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

• 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
• 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

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.

拓扑异构酶是关键的酶，避免和解决核和线粒体基因组中的DNA超焦，结和曲奈烷。此外，TOP3B是唯一作用于DNA和RNA的拓扑异构酶。所有DNA交易都需要拓扑异构酶，尤其是转录和复制，还需要染色质重塑，DNA修复和重组。 TOP1MT是我们发现的所有脊椎动物细胞中存在的线粒体拓扑异构酶（包括人和啮齿动物），对于在组织再生和癌症进展过程中将线粒体DNA拷贝数与细胞增殖至关重要。 我们还发现人和小鼠线粒体含有TOP2。最近发现TOP3B解决了RNA纠缠，对于翻译和R环分辨率至关重要。失活的TOP3B突变与神经退行性疾病和癌症有关。 Top1是广泛使用的抗癌药物的靶标，这些药物在内，包括伊立替康和拓扑克，它们是植物生物碱凸轮甲虫的水溶性衍生物。它们用于治疗卵巢，结肠和肺癌以及血液学和儿科恶性肿瘤。 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减轻这些既定的限制。这导致了我们新颖的Top1靶向抗癌剂（Indenoisoquinolines）的发现。 NCI癌症研究中心与普渡大学的Cushman博士和NCI药物开发计划（DTP）合作，发现，专利并寻求Indenoisoquinolines。我们的两个Indenoisoquinolines，LMP400（Indotecan = NSC 743400）和LMP776（Indimitecan = NSC 725776）在NCI临床中心成功完成了1期临床试验。这些药物现在可用于2期试验。此外，基于最近在美国的NCI临床肿瘤学计划（COP）下，临床试验中的第三个衍生物LMP744在临床试验中表现出了非凡的活性。 LMP Indenoisoquinoline药物开发是我们小组，临床肿瘤学分支（Doroshow博士和Alice Chen的人类临床试验），DTP和SAIC（Hollingshead博士和小鼠模型和药物学生物标志物）的合作。我们的目标是使indenoisoquinolines成为第一个临床非膜非膜片药物。我们还继续将indenoisoquinoline衍生物作为第二代。新系列的化合物包括目前在临床试验中具有特定药代动力学特性的Indenoisoquinolines更有效的化合物。我们正在启动项目，以在递送载体中制定indenoisoquinolines，以增加其在肿瘤中的浓度，同时避免正常组织。这个目标通过靶向药物递送实现了精确医学的目标。在这种情况下，我们最近发现，假定的DNA-RNA旋转酶Schlafen 11（SLFN11）的表达决定了对indenoisoquinolines的反应，并且BRCA缺陷使癌细胞具有对indenoisoquinolines的选择性敏感。因此，在第二阶段临床试验中，SLFN11和同源重组缺陷（HRD）都可以用作生物标志物。因为在肿瘤特异性分子（Cybrexa cbx-12）中，使用了高度有效的凸蛋白衍生物用于抗体 - 毒与偶联物（例如在Enhertu和trodelvy中），并用作细胞毒性有效载荷（Cybrexa cbx-12），我们最近与ExaTaCan and ExaTaTecan进行了与ExaTaTecan的相比， Irinotecan。我们发表的结果表明，exatecan的优越性以及SLFN11和HRD在预测活动中的相关性，以及Exatecan与ATR抑制剂在临床发育中的高度协同作用。我们对拓扑异构酶基本生物学的研究还研究了TOP1作为核糖核酸酶的作用。实际上，当Top1与掺入核糖核苷酸的核糖核苷酸结合到DNA底物时，TOP1CC自发地转化为单链中的糖后，糖的2个羟基消除了TOP1，通过形成2个P-Prime，3-PRIME-CYCLIME核苷酸的TOP1，该核苷酸是由3个P-PRIME-PRIME-ED-TOPS制成的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核糖核苷酸。 我们正在追求这个项目，最近证明，当第二个TOP1站点发生在那些欠混合的核糖核苷酸附近时，Top1可以产生DNA双链断裂。这些新的结果加在一起增加了我们以前的发现，显示了TOP1的重组和潜在的诱变特性。他们还基于TOP1CC修复途径的重要性（包括酪酶-DNA磷酸二酯酶，TDP1和TDP2；请参阅下一个项目）。线粒体型IB拓扑异构酶TOP1MT在我们的实验室中发现。 TOP1MT存在于所有脊椎动物中，并由复制的祖先祖先TOP1基因复制而产生的核基因编码。在我们的实验室中产生的TOP1MT敲除小鼠的生存能力促使我们确定缺乏TOP1MT的其他拓扑异构酶可以补充哪些其他拓扑异构酶。我们发现TOP2A（拓扑异构酶IIα）和TOP2B（拓扑异构酶IIβ）都存在并且在线粒体中起作用。这可以解释我们TOP1MT敲除小鼠的轻度表型。但是，当用TOP2抑制剂阿霉素挑战（在线粒体中积聚并靶向线粒体TOP2B）时，我们的TOP1MT敲除小鼠会产生致命的心脏毒性，并深刻改变线粒体和线粒体DNA。此外，当Top1mt敲除小鼠受到肝毒素（四氯化碳）的挑战时，它们无法迅速再生其肝脏并表现出增加的线粒体。两种表型都表明，在器官需要将线粒体与快速细胞增殖的条件下，TOP1MT对于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负超串联，这意味着Top1MT在放松MTDNA的负超螺旋中具有选择性作用。因此，TOP1MT并不是必需的，但对于在某些代谢条件下的mtDNA复制和结构似乎至关重要。我们还证明了TOP1MT对肿瘤发育的重要性。值得注意的是，此功能不仅是由于Top1MT对MTDNA拷贝数的影响，而且还归因于Top1MT作为线粒体蛋白质合成的辅助因子的非传统函数。