Humans encode 6 topoisomerases: Two type IB: Top1 and Top1mt, two type IIA: Top2A and Top2B, and two type IA: Top3A and Top3B. Topoisomerase enzymes are critical for avoiding DNA untanglements including supercoils, knots and catenanes, and are required for all DNA transactions including transcription and replication. 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 irinotecan and topotecan, which are camptothecin derivatives efficiently used to treat ovarian, colon and lung cancers as well as hematologic and pediatric malignancies. However, camptothecins have well-defined limitations including chemical instability (due to their alpha-hydroxylactone structure), drug efflux by the ABCG2 and ABCB1 plasma membrane transporters, and dose-limiting gastro-intestinal and bone marrow toxicity. We have pursued our discovery and molecular pharmacology of novel Top1-targeted anticancer agents to alleviate these limitations. 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 several advantages over camptothecins: 1/ they are chemically stable and 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 camptothecins indicating a tight fit in the Top1-DNA cleavage complexes;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 -- indimitecan and 743400--indotecan) are in clinical trials at the NCI. This drug development is a collaboration between LMP (our group and Dr. Bonner for gamma-H2AX biomarker), the Clinical Oncology Branch (Dr. Doroshow and Shivaani Kummar for 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 NCI-discovered drugs in the Phase 0/I pipeline with histone gamma-H2AX as a biomarker. We are also continuing to develop indenoisoquinoline derivatives as second generation. The new series is even more potent than the compounds in 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. Regarding the basic biology of topoisomerases, we discovered that, 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, and second because those misincorporation sites give rise to short nucleotide deletions and insertion in a Top1-dependent manner. Together these new results add to our previous findings showing the recombinogenic and potentially mutagenic properties of Top1. We have pursued our studies relating Top1 to transcription. First, we showed the critical relationship between Top1 and transcription stop points that are associated with the formation of alternative DNA structures (guanosine quartets and R-loops) in the negatively supercoiled DNA segments that tend to arise in the wake of transcription complexes. Such negative supercoiling is facilitated by deficiency in Top1, which under normal conditions functions to eliminate the negative supercoiling generated in the wake of moving transcription complexes. We also demonstrated that Top1 stabilization by Top1-targeted drugs (and abnormal DNA structures;see above) induces abnormal splicing, especially in genes that encode splicing factors. Finally, using microarray analysis, we were able to show that the trapping of Top1 cleavage complexes by camptothecins blocks transcription selectively in long transcripts and at intron-exon junctions, which is consistent with the role of Top1 to relieve transcription-induced supercoiling and in splicing. 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). Although TOP1mt knockout mice are viable, we found that they are hypersensitive to the cardiotoxicity of doxorubicin. This is because Top1mt is required to regenerate mitochondrial DNA upon damage by doxorubicin. We also found that Top1mt is required to maintain the normal supercoiling of mitochondrial DNA and acts throughout the mitochondrial genome with preferential sites in the regulatory and replication origin regions. 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 not only explains the mild phenotype of our Top1mt knockout mice but also the cardiotoxicity of doxorubicin because of the trapping of mitochondrial topoisomerase II by doxorubicin. We have begun crossing our Top1mt knockout mice with other genetically altered mice bearing mitochondrial gene alterations.
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