DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

拓扑异构酶是避免或限制细胞核和线粒体基因组中DNA超螺旋、结和链链的关键酶。它们是所有DNA交易，尤其是转录和复制所必需的。我们最近发现，在组织再生过程中，线粒体拓扑异构酶TOP1MT对线粒体DNA拷贝数与细胞增殖的耦合至关重要，人类和小鼠的线粒体也含有TOP2。最近发现，TOP3B可以解决RNA解结问题，对神经元的转录至关重要。TOP3B突变与神经缺陷和神经退行性疾病有关。TOP1是两种广泛使用的抗癌药物伊立替康和拓扑替康的靶点，这两种药物都是喜树碱的衍生物。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），药物通过ABCG2和ABCB1质膜转运体从癌细胞中流出，血液快速清除，以及剂量限制的胃肠道和骨髓毒性，我们开始发现非喜树碱药物，以减轻这些公认的局限性。这导致我们发现了新的靶向top1的抗癌药物（吲哚异喹啉）。与普渡大学的Cushman博士和NCI药物开发计划（DTP）合作，发现了吲哚异喹啉类药物，并获得了专利。我们现在已经确定，吲哚异喹啉类化合物与喜树碱相比具有显著的优势：1 .它们具有化学稳定性，相对容易合成和化学优化；2/它们在特定的基因组位点捕获与喜树碱捕获的不同的TOP1切割复合物；3/其细胞半衰期比喜树碱长得多；4/它们产生的TOP1切割复合物比喜树碱捕获的更稳定，这反映了它们与TOP1- dna切割复合物的紧密配合（界面结合）；它们不是多药耐药外排泵（如ABCB1 （Pgp）、ABCG2 （Mrp/Bcrp）和ABCC1 (Mrp1)）的底物。两种吲哚异喹啉（NSC 725776 - lmp776 -吲哚替康和743400 - lmp400 -吲哚替康）正在NCI临床中心进行临床试验。indotecan的1期临床试验刚刚完成，该药物现在可用于2期试验。此外，基于最近在美国多家兽医诊所的临床肿瘤学项目（COP）下，LMP744在犬类临床试验中表现出显著的活性，第三种衍生物LMP744已被选中用于人体临床试验的开发。这种药物的开发是LMP（我们的团队和Bonner博士研究γ - h2ax生物标志物）、临床肿瘤学分支（Doroshow博士和Alice Chen博士进行人体临床试验）、DTP和SAIC （Hollingshead博士、Parchment博士和Kinders博士研究小鼠模型和药效学生物标志物）以及普渡大学（Mark Cushman博士研究药物合成）之间的合作。我们的目标是使吲哚异喹啉成为第一种非喜树碱药物。我们也在继续开发第二代吲哚异喹啉衍生物。新系列包含比目前临床试验中的吲哚异喹啉类药物更有效的化合物。此外，我们正在启动一个项目，在递送载体中配制吲哚异喹啉，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。近年来，我们对拓扑异构酶的基础生物学研究主要集中在TOP1作为核糖核酸酶的作用上。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和他的团队最近表明，在正常复制过程中，核糖核苷酸很容易错误结合（尤其是在DNA合成的前链上），其次，因为我们已经表明，通过在错误结合的核糖核苷酸的链上进行顺序的TOP1切割，这些错误结合的位点会导致短核苷酸缺失和插入。我们目前正在进行这个项目，我们的初步结果表明，TOP1可以产生DNA双链断裂，当第二个TOP1位点出现在DNA的另一条链上错误结合的核糖核苷酸附近时。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是由一个存在于所有脊椎动物体内的核基因编码的，这个核基因可能是由一个共同的祖先TOP1基因的复制而产生的（在今天的简单脊索动物中发现，在更远的酵母和植物中发现）。我们在实验室产生的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。这一发现仅解释了我们的TOP1mt敲除小鼠的轻度表型。然而，当使用TOP2抑制剂doxorubicin（积聚在线粒体中，可以靶向线粒体TOP2B）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基因敲除小鼠受到肝毒素（四氯化碳）攻击时，我们发现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，在器官需要将其mtDNA质量与快速细胞增殖相结合的条件下，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。

Topoisomerase are critical enzymes to avoid or limit DNA supercoils, knots and catenanes both in the nuclear and mitochondrial genomes. They are required for all DNA transactions, especially transcription and replication. We recently found that TOP1MT, the mitochondrial topoisomerase is critical to couple mitochondrial DNA copy number with cellular proliferation during tissue regeneration, and that human and mouse mitochondria also contain TOP2. TOP3B was recently discovered to resolve RNA untanglements and to be critical for transcription in neurons. TOP3B mutations have been associated with neurological defects and neurodegenerative diseases. TOP1 is the target of two widely used anticancer drugs, irinotecan and topotecan, which are both camptothecin derivatives. 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 the cancer cells by the ABCG2 and ABCB1 plasma membrane transporters, rapid clearance for the blood, and dose-limiting gastro-intestinal and bone marrow toxicity, we initiated the discovery of non-camptothecin drugs that would alleviate these well-established limitations. This led to the discovery of our novel TOP1-targeted anticancer agents (the indenoisoquinolines). The indenoisoquinolines have been discovered, patented and pursued in collaboration with Dr. Cushman at Purdue University and the NCI Drug Development Program (DTP). We have now established that the indenoisoquinolines have significant advantages over the camptothecins: 1/ they are chemically stable and relatively easy to synthesize and chemically optimize; 2/ they trap TOP1 cleavage complexes at specific genomic sites that differ from those trapped by camptothecins; 3/ their cellular half-life is much longer than camptothecins; 4/ the TOP1 cleavage complexes they produce are more stable than those trapped by the camptothecins, which reflects their tight fit in the TOP1-DNA cleavage complexes (interfacial binding); 5/ they are not substrates for the multidrug resistance efflux pumps (such as ABCB1 (Pgp), ABCG2 (Mrp/Bcrp) and ABCC1 (Mrp1)). Two indenoisoquinolines (NSC 725776 -LMP776- indimitecan and 743400 -LMP400- indotecan) are in clinical trials at the NCI clinical center. The phase 1 clinical trial of indotecan has just been completed, and the drug is now available for Phase 2 trials. In addition, a third derivative, LMP744 has been selected for clinical development in human trials, based on the recent finding that LMP744 showed remarkable activity in dog clinical trials under the Clinical Oncology Program (COP) in multiple veterinary clinics across the USA. This drug development is a collaboration between LMP (our group and Dr. Bonner for gamma-H2AX biomarker), the Clinical Oncology Branch (Dr. Doroshow and Alice Chen for the human clinical trials), DTP and SAIC (Dr. Hollingshead, Dr. Parchment and Dr. Kinders for mouse models and pharmacodynamic biomarkers), and Purdue University (Dr. Mark Cushman for drug synthesis). Our goal is to make the indenoisoquinolines the first 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. Moreover, we are initiating a project 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. Our studies on the basic biology of topoisomerases have recently focused on 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 misincorportated ribonucleotide. We are currently pursuing this project and our preliminary results indicate 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 encoded by a nuclear gene present in all vertebrates, which probably arose by duplication of a common ancestral TOP1 gene (found today in simple chordates and more distantly in yeast and plants). The viability of the TOP1mt knockout mice, which we generated in our laboratory prompted us to determine which other topoisomerase 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 finding only explains 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), we found they fail to rapidly regenerate their liver and exhibit increased mitophagy. Both phenotypes suggest that TOP1MT is important for mtDNA replication in conditions where an organ needs to couple its mtDNA mass 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.