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: 7592558
• 负责人: YVES POMMIER
• 金额: $ 73.13万
• 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/7592558
• 关键词: Antineoplastic Agents; Apoptosis; Apoptotic; Area; Arsenic Trioxide; BRCA1 gene; BRCA2 gene; Behavior; Binding; Bioinformatics; Biological; Biological Markers; Bloom Syndrome; CCR; Camptothecin; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Death; Cell Line; Cells; Chromatin; Cisplatin; Class; Clinical; Clinical Protocols; Clinical Trials; Collaborations; Colon Carcinoma; Colorectal; Colorectal Cancer; Complement; Complex; Cyclin-Dependent Kinase Inhibitor; Cyclins; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Single Strand Break; DNA biosynthesis; DNA-dependent protein kinase; Databases; Defect; Dependence; Development; Developmental Therapeutics Program; Down-Regulation; Drug Combinations; ERCC2 gene; Ecteinascidin 743; Etoposide; Event; Ewings sarcoma; Frequencies; Genetic Recombination; Genetic Transcription; Genomic Instability; Genomics; Goals; Guanine; HCT116 Cells; HT29 Cells; Hereditary Disease; Histones; Hypersensitivity; International; Journals; Lesion; Lymphoma; Malignant Childhood Neoplasm; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of testis; Maps; Mediating; Minor Groove; Mismatch Repair; Modification; Molecular; Monitor; Mutation; NSC 686288; Normal Cell; Nuclear; Nucleotide Excision Repair; Ovarian; Paclitaxel; Pathway interactions; Patients; Penetrance; Pharmaceutical Preparations; Pharmacology; Phase; Phosphorylation; Platinum; Poly(ADP-ribose) Polymerases; Preclinical Drug Evaluation; Predisposition; Proteasome Inhibition; Protein Overexpression; Publishing; Recombinant Proteins; Reporting; Resistance; Role; Sampling; Simulate; Site; Small Interfering RNA; Solid Neoplasm; Stimulus; TNFSF10 gene; TP53 gene; Therapeutic; Topoisomerase; Topoisomerase Inhibitors; Topoisomerase-I Inhibitor; Type I DNA Topoisomerases; Vinblastine; Work; Yondelis; adduct; batracylin; cancer cell; cofactor; colon cancer cell line; high throughput screening; human H2AX protein; human TOP1 protein; indolicidin; inhibitor/antagonist; insight; leukemia; malignant breast neoplasm; melanoma; member; novel; polypeptide; pre-clinical; programs; repaired; response; sarcoma; sulfotransferase; therapy resistant; tumor; tyrosyl-DNA phosphodiesterase

Deficiencies in DNA repair (mismatch repair deficiencies in colon cancers, nucleotide excision repair in ovarian and testicular cancers, and in melanomas), deficiencies in cell cycle checkpoints (Rb, p53, BRCA1, BRCA2, and Chk2 deficiencies in solid tumors), and apoptosis (APC mutations in colon cancers, Bcr-Abl recombinations in leukemia, Bcl-2 overexpression in lymphomas) promote cancers. They also contribute to therapeutic responses as normal cells have more robust alternative (redundant) pathways. The Mre11-Rad50-Nbs1 (MRN) complex binds DNA double-strand breaks to repair DNA and activate checkpoints. We recently reported MRN deficiency in 3 of the 7 colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study MRNs involvement in replication-mediated DNA double-strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double-strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ATM-dependent, and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 downregulation by siRNA in HT29 cells inhibited Chk2 activation and downregulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in 3 out of the 4 MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, 6 out of the 7 colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double-strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double-strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers. To integrate these alterations within the emerging cellular network of molecular pathways, we have developed a mapping convention that can be visually represented as molecular interaction maps (MIMs). These maps are being presented by different members of the LMP (Dr. Kohn, Dr. Aladjem, and Dr. Pommier) at different meetings. They are published in international journals with high impact factors and we have developed an interactive Website in collaboration with the LMP Bioinformatic group (Dr. Weinstein) (http://discover.nci.nih.gov/mim). 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 such as aminoflavone. Aminoflavone is beginning clinical trials and we found that aminoflavone induces replication double-strand breaks and histone H2AX phosphorylation (gamma-H2AX). Hence, gamma-H2AX can be used as a biomarker to monitor aminoflavone activity in tumor samples. Using the NCI 60 cell line database we found that sulfotransferase expression is highly correlated with aminoflavone activity and can be used to select patients who should benefit from aminoflavone. We are continuing our studies with ecteinascidin 743 (Et743 - Yondelis) (NSC 648766). Clinical responses to Et743 have been observed in sarcomas, which are notoriously resistant to therapy, as well as in ovarian and breast cancer. Et743 differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively Et743 traps the transcription-coupled NER (TC-NER). Thus, Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability. The complementary between the activities of Et-743 and cisplatin with respect to TC-NER suggests the use of Et743 in cisplatin-resistant tumors and vice-versa. A clinical protocol has been proposed for a clinical trial of Et743 in pediatric cancers (Collaboration with Dr. Frank Balis, CCR, NCI). Further molecular studies are planned to determine the transcription- and the strand-specific-dependence of the DNA single-strand breaks induced by Et-743. We are also looking at TC-NER-dependent transcription inhibition by microarray analyses using NER-deficient, XPD, and XPD-complemented cells and Ewing sarcoma cell lines. Because most cancers have alterations in the cell cycle checkpoint pathways (p53, pRb) and cell cycle machinery (cyclins, cyclin-dependent kinase inhibitors such as p16), we are exploring inhibitors of cell cycle checkpoints as novel anticancer agents. We are investigating the role of Chk2 in cell cycle checkpoint response in cancer cells. We have expressed Chk2 as a recombinant protein and set up a high throughput screen to discover Chk2 inhibitors (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI). We have also set up another mid- high-throughput screen for inhibitors of Tdp1. Tdp1 inhibitors should be synergistic in combination with Top1 inhibitors. Our studies on apoptosis are focused on chromatin modifications. We found that one of the early events in apoptosis is the induction of apoptotic Topoisomerase I-DNA complexes. The apoptotic Topoisomerase I-DNA complexes are induced by a variety of apoptotic stimuli: arsenic trioxide, etoposide, camptothecin, platinum derivatives, taxol, vinblastine. Our working hypothesis that these apoptotic Topoisomerase I-DNA complexes are produced by oxidative lesion of genomic DNA, which trap Topoisomerase I bound to chromatin. Apoptotic Topoisomerase I-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 are focusing on nuclear alterations produced by TRAIL, which is in clinical trials

DNA修复缺陷（结肠癌中的错配修复缺陷、卵巢癌和睾丸癌以及黑色素瘤中的核苷酸切除修复缺陷）、细胞周期检查点缺陷（实体瘤中的Rb、p53、BRCA1、BRCA2和Chk2缺陷）和细胞凋亡缺陷（结肠癌中的APC突变、白血病中的Bcr-Abl重组、淋巴瘤中的Bcl-2过表达）促进癌症的发生。它们也有助于治疗反应，因为正常细胞有更强大的替代（冗余）途径。Mre11-Rad50-Nbs1 （MRN）复合体结合DNA双链断裂修复DNA并激活检查点。我们最近报道了NCI抗癌药物筛选的7个结肠癌细胞系中有3个存在MRN缺陷。为了研究MRNs参与复制介导的DNA双链断裂，我们检测了喜树碱的检查点反应，喜树碱在复制叉与拓扑异构酶I切割复合物碰撞后诱导复制介导的DNA双链断裂。MRN缺陷细胞缺乏Chk2激活，而Chk1激活与MRN无关。Chk2的激活依赖于atm，并与Mre11和Nbs1的磷酸化有关。mrn缺失的HCT116细胞中Mre11的补充恢复了Chk2的激活以及Rad50和Nbs1的水平。相反，HT29细胞中siRNA下调Mre11抑制Chk2激活，下调Nbs1和Rad50。蛋白酶体抑制也恢复了HCT116细胞中的Rad50和Nbs1水平，这表明Mre11稳定了Rad50和Nbs1。由于Chk2水平低，在4个精通mrn的结肠直肠癌细胞系中，有3个细胞系的Chk2激活也存在缺陷。因此，NCI抗癌药物筛选的7个结肠癌细胞系中有6个在响应复制介导的DNA双链断裂时功能上存在chk2缺陷。我们提出Mre11稳定Nbs1和Rad50， MRN激活ATM下游的Chk2，以响应复制介导的DNA双链断裂。HCT116中Chk2缺乏与s期检查点缺陷、G2阻滞延长和喜树碱过敏相关。MRN和Chk2缺陷的高频率可能导致结肠直肠癌中喜树碱的基因组不稳定性和治疗反应。为了将这些变化整合到新兴的细胞分子通路网络中，我们开发了一种作图惯例，可以直观地表示为分子相互作用图（MIMs）。这些地图由LMP的不同成员（Kohn博士、Aladjem博士和Pommier博士）在不同的会议上展示。它们发表在具有高影响因子的国际期刊上，我们与LMP生物信息学小组（Weinstein博士）合作开发了一个互动网站（http://discover.nci.nih.gov/mim）。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。我们专注于改变染色质和细胞周期进程的药物，如氨基黄酮。氨基黄酮已经开始临床试验，我们发现氨基黄酮诱导复制双链断裂和组蛋白H2AX磷酸化（γ -H2AX）。因此，γ - h2ax可作为监测肿瘤样品中氨基黄酮活性的生物标志物。利用NCI 60细胞系数据库，我们发现硫转移酶的表达与氨基黄酮活性高度相关，可以用来选择应该从氨基黄酮中获益的患者。我们正在继续对ecteinascidin 743 (Et743 - Yondelis) （NSC 648766）的研究。对Et743的临床反应已经在肉瘤以及卵巢癌和乳腺癌中观察到，肉瘤是出了名的耐药。Et743不同于其他临床使用的抗癌药物，因为它在DNA次要凹槽的特定鸟嘌呤上形成共价加合物，并且因为它选择性地捕获转录偶联的NER （TC-NER）。因此，Et743定义了一类新的抗癌药物，其中增强的抗增殖活性与增强的细胞dna修复能力并行。Et-743和顺铂在TC-NER方面的互补性表明，Et743可用于顺铂耐药肿瘤，反之亦然。Et743在儿科癌症的临床试验已提出临床方案（与Frank Balis博士合作，CCR， NCI）。进一步的分子研究计划确定Et-743诱导的DNA单链断裂的转录特异性和链特异性依赖。我们还通过使用ner缺陷、XPD和XPD补充细胞和Ewing肉瘤细胞系进行微阵列分析，研究tc - ner依赖性转录抑制。由于大多数癌症在细胞周期检查点途径（p53, pRb）和细胞周期机制（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，我们正在探索细胞周期检查点抑制剂作为新型抗癌药物。我们正在研究Chk2在癌细胞细胞周期检查点反应中的作用。我们已经将Chk2作为重组蛋白表达，并建立了高通量筛选来发现Chk2抑制剂(与dr。Shoemaker and Scudiero， DTP， NCI)。我们还建立了另一个Tdp1抑制剂的中高通量筛选。Tdp1抑制剂与Top1抑制剂联合应具有协同作用。我们对细胞凋亡的研究主要集中在染色质修饰上。我们发现凋亡的早期事件之一是诱导凋亡的拓扑异构酶I-DNA复合物。凋亡拓扑异构酶I-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、长春花碱。我们的工作假设是，这些凋亡的拓扑异构酶I-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拓扑异构酶I。凋亡拓扑异构酶I-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，我们将重点放在临床试验中TRAIL产生的核改变上