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

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）中都有改变，我们正在分析与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修复抑制剂和细胞周期检查点作为新型抗癌药物。DNA修复缺陷不仅易导致癌症（例如BLM、Mre11、着色性干皮病和共济失调毛细血管扩张症），而且在癌细胞对靶向DNA和染色质治疗的反应中也起着重要作用。我们建立了酪氨酸-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，目的是寻找具有治疗潜力的新药。高通量筛选已经与美国国立卫生研究院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）建立。我们还表明，Tdp1抑制剂与top1靶向药物和一系列抗癌药物（包括博莱霉素、依托泊苷和烷基化剂）联合使用时应具有协同作用。我们还表明，Tdp1在DNA损伤时受到调节。ATM和DNA- pk对Tdp1的磷酸化稳定了Tdp1，促进了Tdp1与DNA修复碱基切除因子XRCC1的结合。在最近的一项研究中，我们也发现Tdp1进入线粒体，对线粒体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，因为线粒体产生氧自由基并产生线粒体DNA损伤，这是由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网站上获得），首次显示了Chk2（通过蛋白质组学、磷蛋白质组学、基因表达和外显子组测序确定）在NCI-DTP筛选的60个细胞系中的差异状态。这些数据表明，癌细胞根据其Chk2状态至少属于两种类型之一。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。另一组由内源性Chk2被ATM/ATR/DNA-PK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才能存活。我们还建立了一个高通量筛选发现Chk2抑制剂(与博士合作。Shoemaker和Scudiero， DTP， NCI，以及Provid Pharma的同事)发现了一个新的Chk2抑制剂家族，双胍基腙。这些新药作为竞争性ATP抑制剂对抗Chk2。与David Waugh （CCR）合作合成了类似物并与Chk2共结晶。已经开发了细胞测定方法来测量Chk2在细胞中的抑制作用，并确定Chk2抑制剂是否可以与Top1抑制剂和其他目前可用的化疗药物（微管抑制剂）协同作用。我们提出Chk2抑制剂可以选择性地抑制过度表达活化Chk2的肿瘤细胞。为了从全球系统生物学的角度研究癌症的通路，我们正在与NCI发展治疗计划（DTP）的同事和CCR的Meltzer小组合作，调查和描述来自NCI发展治疗计划（DTP）的60种癌细胞系（NCI-60）。这个数据库在世界上是独一无二的，因为它包含了超过16000种药物的活性模式，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）编码序列的全序列测序。我们的数据库将在LMP基因组学和生物信息学集团（GBG）的网站上提供：http://discover.nci.nih.gov我们的第一篇手稿发表后。该项目利用了构成DTP药物筛选的60种癌细胞系的独特数据库。这些数据库包括所有基因和所有外显子的几个基因表达平台（Affymetrix和Agilent）。它们还包括高分辨率SNIPs，阵列CGH， SKY和染色体参数。今年，我们实现了两个新的数据库：一个包含任何给定基因的整个mRNA表达数据的z-score工具，以及一个高分辨率阵列CGH （NimbleGen平台）。这个项目是CCR和DCTD的合作项目。它的独特性在于LMP-GBG的高质量基因组数据库和DCTD产生的药物反应。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。这提供了独特的方法来关联药物反应与特定的基因和基因对基因。我们正在研究几种临床前和早期临床开发的新药，包括nci -发育治疗项目（DTP）的药物。我们专注于研究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的分子靶点，这是一种产生染色质凝结的海洋天然产物。我们还表征了新型聚磷酸核糖聚合酶（PARP）抑制剂Veliparib （ABT-888）的分子药理学，并阐明了PARP和Tdp1在拓扑异构酶诱导的DNA损伤修复中的密切关系。我们对细胞凋亡的研究主要集中在染色质修饰上。我们是第一个证明凋亡的早期事件之一是诱导凋亡拓扑异构酶I-DNA复合物。我们和其他人发现，凋亡的Top1-DNA复合物可由多种凋亡刺激诱导：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，它将Top1束缚在染色质上。凋亡的Top1-DNA复合体反过来激活额外的凋亡反应/途径，并可能代表一个不可逆的凋亡激活环。为了进一步阐明凋亡程序诱导的分子事件，我们最近的研究集中在临床试验中TRAIL产生的核改变上。我们首次报道了在早期细胞凋亡中诱导一种新的染色质改变：凋亡环。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。从基本观点来看，凋亡环可以用来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，凋亡环可以用来评分对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。

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.