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

基本信息

批准号：
10925958
负责人：
YVES POMMIER
金额：
$ 231.16万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10925958
关键词：
APEXL2 Gene Acyclovir Advanced Malignant Neoplasm Antineoplastic Agents Antineoplastic Combined Chemotherapy Protocols Apoptosis Ataxia Telangiectasia Binding Biochemical Biological Assay Biology CCR CDT1 Gene CHEK1 gene Cancer cell line Cell Cycle Checkpoint Cell Death Cell Line Cells Chromatin Clinic Clinical Clinical Pharmacology Clinical Trials Collaborations Communities Complex Crystallography Cytarabine DNA DNA Damage DNA Repair DNA Synthesis Inhibitors DNA replication fork DNA-protein crosslink Databases Digestion Drug Combinations ERCC1 gene Enzymes Excision FDA approved Genes Genomics Glycoside Hydrolases Histone Deacetylase Inhibitor Hydrolysis Immune checkpoint inhibitor Induction of Apoptosis Knock-out Licensing Factor Malignant neoplasm of ovary Manuscripts Mediating Methylation Molecular Molecular Mechanisms of Action Mutation National Center for Advancing Translational Sciences Online Systems Organoids Pathway interactions Patient Selection Patients Peptide Hydrolases Pharmaceutical Preparations Pharmacogenomics Pharmacology Phosphodiesterase Inhibitors Platinum Poly(ADP-ribose) Polymerase Inhibitor Post-Translational Protein Processing Proteolysis Publishing RNA Rationalization Recombinants Regulation Replication Licensing Research Resistance Resources Site Stress Structure Sumoylation Pathway TOP1 gene TOP2A gene Testing Time Tissues Topoisomerase Topoisomerase Inhibitors Tubulin Tumor Suppressor Proteins Tyrosine Ubiquitination Virus Replication Work Zidovudine adduct anticancer research arginine methyltransferase base biomarker signature cancer therapy clinical development clinically relevant drug candidate drug response prediction drug testing endonuclease genomic data genomic predictors genomic signature homologous recombination improved inhibitor kinase inhibitor malignant breast neoplasm mitochondrial genome molecular modeling multicatalytic endopeptidase complex novel nuclease patient biomarkers patient response precision medicine predicting response predictive marker prevent protein kinase inhibitor proteostasis proteotoxicity public database recruit repaired replication stress response response biomarker small cell lung carcinoma targeted agent temozolomide therapeutic target tool tumor tyrosyl-DNA phosphodiesterase ubiquitin ligase web app web site

项目摘要

We are pursuing complementary projects to elucidate the molecular pharmacology of clinically relevant inhibitors of topoisomerases, DNA repair and cell cycle checkpoints. Project #1. Repair of topoisomerase cleavage complexes (TOPccs) by tyrosyl-DNA-phosphodiesterases (TDPs), endonucleases and SUMOylation/ubiquitylation/PARylation Aim 1:Post-translational modifications of TOPccs: TOPccs are excised from DNA 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 TOPcc by nucleases (Mre11, XPF-ERCC1, XPG, FEN1, APE2...). 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. We have also discovered that, unique to TOP1, TOP1ccs are rapidly PARylated by PARP1 and de-PARylated by PARG (PolyADPribose Glycohydrolase). Thus, PARylation regulates the proteolytic digestion of TOP1ccs by preventing their excessive proteasomal degradation while recruiting TDP1. 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 demonstrated that TDP2 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. Our recent studies show that TDP2 also excises TOP3Bccs both from DNA and RNA, and that TDP2 excises TOP3Accs after their proteolysis by the replication-associated protease Spartan (SPRTN). 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. 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. We published the first crystal structures of TDP1 inhibitors in complex with their target enzyme. 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. PARP inhibitors (olaparib, rucaparib, niraparib and talazoparib) are FDA-approved. 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 recently showed that the DNA-protein crosslink protease (Spartan: SPTN), TDPs and ubiquitination are involved for the removal of trapped PARP1 and that ubiquitylation of PARP1 by the SUMO-dependent ubiquitin ligase RNF4 precedes the eviction of PARP1 from chromatin. Project #3. Patient-derived cancer cell lines and organoids to discover and validate novel genomic predictive biomarkers for patient selection and rational drug combinations with TOP1, PARP and DNA damage response (DDR) and cell cycle checkpoint (ATR) inhibitors as part of CellMiner 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 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 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). 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 published at Cell Press. We have also integrated in the CellMinerCDB database the response of 183 cancer cell lines to 2,650 drugs tested at the National Center for Advancing Translational Sciences (NCATS. These publicly available database and website have been published in Cancer Research and highlighted by the CCR Press Office. Project #4. Schlafen 11 (SLFN11) a predictive biomarkers of response to DNA damaging drugs: We discovered SLFN11 as dominant predictor of response to DNA and replication damaging drugs. 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 that SLFN11 is recruited to DNA damage sites and to stressed replication forks by binding to RPA and the replicative CMG complex, opening chromatin, inducing the immediate early response (EIR) stress genes, and promoting the degradation of the replication licensing factor CDT1. This year, we showed that SLFN11 regulating protein homeostasis and proteotoxic stress. 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 inactivation in approximately 50% of all cancer cell lines and patient tumors can be reversed by treatment with histone deacetylase (HDAC) inhibitors to overcome 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的临床相关抑制剂的分子药理学。项目＃1。通过酪糖基-DNA-磷酸二酯酶（TDP），核酸内切酶和sumoylation/ubiquityLation/parylation/parylation/parylation Aim 1：TOPCC的转基因修饰（TOPCC）从TOPCC中切除DNA的两种催化器：1/1：拓扑异构酶和DNA末端因酪酶-DNA-磷酸二酯酶（TDP1和TDP2）破裂； 2/ DNA片段的核酸内切酶通过核酸酶（Mre11，XPF-ERCC1，XPG，FEN1，APE2 ...）与TOPCC的DNA片段裂解。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。我们还发现，TOP1独有的TOP1CC被PARP1迅速对其进行了pary，并被PARG（polyadpribose glydrololose）脱脂。因此，parylation通过在募集TDP1的同时防止其过量的蛋白酶体降解来调节TOP1CC的蛋白水解消化。 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。我们证明了TDP2在线粒体基因组中删除了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。我们最近的研究表明，TDP2还从DNA和RNA中对TOP3BCC进行了启动，并且TDP2在通过复制相关的蛋白酶spartan（SPRTN）蛋白水解后的TOP3ACC进行了top3ACC。 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。我们正在使用重组TDP酶的生化测定。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药物候选物的分子药理学。我们发表了TDP1抑制剂的第一个晶体结构及其靶酶。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。 PARP抑制剂（Olaparib，Rucaparib，Niraparib和Talazoparib）是FDA批准的。 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）。我们最近表明，DNA-蛋白交联蛋白酶（Spartan：SPTN），TDP和泛素化涉及去除被困的PARP1，并且SUMO依赖性的泛素性依比素连接酶RNF4对PARP1的泛素化涉及PARP1，这是PARP1从染色蛋白中驱逐的驱逐。项目＃3。患者衍生的癌细胞系和类器官，以发现和验证与TOP1，PARP和DNA损伤响应（DDR）和细胞周期检查点（ATR）抑制剂（ATR）抑制剂的患者选择和合理药物组合的新型基因组预测生物标志物，作为细胞计算机的一部分，目前缺乏预测性生物标志物在较广泛使用DNA的抗DNA抗抗抗DNA的抗逆抗抗性和抗DNA抗抗抗Antigart的抗逆转录行为之间，并不是针对DNA抗DNA的响应式抗抗DNA的抗逆转录行为。确定DDR的决定因素，以预测药物反应和合理化药物组合。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）。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。我们为患者来源的小细胞肺癌（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 Minna（UTSW）合作。手稿和资源已在Cell Press发表。我们还将183个癌细胞系对2,650种药物的反应集成到国家前进的转化科学中心（NCATS。这些公开可用的数据库和网站）在癌症研究中发表，并由CCR Press Offect发表，并在CCR新闻办公室发表。项目＃4。对DNA的反应和重复损害药物的主要预测是对TOP1，TOP2，PARP抑制剂，DNA合成抑制剂和铂衍生物的反应为了阐明SLFN11作用和调节的分子机制，以及与患者的反应和原理药物组合的相关性。因子CDT1。今年，我们表明SLFN11调节蛋白质稳态和蛋白质毒性应激。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％的癌细胞系中，SLFN11灭活和患者肿瘤可以通过用组蛋白脱乙酰基酶（HDAC）抑制剂治疗来逆转以克服对DNA靶向抗癌药物的耐药性。

项目成果

期刊论文数量（84）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

RNAi screening identifies TAK1 as a potential target for the enhanced efficacy of topoisomerase inhibitors.

DOI：
10.2174/156800911797264734
发表时间：
2011-10
期刊：
Current cancer drug targets
影响因子：
3
作者：
Martin SE;Wu ZH;Gehlhaus K;Jones TL;Zhang YW;Guha R;Miyamoto S;Pommier Y;Caplen NJ
通讯作者：
Caplen NJ

Zalypsis (PM00104) is a potent inducer of gamma-H2AX foci and reveals the importance of the C ring of trabectedin for transcription-coupled repair inhibition.

DOI：
10.1158/1535-7163.mct-09-0336
发表时间：
2009-07
期刊：
Molecular cancer therapeutics
影响因子：
5.7
作者：
Guirouilh-Barbat J;Antony S;Pommier Y
通讯作者：
Pommier Y

Deazaflavin Inhibitors of Tyrosyl-DNA Phosphodiesterase 2 (TDP2) Specific for the Human Enzyme and Active against Cellular TDP2.

DOI：
10.1021/acschembio.5b01047
发表时间：
2016-07-15
期刊：
ACS chemical biology
影响因子：
4
作者：
Marchand C;Abdelmalak M;Kankanala J;Huang SY;Kiselev E;Fesen K;Kurahashi K;Sasanuma H;Takeda S;Aihara H;Wang Z;Pommier Y
通讯作者：
Pommier Y

Report on the first SLFN11 monothematic workshop: from function to role as a biomarker in cancer.

DOI：
10.1186/s12967-017-1296-3
发表时间：
2017-10-02
期刊：
Journal of translational medicine
影响因子：
7.4
作者：
Ballestrero A;Bedognetti D;Ferraioli D;Franceschelli P;Labidi-Galy SI;Leo E;Murai J;Pommier Y;Tsantoulis P;Vellone VG;Zoppoli G
通讯作者：
Zoppoli G

Phosphatase 1 Nuclear Targeting Subunit, a Novel DNA Repair Partner of PARP1

磷酸酶 1 核靶向亚基，PARP1 的新型 DNA 修复伙伴