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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解决核基因组和线粒体基因组中 DNA 超螺旋、结和索链的关键酶。此外，TOP3B 是唯一同时作用于 DNA 和 RNA 的拓扑异构酶。所有 DNA 处理（尤其是转录和复制）以及染色质重塑、DNA 修复和重组都需要拓扑异构酶。我们发现，TOP1MT 是所有脊椎动物细胞（包括人类和啮齿动物）中存在的线粒体拓扑异构酶，它对于组织再生和癌症进展过程中线粒体 DNA 拷贝数与细胞增殖的耦合至关重要。我们还发现人和小鼠的线粒体含有 TOP2。最近发现 TOP3B 可以解决 RNA 缠结，并且对于翻译和 R 环解析至关重要。 TOP3B 突变失活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，包括伊立替康和托泊替康，它们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-羟基内酯）、药物通过 ABCG2 和 ABCB1 质膜转运蛋白从癌细胞中流出、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缓解这些既定的限制。这导致我们发现了新型 TOP1 靶向抗癌药物（茚并异喹啉）。茚并异喹啉是由 NCI 癌症研究中心与普渡大学 Cushman 博士和 NCI 药物开发计划 (DTP) 合作发现、申请专利并进行开发的。我们的两种茚并异喹啉，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。这些药物现已可用于二期试验。此外，第三种衍生物LMP744正在临床试验中，基于最近的发现，它在美国NCI临床肿瘤学计划（COP）下的兽医临床试验中显示出显着的活性。 LMP 茚并异喹啉药物的开发是我们小组、临床肿瘤学分部（Doroshow 博士和 Alice Chen 博士负责人体临床试验）、DTP 和 SAIC（Hollingshead 博士和 Parchment 博士负责小鼠模型和药效生物标志物）之间的合作。我们的目标是使茚并异喹啉成为第一个临床非喜树碱药物。我们还继续开发第二代茚并异喹啉衍生物。新系列包含的化合物比目前临床试验中的茚并异喹啉更有效，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茚并异喹啉的项目，以增加其在肿瘤中的浓度，同时不影响正常组织。这一目标通过靶向药物输送实现了精准医疗的目标。在此背景下，我们最近发现假定的 DNA-RNA 解旋酶 Schlafen 11 (SLFN11) 的表达决定了对茚并异喹啉的反应，并且 BRCA 缺陷使癌细胞对茚并异喹啉选择性敏感。因此，SLFN11和同源重组缺陷（HRD）都可以作为二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缀合物的弹头（例如 Enhertu 和 Trodelvy）以及肿瘤特异性递送大分子 (Cybrexa CBX-12) 中的细胞毒性有效负载，因此我们最近使用 exatecan 进行了分子药理学研究，并与拓扑替康和 SN-38（喜树碱的活性代谢物）进行了比较。伊立替康。我们发表的结果证明了 exatecan 的优越性以及 SLFN11 和 HRD 在预测活性方面的相关性，以及 exatecan 与 ATR 抑制剂组合在临床开发中的高度协同作用。我们对拓扑异构酶基础生物学的研究还研究了 TOP1 作为核糖核酸酶的作用。事实上，当 TOP1 与带有错误掺入的核糖核苷酸的 DNA 底物结合时，在糖的 2-prime-羟基基团通过在最初由 TOP1 产生的断裂的 3-prime 末端形成 2-prime、3-prime-环状核苷酸消除 TOP1 后，TOP1cc 会自发转化为单链断裂。这一发现很重要，原因有二：首先，因为 Thomas Kunkel 和他的团队（我们的合作者之一）最近表明，核糖核苷酸在正常复制过程中很容易被错误掺入（特别是在 DNA 合成的前导链上），其次因为我们已经表明，通过在错误掺入链上连续 TOP1 切割，这些错误掺入位点会导致短核苷酸缺失和插入。核糖核苷酸。我们正在开展这个项目，最近证明，当第二个 TOP1 位点出现在 DNA 相对链上那些错误掺入的核糖核苷酸附近时，TOP1 可以产生 DNA 双链断裂。这些新结果共同补充了我们之前的发现，显示了 TOP1 的重组和潜在诱变特性。它们还强调了 TOP1cc 修复途径的重要性（包括酪氨酰 DNA 磷酸二酯酶、TDP1 和 TDP2；请参阅下一个项目）。线粒体IB型拓扑异构酶TOP1MT是我们实验室发现的。 TOP1MT 存在于所有脊椎动物中，由一个核基因编码，该核基因是通过复制共同祖先 TOP1 基因而产生的。我们实验室培育的 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 TOP1MT 的缺乏。我们发现 TOP2A（拓扑异构酶 II α）和 TOP2B（拓扑异构酶 II β）均存在于线粒体中并发挥功能。这可以解释我们的 TOP1MT 敲除小鼠的温和表型。然而，当受到 TOP2 抑制剂阿霉素（其在线粒体中积累并可靶向线粒体 TOP2B）的挑战时，我们的 TOP1MT 敲除小鼠会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）攻击时，它们无法快速再生肝脏并表现出线粒体自噬增加。两种表型都表明，在器官需要将其线粒体与快速细胞增殖结合的条件下，TOP1MT 对于 mtDNA 复制非常重要。此外，TOP1MT 敲除小鼠产生的小鼠胚胎成纤维细胞的 mtDNA 负超螺旋增加，这意味着 TOP1MT 在放松 mtDNA 负超螺旋方面具有选择性作用。因此，TOP1MT 并不是必需的，但在某些代谢条件下对于 mtDNA 的复制和结构似乎至关重要。我们还证明了 TOP1MT 对于肿瘤发展的重要性。值得注意的是，这一功能不仅是由于 TOP1MT 对 mtDNA 拷贝数的影响，而且还由于 TOP1MT 作为线粒体蛋白质合成的辅助因子的非典型功能。