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

我们正在进行补充项目，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。目的1:TOPccs的翻译后修饰：TOPccs主要通过两种机制从DNA上切除：1/酪氨酸-DNA-磷酸二酯酶（TDP1和TDP2）水解拓扑异构酶催化酪氨酸与DNA断裂端之间的共价键；2/内切酶通过核酸酶（Mre11, XPF-ERCC1， XPG, FEN1, APE2…）切割靠近TOPcc的DNA片段。由于TDP1和TDP2切割的共价拓扑异构酶-酪氨酸- dna键位于TOPccs的深处，因此TOPccs需要被蛋白水解和/或变性以提供tdp （TDP1和TDP2）的通路。我们正在研究TOPccs的蛋白水解途径。我们的研究结果证明了sumo化和泛素化途径的快速参与，这反过来又驱动蛋白酶体介导的拓扑异构酶降解。我们还发现，TOP1特有的top1cc被PARP1快速聚合，并被PARG （PolyADPribose Glycohydrolase）去聚合。因此，PARylation通过阻止top1cc过度的蛋白酶体降解来调节top1cc的蛋白水解消化，同时募集TDP1。目的2:TDPs生物学：TDP1和TDP2分别优先修复TOP1cc和TOP2cc。除了TOP1cc外，TDP1还能去除3'-DNA末端的受损和非规范碱基和加合物。这就解释了为什么缺乏TDP1使细胞不仅对TOP1抑制剂敏感，而且对替莫唑胺、阿糖胞苷、齐多夫定（AZT）和阿昔洛韦也敏感。我们证明了TDP2去除线粒体基因组中的TOP2cc，并首次证明精氨酸甲基转移酶（PRMT5）通过直接结合和甲基化TDP1来激活TDP1。我们最近的研究表明，TDP2也从DNA和RNA中切除top3bcc，并且TDP2在复制相关蛋白酶SPRTN （Spartan）水解后切除TOP3Accs。目标3:TDP的药理学和靶向：靶向TDP的基本原理是基于TDP在DNA修复和病毒复制中的重要性，以及TDP抑制剂在抗癌药物组合中的潜力。我们使用重组TDP酶进行生化检测。我们还利用TDP1和TDP2敲除细胞系、晶体学测定和分子模型来研究候选药物的分子药理学。我们首次发表了TDP1抑制剂与靶酶复合物的晶体结构。项目# 2。PARP抑制剂捕获PARP：分子机制和翻译意义PARP抑制剂代表了针对DNA损伤反应的最先进的癌症治疗方法。PARP抑制剂（olaparib, rucaparib， niraparib和talazoparib）是fda批准的。PARP抑制剂是第一个利用合成致死性概念治疗同源重组缺乏症（HRD）的临床药物。我们的研究揭示了“PARP诱捕”是解释PARP抑制剂作为抗癌药物作用的分子机制的关键机制。这一发现以及我们在talazoparib上的工作促成了talazoparib在2018年被批准用于乳腺癌和卵巢癌。我们的研究重点是与替莫唑胺和TOP1抑制剂（包括我们的非喜树碱吲哚异喹啉TOP1抑制剂）联合使用最具协同作用；2/同源重组后PARP抑制剂应答的修复机制和决定因素（HR; BRCAness）。我们最近发现dna -蛋白交联蛋白酶（SPTN）、TDPs和泛素化参与了被困PARP1的去除，并且sumo依赖的泛素连接酶RNF4对PARP1的泛素化先于PARP1从染色质中移除。项目# 3。患者来源的癌细胞系和类器官，以发现和验证新的基因组预测生物标志物，用于患者选择和合理的药物组合。PARP和DNA损伤反应（DDR）和细胞周期检查点（ATR）抑制剂作为CellMiner的一部分，目前缺乏广泛使用的DNA靶向抗癌治疗的预测性生物标志物，以及它们的主要靶点与细胞反应之间缺乏直接相关性，因此需要确定DDR决定因素来预测药物反应和合理的药物组合。利用广泛的NCI-60药物数据库（40,000种药物，包括FDA批准的药物和临床研究药物）、全基因组数据和我们的CellMiner设备，我们发现了几种新的dna靶向药物预测生物标志物：SLX4 （FANCP）突变、ATAD5 （ELG1）突变和SLFN11 （Schlafen 11）表达。我们已经将这些分析扩展到组织特异性癌细胞系数据库（NCI小细胞肺癌）和更大的数据库（CCLE: MIT-Broad研究所和CGP: MGH-Sanger），以及CCR临床试验以测试预测性生物标志物特征。这些癌细胞系数据库已经通过CellMiner基于网络的应用程序（http://discover.nci.nih.gov/cellminercdb）广泛免费地提供给研究界。我们与NCI-DTP （Beverly Teicher分子药理学组）和John Minna （UTSW）合作，为患者源性小细胞肺癌（SCLC）创建了一个新的数据库和基于网络的药物基因组学工具：SCLC- cellminer。手稿和资料已在细胞出版社出版。我们还在CellMinerCDB数据库中整合了183个癌细胞系对国家促进转化科学中心（NCATS）测试的2650种药物的反应。这些公开可用的数据库和网站已发表在《癌症研究》杂志上，并由CCR新闻办公室重点介绍。项目# 4。Schlafen 11 （SLFN11）是对DNA损伤药物反应的预测性生物标志物：我们发现SLFN11是对DNA和复制损伤药物反应的主要预测因子。SLFN11决定对TOP1、TOP2、PARP抑制剂、DNA合成抑制剂和铂衍生物的反应，而不是对微管蛋白或蛋白激酶抑制剂或凋亡诱导药物的反应。SLFN11在大约50%的癌细胞系中失活，使它们能够抵抗DNA损伤剂。我们的目的是阐明SLFN11的作用和调控的分子机制，以及与患者反应和基本药物组合的相关性。我们发现SLFN11通过结合RPA和复制CMG复合体，打开染色质，诱导即时早期反应（EIR）应激基因，并促进复制许可因子CDT1的降解，被招募到DNA损伤位点和应激复制叉上。今年，我们发现SLFN11调节蛋白质稳态和蛋白质毒性应激。我们提出SLFN11作为细胞复制应激的“限制性因子”和“潜在的肿瘤抑制因子”。我们还证明，大约50%的癌细胞系和患者肿瘤中的SLFN11失活可以通过组蛋白去乙酰化酶（HDAC）抑制剂治疗来逆转，以克服对dna靶向抗癌药物的耐药性。

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.