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抑制剂的临床相关抑制剂的分子药理学。项目# 1。酪氨酸-DNA-磷酸二酯酶（TDPs）、核酸内切酶和SUMOylation/泛素化对拓扑异构酶裂解复合物（TOPccs）的修复目的1:TOPccs的翻译后修饰：TOPccs主要通过两种机制被切除：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…）切割靠近TOP1cc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。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。我们正在研究TDP1是如何被调节和招募到DNA受损部位的。我们之前报道过TDP1与PARP1偶联，抑制PARP1会导致TDP1失活。此外，我们之前已经确定TDP1分别在核基因组和线粒体基因组中切除TOP1cc和TOP1MTcc。我们证明了TDP2也能去除线粒体基因组中的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TDP1来激活TDP1。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。为此，我们使用重组TDP酶进行生化分析。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。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。最近有四种抑制剂（olaparib, rucaparib， niraparib和talazoparib）被批准。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）。我们目前的研究重点是dna -蛋白交联蛋白酶（SPTN）、TDPs和泛素化在去除被困PARP中的作用。项目# 3。患者来源的癌细胞系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DAN损伤反应（DDR）抑制剂目前缺乏用于广泛应用的DNA靶向抗癌治疗的预测性生物标志物，其主要靶点与细胞反应之间缺乏直接相关性，因此需要确定新的DNA损伤反应（DDR）决定因素，以预测药物反应并使药物组合合理。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。我们现在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。在过去的一年里，我们与NCI-DTP （Beverly Teicher分子药理学组）和John Minna （UTSW）合作，建立了一个新的数据库和基于网络的患者源性小细胞肺癌（SCLC）药物基因组学工具：SCLC- cellminer。手稿和资源已存放在BioRxiv和修订中在细胞出版社。项目# 4。Schlafen 11 （SLFN11）是对DNA损伤药物反应的预测性生物标志物：分子机制和翻译意义SLFN11是通过我们的NCI-60分析和CCLE团队并行发现的。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。我们通过证明SLFN11通过结合RPA和复制CMG复合体被招募到DNA损伤位点和应激复制叉，发现了SLFN11的关键分子作用机制。在此过程中，SLFN11阻断了延长复制子并不可逆地阻断了复制。我们还发现SLFN11诱导染色质可及性和立即早期反应（EIR）应激基因的诱导。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，SLFN11在大约50%的所有癌细胞系和患者肿瘤中是表观遗传失活的，并且用组蛋白去乙酰化酶（HDAC）抑制剂治疗可以重新激活SLFN11的表达并克服对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 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.