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DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

DNA 拓扑异构酶作为抗癌药物的核和线粒体靶标

基本信息

批准号：
10702291
负责人：
YVES POMMIER
金额：
$ 93.56万
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/10702291
关键词：
ABCB1 gene ABCG2 gene Antibody-drug conjugates Antineoplastic Agents Binding Biochemistry Biological Biological Markers Biology Blood Bone Marrow Camptothecin Carbon Tetrachloride Cardiotoxicity Catenanes Cell Proliferation Cell membrane Cells Chemicals Chromatin Chromosome Segregation Clinic Clinical Clinical Oncology Clinical Trials Collaborations Colon Carcinoma Complement Complex Cyclic Nucleotides DNA DNA Damage DNA Double Strand Break DNA Repair DNA Structure DNA Topoisomerases DNA biosynthesis DNA copy number DNA topoisomerase II alpha Development Diarrhea Dose-Limiting Doxorubicin Drug Delivery Systems Drug Efflux Drug Kinetics Drug Targeting ERCC1 gene Embryo Enzymes Epirubicin Etoposide Exatecan Exhibits Fibroblasts Generations Genes Genetic Recombination Genetic Transcription Genome Genome Stability Goals Hematologic Neoplasms Human Idarubicin Immune System Diseases Intercalating Agents Knockout Mice Laboratories Legal patent Lesion Liver Malignant Childhood Neoplasm Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of ovary Mediating Membrane Transport Proteins Metabolic Mitochondria Mitochondrial DNA Mitochondrial Proteins Mitoxantrone Molecular Mus Mutation NCI Center for Cancer Research Natural regeneration Neurodegenerative Disorders Normal tissue morphology Nuclear Nucleotides Organ Ovarian Carcinoma Pancreatic ribonuclease Pathway interactions Pharmaceutical Preparations Pharmacology Pharmacology Study Phase I Clinical Trials Phase II Clinical Trials Phenotype Plant alkaloid Poison Post-Translational Protein Processing Program Development Property Protein Biosynthesis Publishing RNA RNA Helicase Resolution Ribonucleotides Rodent Role SN-38 Series Site Sumoylation Pathway Superhelical DNA TOP1 gene TOP2A gene Topoisomerase Topoisomerase II Topoisomerase III Topoisomerase Inhibitors Topotecan Toxic effect Toxin Transact Translations Type I DNA Topoisomerases Universities Vertebrates Water adduct anti-cancer therapeutic base cancer cell chromatin remodeling clinical center clinical development cofactor cytotoxic delivery vehicle drug development endonuclease homologous recombination hydroxyl group inhibitor insight irinotecan lung Carcinoma macromolecule mitochondrial genome mouse model novel oncology program patient biomarkers patient stratification pharmacodynamic biomarker phase II trial precision medicine repaired response sugar synergism therapeutic target tissue regeneration topoisomerase IIIalpha transcriptome tumor tumor progression tyrosyl-DNA phosphodiesterase

项目摘要

Topoisomerases are critical enzymes that avoid and resolve DNA supercoils, knots and catenanes both in the nuclear and mitochondrial genomes. In addition, TOP3B is the only topoisomerase acting both on DNA and RNA. Topoisomerases are required for all DNA transactions, especially transcription and replication, but also chromatin remodeling, DNA repair and recombinations. TOP1MT, the mitochondrial topoisomerase present in all vertebrate cells (including humans and rodents), which we discovered, is critical to couple mitochondrial DNA copy number with cellular proliferation during tissue regeneration and cancer progression. We also discovered that human and mouse mitochondria contain TOP2. TOP3B was recently discovered to resolve RNA entanglements and to be critical for translation and R-loop resolution. Inactivating TOP3B mutations have been associated with neurodegenerative diseases and cancer. TOP1 is the target of widely used anticancer drugs including irinotecan and topotecan, which are water-soluble derivatives of the plant alkaloid camptothecin. They are used to treat ovarian, colon and lung cancers as well as hematologic and pediatric malignancies. Based on the fact that camptothecins have limitations including chemical instability (due to their alpha-hydroxylactone), drug efflux from cancer cells by the ABCG2 and ABCB1 plasma membrane transporters, rapid clearance for the blood, dose-limiting bone marrow toxicity, and severe diarrhea in the case of irinotecan, we initiated the discovery of non-camptothecin drugs to alleviate these established limitations. This led to the discovery of our novel TOP1-targeted anticancer agents (the indenoisoquinolines). The indenoisoquinolines have been discovered, patented, and pursued by the NCI Center for Cancer Research in collaboration with Dr. Cushman at Purdue University and the NCI Drug Development Program (DTP). Two of our indenoisoquinolines, LMP400 (Indotecan = NSC 743400) and LMP776 (Indimitecan = NSC 725776) successfully completed Phase 1 clinical trial at the NCI Clinical Center. The drugs are now available for Phase 2 trials. In addition, a third derivative, LMP744 in clinical trial, based on the recent finding that it shows remarkable activity in veterinary clinical trials under the NCI Clinical Oncology Program (COP) across the USA. The LMP indenoisoquinoline drug development is a collaboration between our group, the Clinical Oncology Branch (Dr. Doroshow and Alice Chen for the human clinical trials), DTP and SAIC (Dr. Hollingshead, and Dr. Parchment for mouse models and pharmacodynamic biomarkers). Our goal is to make the indenoisoquinolines the first clinical non-camptothecin drugs. We are also continuing to develop indenoisoquinoline derivatives as second generation. The new series encompasses compounds that are even more potent than the indenoisoquinolines presently in clinical trials, and which have specific pharmacokinetic properties. We are initiating projects to formulate the indenoisoquinolines in delivery vectors to increase their concentration in tumors while sparing normal tissues. This aim meets the goal of precision medicine by targeted drug delivery. In this context, we recently found that expression of the putative DNA-RNA helicase Schlafen 11 (SLFN11) determines response to the indenoisoquinolines and that BRCA-deficiencies render cancer cells selectively sensitive to the indenoisoquinolines. Hence, both SLFN11 and homologous recombination deficiencies (HRD) could serve as biomarkers in the Phase 2 clinical trials. Because highly potent camptothecin derivatives are being used a warhead for antibody-drug-conjugates (such as in Enhertu and Trodelvy) and as cytotoxic payloads in tumor-specific delivery macromolecules (Cybrexa CBX-12), we have recently performed molecular pharmacology studies with exatecan in comparison with topotecan and SN-38, the active metabolite of irinotecan. Our published results demonstrate the superiority of exatecan and the relevance of SLFN11 and HRD for predicting activity, as well as the high synergy of exatecan in combination with ATR inhibitors in clinical development. Our studies on the basic biology of topoisomerases also study the role of TOP1 as a ribonuclease. Indeed, when TOP1 binds to a DNA substrate with a misincorporated ribonucleotide, the TOP1cc is spontaneously converted into a single-strand break after the 2-prime-hydroxyl group of the sugar eliminate TOP1 by forming a 2-prime,3-prime-cyclic nucleotide at the 3-prime-end of the break that was initially made by TOP1. This finding is important for two reasons: first, because Thomas Kunkel and his group, one of our collaborators, have recently shown that ribonucleotides are readily misincorporated during normal replication (especially on the leading strand for DNA synthesis), and second because we have shown that those misincorporation sites give rise to short nucleotide deletions and insertion, by sequential TOP1 cleavage on the strand with the misincorporated ribonucleotide. We are pursuing this project and recently demonstrated that TOP1 can generate DNA double-strand breaks when a second TOP1 site occurs in the vicinity of those misincorporated ribonucleotide on the opposite strand of DNA. Together these new results add to our previous findings showing the recombinogenic and potentially mutagenic properties of TOP1. They also underpin the importance of TOP1cc repair pathways (including the tyrosyl-DNA phosphodiesterases, TDP1 and TDP2; see next project). Mitochondrial type IB topoisomerase, TOP1MT, was discovered in our laboratory. TOP1MT is present in all vertebrates and is encoded by a nuclear gene that arose by duplication of a common ancestral TOP1 gene. The viability of the TOP1MT knockout mice, which were generated in our laboratory prompted us to determine which other topoisomerases could complement for lack of TOP1MT. We found that both TOP2A (topoisomerase II alpha) and TOP2B (topoisomerase II beta) are present and functional in mitochondria. This may explain the mild phenotype of our TOP1MT knockout mice. However, when challenged with the TOP2 inhibitor doxorubicin, which accumulates in mitochondria and can target mitochondrial TOP2B, our TOP1MT knockout mice develop lethal cardiotoxicity with profound alterations of mitochondria and mitochondrial DNA. Furthermore, when TOP1MT knockout mice are challenged with a liver toxin (carbon tetrachloride), they fail to rapidly regenerate their liver and exhibit increased mitophagy. Both phenotypes suggest that TOP1MT is important for mtDNA replication under conditions where an organ needs to couple its mitochondria with rapid cellular proliferation. In addition, mouse embryonic fibroblasts generated from TOP1MT knockout mice have increased mtDNA negative supercoiling, implying a selective role for TOP1MT in relaxing the negative supercoiling of mtDNA. Thus, TOP1MT is not essential but appears to be crucial for mtDNA replication and structure in certain metabolic conditions. We also demonstrated the importance of TOP1MT for tumor development. Notably, this function is not only due to the impact of TOP1MT on mtDNA copy number but also to a non-canonical function of TOP1MT as a cofactor for protein synthesis in mitochondria.

拓扑异构酶是避免和分解核和线粒体基因组中的DNA超螺旋、结和链链的关键酶。此外，TOP3B是唯一同时作用于DNA和RNA的拓扑异构酶。拓扑异构酶是所有DNA交易，尤其是转录和复制，以及染色质重塑，DNA修复和重组所必需的。我们发现，所有脊椎动物细胞（包括人类和啮齿动物）中都存在线粒体拓扑异构酶TOP1MT，在组织再生和癌症进展过程中，它对线粒体DNA拷贝数与细胞增殖的耦合至关重要。我们还发现人类和小鼠的线粒体含有TOP2。最近发现，TOP3B可以解决RNA缠结，对翻译和r环的解析至关重要。失活的TOP3B突变与神经退行性疾病和癌症有关。TOP1是广泛应用的抗癌药物的靶点，包括伊立替康和拓扑替康，这两种药物都是植物生物碱喜树碱的水溶性衍生物。它们用于治疗卵巢癌、结肠癌和肺癌以及血液学和儿科恶性肿瘤。基于喜树碱的局限性，包括化学不稳定性（由于其α -羟内酯），ABCG2和ABCB1质膜转运蛋白从癌细胞外排，血液快速清除，剂量限制性骨髓毒性以及伊立替康的严重腹泻，我们开始发现非喜树碱药物来缓解这些既定的局限性。这导致我们发现了新的靶向top1的抗癌药物（吲哚异喹啉）。吲哚异喹啉类药物是由NCI癌症研究中心与普渡大学的库什曼博士以及NCI药物开发项目（DTP）合作发现并申请专利的。我们的两种吲哚异喹啉药物LMP400 （Indotecan = NSC 743400）和LMP776 （Indimitecan = NSC 725776）在NCI临床中心成功完成了i期临床试验。这些药物现已进入第二阶段试验。此外，基于最近在美国NCI临床肿瘤学计划（COP）的兽医临床试验中显示出显着活性的最新发现，第三种衍生物LMP744正在临床试验中。LMP吲哚异喹啉药物开发是我们集团、临床肿瘤学分支（人类临床试验的Doroshow博士和Alice Chen博士）、DTP和SAIC（小鼠模型和药效学生物标志物的Hollingshead博士和Parchment博士）之间的合作。我们的目标是使吲哚异喹啉类药物成为临床第一种非喜树碱类药物。我们也在继续开发第二代吲哚异喹啉衍生物。新系列包括比目前临床试验中的吲哚异喹啉类更有效的化合物，并且具有特定的药代动力学特性。我们正在启动在递送载体中配制吲哚异喹啉的项目，以增加其在肿瘤中的浓度，同时保留正常组织。这一目标通过靶向给药实现了精准医疗的目标。在这种情况下，我们最近发现DNA-RNA解旋酶Schlafen 11 （SLFN11）的表达决定了对吲哚异喹啉的反应，并且brca缺陷使癌细胞对吲哚异喹啉选择性敏感。因此，SLFN11和同源重组缺陷（HRD）都可以作为2期临床试验的生物标志物。由于高效喜树碱衍生物被用作抗体-药物偶联物的战斗部（如Enhertu和Trodelvy）和肿瘤特异性递送大分子（Cybrexa CBX-12）的细胞毒性有效载荷，我们最近对艾替康与拓扑替康和伊立替康的活性代谢物SN-38进行了分子药理学研究。我们发表的研究结果证明了exatecan的优势，以及SLFN11和HRD在预测活性方面的相关性，以及exatecan与ATR抑制剂联合在临床开发中的高度协同作用。我们对拓扑异构酶的基础生物学研究也研究了TOP1作为核糖核酸酶的作用。事实上，当TOP1与一个错误结合的核糖核苷酸结合到DNA底物上时，在糖的2 ‘端羟基通过在最初由TOP1产生的断裂的3 ’端形成2 ‘,3 ’端的环核苷酸消除TOP1后，TOP1cc自发地转化为单链断裂。这一发现之所以重要，有两个原因：首先，我们的合作者之一Thomas Kunkel和他的团队最近发现，核糖核苷酸在正常复制过程中很容易错配（尤其是在DNA合成的前导链上），其次，因为我们已经证明，这些错配位点通过对错配核糖核苷酸的链进行顺序的TOP1切割，会导致短核苷酸缺失和插入。我们正在进行这个项目，并且最近证明，当第二个TOP1位点出现在DNA相反链上错误结合的核糖核苷酸附近时，TOP1可以产生DNA双链断裂。总之，这些新的结果增加了我们之前的发现，显示了TOP1的重组和潜在的诱变特性。它们也支持了TOP1cc修复途径的重要性（包括酪氨酸- dna磷酸二酯酶，TDP1和TDP2；见下一个项目）。我们实验室发现了线粒体IB型拓扑异构酶TOP1MT。TOP1MT存在于所有脊椎动物中，由一个核基因编码，该核基因是由共同祖先的TOP1基因复制而产生的。在我们实验室产生的TOP1MT敲除小鼠的生存能力促使我们确定哪些其他拓扑异构酶可以补充缺乏TOP1MT。我们发现TOP2A（拓扑异构酶II α）和TOP2B（拓扑异构酶II β）都存在于线粒体中并起作用。这可能解释了我们的TOP1MT敲除小鼠的温和表型。然而，当使用TOP2抑制剂多柔比星（doxorubicin）攻击时，我们的TOP1MT敲除小鼠产生致命的心脏毒性，线粒体和线粒体DNA发生深刻改变。此外，当TOP1MT敲除小鼠受到肝毒素（四氯化碳）的攻击时，它们不能迅速再生肝脏，并表现出线粒体自噬增加。这两种表型表明，在器官需要将线粒体与快速细胞增殖结合的条件下，TOP1MT对mtDNA复制很重要。此外，由TOP1MT敲除小鼠产生的小鼠胚胎成纤维细胞增加了mtDNA负超卷曲，这意味着TOP1MT在放松mtDNA负超卷曲方面具有选择性作用。因此，在某些代谢条件下，TOP1MT不是必需的，但似乎对mtDNA的复制和结构至关重要。我们还证明了TOP1MT对肿瘤发展的重要性。值得注意的是，这种功能不仅是由于TOP1MT对mtDNA拷贝数的影响，而且还由于TOP1MT作为线粒体中蛋白质合成的辅助因子的非规范功能。