密切关系。我们对细胞凋亡的研究主要集中在染色质修饰上。我们是第一个证明细胞凋亡的早期事件之一是诱导凋亡拓扑异构酶 I-DNA 复合物的人。我们和其他人发现凋亡的 Top1-DNA 复合物是由多种凋亡刺激物诱导的：三氧化二砷、依托泊苷、喜树碱、铂衍生物、紫杉醇和长春花碱。我们的工作假设是，这些凋亡的 Top1-DNA 复合物是由基因组 DNA 的氧化损伤产生的，它捕获了与染色质结合的 Top1。凋亡 Top1-DNA 复合物反过来激活额外的凋亡反应/途径，并可能代表不可逆的凋亡激活循环。为了进一步阐明细胞凋亡程序诱导的分子事件，我们最近的研究重点是 TRAIL 产生的核改变，该药物正在进行临床试验。我们是第一个报告早期细胞凋亡中诱导新的染色质改变：凋亡环的人。我们已经证明凋亡环包含 DNA 损伤反应 (DDR) 蛋白的一个子集。这可能有两个含义。从基本角度来看，凋亡环可用于更好地理解程序性细胞死亡过程中发生的染色质变化。从翻译的角度来看，凋亡环可用于对对 TRAIL 和其他通过诱导细胞凋亡来对抗癌细胞的药物有反应的肿瘤进行评分。