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.
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