DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

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、结和链链链的关键酶。此外，TOP3B是唯一一种同时作用于DNA和RNA的双拓扑异构酶。拓扑异构酶是所有DNA交易，特别是转录和复制，以及染色质重塑，DNA修复和重组所必需的。我们之前发现的脊椎动物细胞（包括人类和啮齿动物）线粒体拓扑异构酶TOP1MT在组织再生和癌症进展过程中对线粒体DNA拷贝数与细胞增殖的耦合以及线粒体蛋白翻译至关重要。我们还发现人类和小鼠的线粒体含有TOP2。TOP3B是唯一能够分解RNA解结的拓扑异构酶。失活的TOP3B突变与神经退行性疾病和癌症有关。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/它们在与喜树碱不同的特定基因组位点捕获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。我们的两种吲哚异喹啉药物LMP400 （Indotecan = NSC 743400）和LMP776 （Indimitecan = NSC 725776）最近在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。该药物的开发是DTB（美国），临床肿瘤学分支（多罗西奥博士和爱丽丝陈博士进行人体临床试验），DTP和SAIC （Hollingshead博士，Parchment博士和Kinders博士进行小鼠模型和药效学生物标志物）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，推定的DNA-RNA解旋酶Schlafen 11 （SLFN11）的表达决定了对吲哚异喹啉和其他TOP1抑制剂的反应，并且brca缺陷使癌细胞对吲哚异喹啉和现有的TOP1抑制剂选择性敏感。因此，SLFN11和同源重组缺陷（HRD）和BRCAness都可以作为2期临床试验的生物标志物。我们对拓扑异构酶的基础生物学研究最近确定了TOP1是一种核糖核酸酶。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，在正常复制过程中，核糖核苷酸很容易错配（尤其是在DNA合成的前导链上）；其次，我们已经证明，通过对错配核糖核苷酸的链进行顺序的TOP1切割，这些错配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，我们最近证明了TOP1可以产生DNA双链断裂，当第二个TOP1位点出现在DNA相反链上那些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。核糖核苷酸上的TOP1切割也可能是核糖核苷酸切除修复的另一种途径，我们最近与Shunichi Takeda和Samuel Wilson合作，表明TOP1切割复合物可以作为核苷酸切除修复的备用途径。在过去的一年里，我们对TOP2在构建基因组和调节转录中的重要作用做出了贡献。与Andre Nussenzweig在CCR合作出版了两份手稿。线粒体拓扑异构酶，TOP1mt，是在我们实验室发现的。TOP1mt由存在于所有脊椎动物体内的核基因编码。它可能是由一个共同祖先的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）。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缺乏Top1mt的其他拓扑异构酶。我们发现TOP2A（拓扑异构酶II α）和TOP2B（拓扑异构酶II β）都在线粒体中，这解释了我们的Top1mt敲除小鼠的温和表型。因此，当使用TOP2抑制剂doxorubicin（在线粒体中积累并靶向线粒体TOP2B）攻击时，我们的Top1mt敲除小鼠产生了致命的心脏毒性，线粒体和线粒体DNA发生了深刻的改变。为了确定TOP1mt的具体功能，我们用肝脏毒素（四氯化碳）刺激TOP1mt敲除小鼠。我们的Top1mt基因敲除小鼠不能迅速再生肝脏，并表现出线粒体自噬增加。这两种表型表明，当器官需要将其mtDNA质量与快速细胞增殖相结合时，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。我们最近的研究表明，在小鼠模型中，TOP1mt促进了癌细胞的生长，并且这种功能与TOP1mt先前未被敲除的完全激活线粒体蛋白翻译的作用有关。因此，TOP1MT不是必需的，但在某些代谢条件下，对mtDNA的复制和结构以及mtRNA的翻译似乎至关重要。

Topoisomerase are critical enzymes avoiding and resolving DNA supercoils, knots and catenanes both in the nuclear and mitochondrial genomes. In addition, TOP3B is the only dual topoisomerase acting both on DNA and RNA. Topoisomerases are required for all DNA transactions, especially transcription and replication, but also for chromatin remodeling, DNA repair and recombinations. TOP1MT, the mitochondrial topoisomerase of vertebrate cells (including humans and rodens), which we discovered earlier, is critical to couple mitochondrial DNA copy number with cellular proliferation during tissue regeneration and cancer progression and for mitochondrial protein translation. We also discovered that human and mouse mitochondria contain TOP2. TOP3B is the only topoisomerase for the resolution of RNA untanglements. Inactivating TOP3B mutations have been associated with neurodegenerative diseases and cancer. TOP1 is the target of two widely used anticancer drugs, irinotecan and topotecan, which are both 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). We have now established that the indenoisoquinolines have significant advantages over the camptothecins: 1/ they are chemically stable and relatively easy to synthesize and optimize chemically; 2/ they trap TOP1 cleavage complexes at specific genomic sites that differ from the 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 of our indenoisoquinolines, LMP400 (Indotecan = NSC 743400) and LMP776 (Indimitecan = NSC 725776) recently 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 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 the DTB (us), 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). Our goal is to make the indenoisoquinolines the first clinical non-camptothecin drugs. We are also developing second generation indenoisoquinoline derivatives. 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, as well as other TOP1 inhibitors and that BRCA-deficiencies render cancer cells selectively sensitive to the indenoisoquinolines and the existing TOP1 inhibitors. Hence, both SLFN11 and homologous recombination deficiencies (HRD) and BRCAness could serve as a biomarkers in the Phase 2 clinical trials. Our studies on the basic biology of topoisomerases have recently established 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, ribonucleotides are readily misincorporated during normal replication (especially on the leading strand for DNA synthesis), and second, 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 we 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. TOP1 cleavage at ribonucleotides could also be an alternative pathway for ribonucleotide excision repair and, in collaboration with Shunichi Takeda and Samuel Wilson, we have recently shown that TOP1 cleavage complexes can act as a backup pathway for nucleotide excision repair. In the past year, we have contributed to the understanding of the important role of TOP2 in scaffolding the genome and regulating transcription. Two manuscripts have been published in collaboration with Andre Nussenzweig in CCR. The mitochondrial topoisomerase, TOP1mt, was discovered in our laboratory. TOP1mt is encoded by a nuclear gene present in all vertebrates. It 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 were generated in our laboratory prompted us to determine which other topoisomerase complement for lack of TOP1mt. We found that both TOP2A (topoisomerase II alpha) and TOP2B (topoisomerase II beta) are in mitochondria, explaining the mild phenotype of our Top1mt knockout mice. Accordingly, when challenged with the TOP2 inhibitor doxorubicin, which accumulates in mitochondria and targets mitochondrial TOP2B, our Top1mt knockout mice developed lethal cardiotoxicity with profound alterations of mitochondria and mitochondrial DNA. To determine the specific functions of TOP1mt, we challenged our Top1mt knockout mice with a liver toxin (carbon tetrachloride). Our Top1mt knockout mice fail to rapidly regenerate their liver and exhibit increased mitophagy. Both phenotypes suggest that TOP1MT is important for mtDNA replication when organs need 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. Our more recent study showed that TOP1mt promotes cancer cell growth in murine models and that this function is related to a previously unknockown role of TOP1mt to fully enable mitochondrial protein translation. Thus, TOP1MT is not essential but appears to be crucial for mtDNA replication and structure in certain metabolic conditions and for mtRNA translation.