DNA Topoisomerases as nuclear and mitochondrial targets of Anticancer Drugs

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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.