DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

Division Of Basic Sciences - Nci

Investigators

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Abstract

Topoisomerases are critical enzymes that avoid and remove 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 genomic instability, neurodegenerative diseases, immune disorders, 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 is completing Phase 1 clinical trial at the NCI, based on its 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). The drugs are licensed to Gibson Oncology. Our goal is to make the indenoisoquinolines the first clinical non-camptothecin drugs. This aim meets the goal of precision medicine. In this context, we recently found that expression of the putative DNA-RNA helicase-nuclease 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 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 ribonucleotide excision enzyme. 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 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 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 crucial for mtDNA replication and structure in certain metabolic stress 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. This year we identified TOP1MT deleterious mutations in patients in autoimmune disorders and proposed that release of mitochondrial DNA due to inefficient TOP1MT leads to STING-GAS activation and immune dysregulation. In the past year, we have initiated new projects on the two human Type IA topoisomerases, topoisomerase III-alpha (TOP3A) and -beta (TOP3B). We reported that TOP3A activity is coordinated with DNA replication and propose it is part of the replisome to remove precatenanes behind the replication fork. We also reported that TOP3B activity is required to reduce RNA-DNA hybrid structures, referred to as "R-loops", which otherwise produce genomic instability.