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: 10262019
• 负责人: YVES POMMIER
• 金额: $ 197.05万
• 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/10262019
• 关键词: Acyclovir; Advanced Malignant Neoplasm; Antibody-drug conjugates; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Ataxia Telangiectasia; Binding; Biochemical; Biological Assay; Biology; CCR; CHEK1 gene; Camptothecin; Cancer cell line; Cell Cycle Checkpoint; Cell Death; Cell Line; Cell Survival; Cells; Chromatin; Cleaved cell; Clinic; Clinical; Clinical Pharmacology; Clinical Trials; Collaborations; Communities; Complex; Coupled; Cytarabine; DNA; DNA Damage; DNA Repair; DNA Synthesis Inhibitors; DNA replication fork; DNA-protein crosslink; Databases; Deposition; Development; Drug Combinations; ERCC1 gene; Enzymes; Epigenetic Process; Excision; FDA approved; Genes; Genomics; Histone Deacetylase; Histone Deacetylase Inhibitor; Hydrolysis; Immune checkpoint inhibitor; Institutes; Knock-out; Malignant neoplasm of ovary; Manuscripts; Mediating; Molecular; Molecular Mechanisms of Action; Mutation; Normal tissue morphology; Nuclear; Online Systems; Pathway interactions; Patient Selection; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacogenomics; Pharmacology; Phosphodiesterase Inhibitors; Plant Roots; Platinum; Poly(ADP-ribose) Polymerases; Post-Translational Protein Processing; Recombinants; Regulation; Replicon; Reporting; Research; Resistance; Resources; Role; Schedule; Site; TOP2A gene; Testing; Therapeutic Index; Time; Tissues; Topoisomerase; Topoisomerase Inhibitors; Topoisomerase-I Inhibitor; Toxic effect; Tubulin; Tumor Suppressor Proteins; Type I DNA Topoisomerases; Tyrosine; Ubiquitination; Virus Replication; Work; Zidovudine; adduct; arginine methyltransferase; base; biological adaptation to stress; cancer therapy; clinical development; clinical investigation; clinically relevant; drug candidate; drug response prediction; endonuclease; genomic data; genomic predictors; genomic signature; homologous recombination; improved; inhibitor/antagonist; kinase inhibitor; lung small cell carcinoma; malignant breast neoplasm; mitochondrial genome; molecular modeling; multicatalytic endopeptidase complex; novel; nuclease; patient biomarkers; patient response; precision medicine; predicting response; predictive marker; predictive test; protein kinase inhibitor; recruit; repaired; replication stress; response; response biomarker; targeted agent; temozolomide; therapeutic target; tool; tumor; tyrosyl-DNA phosphodiesterase

We are pursuing complementary projects to elucidate the molecular pharmacology of clinically relevant inhibitors of topoisomerases, DNA repair and cell cycle checkpoint inhibitors. Project #1. Repair of topoisomerase cleavage complexes (TOPccs) by tyrosyl-DNA-phosphodiesterases (TDPs), endonucleases and SUMOylation/ubiquitylation Aim 1:Post-translational modifications of TOPccs: TOPccs are excised by two main mechanisms: 1/ hydrolysis of the covalent linkage between the catalytic tyrosine of topoisomerases and the DNA broken end by tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2); 2/ endonuclease cleavage of the DNA fragment adjacent to the TOP1cc by nucleases (Mre11, XPF-ERCC1, XPG...). Because the covalent topoisomerase-tyrosyl-DNA bonds to be cleaved by TDP1 and TDP2 are deep within the TOPccs, TOPccs need to be proteolyzed and/or denatured to provide access to the TDPs (TDP1 and TDP2). We are studying the proteolytic pathways for TOPccs. Our results demonstrate the rapid engagement of the SUMOylation and ubiquitylation pathways, which, in turn drive proteasome-mediated topoisomerase degradation. Aim 2: Biology of TDPs: TDP1 and TDP2 preferentially repair TOP1cc and TOP2cc, respectively. In addition to TOP1cc, TDP1 removes damaged and non-canonical bases and adducts from 3'-DNA ends. This explains why lack of TDP1 sensitizes cells not only to TOP1 inhibitors but also to temozolomide, cytarabine, zidovudine (AZT) and acyclovir. We are studying how TDP1 is regulated and recruited to DNA damaged sites. We previously reported that TDP1 is coupled with PARP1 and that inhibiting PARP1 results in TDP1 inactivation. Also, we previously established that TDP1 excises TOP1cc and TOP1MTcc both in the nuclear and mitochondrial genomes, respectively. We demonstrated that TDP2 also removes TOP2cc in the mitochondrial genome and showed for the first time that the arginine methyltransferase (PRMT5) activates TDP1 by directly binding and methylating TDP1. Aim 3: Pharmacology and targeting of TDPs: The rationale for targeting TDPs is rooted in the emerging importance of TDPs for DNA repair and viral replication, and the potential of TDP inhibitors for anticancer drug combinations. To do so, we are using biochemical assays with recombinant TDP enzymes. We are also taking advantage of TDP1 and TDP2 knockout cell lines, crystallographic determinations and molecular modeling to study the molecular pharmacology of the drug candidates. Project #2. PARP trapping by PARP inhibitors: molecular mechanisms and translational implications PARP inhibitors represent the most advanced cancer therapeutics targeting the DNA damage response. Four inhibitors (olaparib, rucaparib, niraparib and talazoparib) have been approved recently. PARP inhibitors are the first drugs to exploit the concept of synthetic lethality for homologous recombination deficiency (HRD) in the clinic. Our studies revealed 'PARP trapping' as a key mechanism explaining the molecular mechanism of action of PARP inhibitors as anticancer agents. This discovery and our work with talazoparib contributed to the approval of talazoparib for breast and ovarian cancer in 2018. Our studies focus on 1/ the most synergistic combinations with temozolomide and with TOP1 inhibitors, including our non-camptothecin indenoisoquinoline TOP1 inhibitors; 2/ the repair mechanisms and determinants of response to PARP inhibitors beyond homologous recombination (HR; BRCAness). We are currently focusing on the roles of the DNA-protein crosslink protease (Spartan: SPTN), TDPs and ubiquitination for the removal of trapped PARP. Project #3. Patient-derived cancer cell lines to discover and validate novel genomic predictive biomarkers for patient selection and rational drug combinations with TOP1, PARP and DAN damage response (DDR) inhibitors The current lack of predictive biomarkers for widely used DNA-targeted anticancer therapies and the lack of direct correlation between their primary targets and cellular response warrant the need to identify novel DNA damage response (DDR) determinants for predicting drug responses and rationalizing drug combinations. Taking advantage of the extensive NCI-60 drug database ( 40,000 drugs including FDA approved and investigational clinical drugs), whole genomic data and our CellMiner facility, we discovered several novel predictive biomarkers for DNA-targeted agents: SLX4 (FANCP) mutations, ATAD5 (ELG1) mutations, and SLFN11 (Schlafen 11) expression. We have now extended these analyses to tissue-specific cancer cell line databases (NCI Small Cell Lung Cancers), and larger databases (CCLE: MIT-Broad Institute and CGP: MGH-Sanger), and to CCR clinical trials to test predictive biomarker signatures. Those cancer cell line databases have been made widely and freely available to the research community via CellMiner web-based application (http://discover.nci.nih.gov/cellminercdb). During this past year, we have generated a novel database and web-based pharmacogenomic tool for patient-derived small cell lung cancers (SCLC): SCLC-CellMiner in collaboration with the NCI-DTP (Beverly Teicher Molecular Pharmacology group) and John Minna (UTSW). The manuscript and resource have been deposited in BioRxiv and under revision at Cell Press. Project #4. Schlafen 11 (SLFN11) a predictive biomarkers for response to DNA damaging drugs: molecular mechanisms and translational implications SLFN11 as dominant predictor of response to DNA damaging drugs was discovered through our NCI-60 analyses and in parallel by the CCLE teams. SLFN11 determines response to TOP1, TOP2, PARP inhibitors, DNA synthesis inhibitors and platinum derivatives but not to tubulin or protein kinase inhibitors or apoptosis-inducing drugs. SLFN11 is inactivated in approximately 50% of cancer cells lines, making them resistant to DNA damaging agents. Our aims are to elucidate the molecular mechanism of SLFN11 action and regulation, and relevance for patient responses and rationale drug combinations. We discovered a key molecular mechanism of action of SLFN11 by demonstrating that SLFN11 is recruited to DNA damage sites and to stressed replication forks by binding to RPA and the replicative CMG complex. In doing so, SLFN11 blocks elongating replicons and irreversibly blocks replication. We also showed that SLFN11 induces chromatin accessibility and induction of the immediated early response (EIR) stress genes. We propose that SLFN11 acts as a "Restriction Factor" for cells with replicative stress and as "potential tumor suppressor". We have also demonstrated that SLFN11 is inactivated epigenetically in approximately 50% of all cancer cell lines and patient tumors, and that treatment with histone deacetylase (HDAC) inhibitors reactivates SLFN11 expression and overcomes resistance to DNA-targeted anticancer drugs.

我们正在追求补充项目，以阐明拓扑异构酶，DNA修复和细胞周期检查点抑制剂的临床相关抑制剂的分子药理学。项目＃1。 Repair of topoisomerase cleavage complexes (TOPccs) by tyrosyl-DNA-phosphodiesterases (TDPs), endonucleases and SUMOylation/ubiquitylation Aim 1:Post-translational modifications of TOPccs: TOPccs are excised by two main mechanisms: 1/ hydrolysis of the covalent linkage between the catalytic tyrosine of topoisomerases and the DNA酪酶-DNA-磷酸二酯酶（TDP1和TDP2）破裂； 2/ DNA片段的核酸内切酶裂解与TOP1CC相邻的核酸酶（MRE11，XPF-ERCC1，XPG ...）。由于要被TDP1和TDP2裂解的共价拓扑异构酶 - 乙酰-DNA键在TOPCC内深处，因此TOPCC需要蛋白水解和/或变性，以提供对TDPS的访问（TDP1和TDP2）。我们正在研究TOPCC的蛋白水解途径。我们的结果表明，Sumoylation和Ubiquitylation途径的快速参与，这又驱动蛋白酶体介导的拓扑异构酶降解。 AIM 2：TDP的生物学：TDP1和TDP2优先修复TOP1CC和TOP2CC。除TOP1CC外，TDP1还取出了3'-DNA末端的损坏和非典型的碱基和加合物。这解释了为什么缺乏TDP1不仅会使细胞不仅对TOP1抑制剂，而且还对替莫唑胺，Cytarabine，Zidovudine（AZT）和Acyclovir敏感。我们正在研究如何调节TDP1并招募到DNA损坏的位点。我们先前报道了TDP1与PARP1结合，并且抑制PARP1导致TDP1失活。此外，我们先前确定TDP1分别在核和线粒体基因组中分别在核和线粒体基因组中造成TOP1CC和TOP1MTCC。我们证明TDP2还去除了线粒体基因组中的TOP2CC，并首次表明精氨酸甲基转移酶（PRMT5）通过直接结合和甲基化的TDP1激活TDP1。 AIM 3：TDP的药理学和靶向靶向：靶向TDP的基本原理植根于TDP对DNA修复和病毒复制的新兴重要性，以及TDP抑制剂对于抗癌药物组合的潜力。为此，我们使用重组TDP酶的生化测定法。我们还利用TDP1和TDP2基因敲除细胞系，晶体学测定和分子建模来研究候选药物的分子药理学。项目＃2。 PARP抑制剂的PARP捕获：分子机制和翻译意义PARP抑制剂代表针对DNA损伤反应的最先进的癌症治疗剂。最近已批准了四种抑制剂（Olaparib，Rucaparib，Niraparib和Talazoparib）。 PARP抑制剂是第一个利用诊所中同源重组缺乏（HRD）的合成致死性概念的药物。我们的研究表明，“ PARP捕获”是一种关键机制，解释了PARP抑制剂作为抗癌剂的分子机理。这一发现以及我们与Talazoparib的工作在2018年有助于塔拉唑帕里进行乳腺癌和卵巢癌的批准。我们的研究重点是1/与替莫唑胺和TOP1抑制剂的最协同组合，包括我们的非蛋白酶Indenoisoisoisoquinoline top1抑制剂； 2/对PARP抑制剂反应的修复机制和决定因素超出了同源重组（HR； BRCANESS）。我们目前正在关注DNA-蛋白交联蛋白酶（Spartan：SPTN），TDP和泛素化的作用，以去除被困的PARP。项目＃3。患者来源的癌细胞系可发现和验证新型的基因组预测生物标志物，用于与TOP1，PARP和DAN损伤响应（DDR）抑制剂的患者选择和合理的药物组合抑制剂，目前缺乏预测性生物标志物，无法广泛使用DNA靶向抗癌疗法，以及用于预测其主要靶标的dna Resports（DNA）的直接相关性（DNA）识别DNA的损害（DNA），以确定DNA的降低（DNA）。合理化药物组合。利用广泛的NCI-60药物数据库（包括FDA批准和研究临床药物在内的40,000种药物），整个基因组数据和我们的CellMiner设施，我们发现了几种用于DNA靶向药物的新型预测性生物标志物：SLX4（SLX4（FANCP）突变，ATAD5（ELG1）突变和Sllfn11（Schlfen11）。现在，我们已经将这些分析扩展到组织特异性的癌细胞系数据库（NCI小细胞肺癌），以及较大的数据库（CCLE：MIT-BROAD INSTITUTE和CGP：MGH-SANGER），以及CCR临床试验以测试预测性生物标志物。这些癌症细胞系数据库已通过基于CellMiner Web的应用程序（http://discover.nci.nih.gov/cellminercdb）广泛，可以自由地向研究社区提供。在过去的一年中，我们为患者衍生的小细胞肺癌（SCLC）生成了一种新型的数据库和基于Web的药物基因组学工具：SCLC-Cellminer与NCI-DTP（Beverly Teicher Molecular Pharmagology Group）和John John Minna（UTSW）合作。手稿和资源已沉积在Biorxiv和Cell Press的修订中。项目＃4。 Schlafen 11（SLFN11）对DNA损害药物的反应的预测生物标志物：通过我们的NCI-60分析和CCLE团队并行发现了分子机制和转化含义SLFN11作为对DNA损害药物反应的主要预测指标。 SLFN11确定对TOP1，TOP2，PARP抑制剂，DNA合成抑制剂和铂衍生物的反应，但不对微管蛋白或蛋白激酶抑制剂或凋亡诱导药物的反应。在大约50％的癌细胞系中，SLFN11灭活，使其对DNA损伤剂有抵抗力。我们的目的是阐明SLFN11作用和调节的分子机制，以及与患者反应和原理药物组合的相关性。我们通过证明SLFN11被募集到DNA损伤位点，并通过与RPA结合和复制性CMG复合物来发现SLFN11的关键分子机理。这样，SLFN11阻止了延长的复制子，并且不可逆地阻止了复制。我们还表明，SLFN11诱导染色质的可及性和诱导直接的早期反应（EIR）应激基因。我们建议SLFN11充当具有复制应激和“潜在肿瘤抑制剂”的细胞的“限制因素”。我们还证明，在大约50％的癌细胞系和患者肿瘤中，SLFN11在表观遗传上被灭活，并且用组蛋白脱乙酰基酶（HDAC）抑制剂治疗可重新激活SLFN11的表达，并克服了对DNA靶向抗癌抗癌药物的耐药性。