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

我们正在进行三个互补的项目，以阐明拓扑异构酶和聚（ADPribose）聚合酶（PARP）抑制剂的临床相关抑制剂的分子药理学。一号工程。目的1：TDP的生物学：TDP 1和TDP 2分别优先修复TOP 1cc和TOP 2cc。除了TOP1 cc之外，TDP 1还从3 '-DNA末端去除受损的和非规范的碱基和加合物。这解释了为什么缺乏TDP 1不仅使细胞对TOP1抑制剂敏感，而且使细胞对替莫唑胺、阿糖胞苷、齐多夫定（AZT）和阿昔洛韦敏感。我们正在研究TDP 1是如何被调节和招募到DNA损伤位点的。我们最近报道了TDP 1与PARP 1偶联，并且抑制PARP 1导致TDP 1失活。由于TDP 1在细胞核和线粒体基因组中都切除了TOP1 cc，我们目前正在研究TDP 2是否也在线粒体基因组中去除了TOP2 cc。我们还使用敲除细胞系，定点诱变和结晶学来阐明TDP的生物学。目标二：TDP的药理学和靶向：靶向TDP的基本原理植根于TDP对DNA修复和病毒复制的重要性，以及TDP抑制剂用于抗癌药物组合的潜力。为此，我们正在使用重组TDP酶的生化测定。我们还利用TDP 1和TDP 2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。二号工程。PARP抑制剂捕获PARP：分子机制和翻译影响PARP抑制剂代表了靶向DNA损伤反应的最先进的癌症治疗方法，其中一种抑制剂奥拉帕尼已经获得批准，其他几种药物处于后期开发阶段。PARP抑制剂是第一种利用同源重组缺陷（HRD）临床合成致死性概念的药物。了解PARP抑制剂的作用机制是成功部署这些药物的关键。我们的研究集中在“PARP捕获”作为这些抑制剂的药理学的一个组成部分。目标1：PARP药理学：我们的研究重点是PARP抑制剂之间在PARP捕获方面的分子作用机制差异，以及这对单药治疗活性和与化疗药物联合治疗的意义。我们正在临床前模型中研究两种最具协同作用的组合：与替莫唑胺和与TOP 1抑制剂，包括我们的非喜树碱茚并异喹啉TOP 1抑制剂（见上文）。第三个项目。药物反应的基因组决定因素和用于患者选择的预测生物标志物的临床前模型以及与TOP1和PARP抑制剂的合理药物组合广泛使用的DNA和染色质靶向药物的活性与其主要靶点之间的简单关系的不合理性保证了需要鉴定用于预测药物反应和合理化药物组合的新型DNA损伤反应（DDR）决定因素。目标1：基于CellMiner使用癌细胞系数据库挖掘药物反应：利用广泛的NCI-60药物数据库（40，000种药物，包括FDA批准的和研究性临床药物），全基因组数据和我们的CellMiner设施，我们发现了几种新的DNA靶向药物的预测生物标志物：SLX 4（FANCP）突变、ATAD 5（ELG 1）突变和SLFN 11（Schlafen 11）表达。我们正在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 Institute和CGP：MGH-Sanger），以及CCR临床试验，以测试预测性生物标志物特征。目的2：SLFN 11作为DNA损伤药物反应的预测生物标志物是通过NCI-60分析发现的，并由CCLE团队平行发现。SLFN 11决定对TOP 1、TOP 2、PARP抑制剂、DNA合成抑制剂和铂衍生物的反应，但不决定对微管蛋白或蛋白激酶抑制剂或凋亡诱导药物的反应。SLFN 11在大约50%的癌细胞系中失活，使它们对DNA损伤剂具有抗性。我们的目的是阐明SLFN 11作用和调节的分子机制，以及与患者反应和合理药物组合的相关性。

We are pursuing three complementary projects to elucidate the molecular pharmacology of clinically relevant inhibitors of topoisomerases and poly(ADPribose) polymerase (PARP) inhibitors. Project #1. Repair of topoisomerase cleavage complexes by tyrosyl-DNA-phosphodiesterases (TDPs) Aim 1: 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 recently reported that TDP1 is coupled with PARP1 and that inhibiting PARP1 results in TDP1 inactivation. Because TDP1 excises TOP1cc both in the nuclear and mitochondrial genomes, we are currently examining whether TDP2 also removes TOP2cc in the mitochondrial genome. We are also using knockout cell lines, site-directed mutagenesis and crystallography to elucidate the biology of TDPs. Aim 2: 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, with one inhibitor, olaparib already an approved medicine and several others in late stage development. PARP inhibitors are the first drugs to exploit the concept of synthetic lethality for homologous recombination deficiency (HRD) in the clinic. Understanding the mechanism(s) of action of PARP inhibitors is key to the successful deployment of these agents. Our studies focus on 'PARP trapping' as an integral component of the pharmacology of these inhibitors. Aim 1: PARP pharmacology: Our studies focus on the differences in the molecular mechanisms of action between PARP inhibitors with respect to PARP trapping and what this means for both monotherapy activity and combination with chemotherapeutic agents. We are investigating in preclinical models the two most synergistic combinations: with temozolomide and with TOP1 inhibitors, including our non-camptothecin indenoisoquinoline TOP1 inhibitors (see above). Project #3. Genomic determinants of drug response and preclinical models for predictive biomarkers for patient selection and rational drug combinations with TOP1 and PARP inhibitors The insufficiencies of simple relationships between the activity of widely used DNA- and chromatin-targeted agents and their primary targets warrant the need to identify novel DNA damage response (DDR) determinants for predicting drug responses and rationalizing drug combinations. Aim 1: Use cancer cell line databases to mine drug responses based on CellMiner: 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 are extending 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. Aim 2: SLFN11 as predictive biomarkers for response to DNA damaging drugs was discovered through 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.