We have pursued our discovery and molecular pharmacology of novel topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors to alleviate the limitations of camptothecins, doxorubicin and etoposide while retaining their potent antitumor activity. The indenoisoquinolines have been discovered and pursued in collaboration with Dr. Cushman at Purdue University. Two indenoisoquinolines (NSC 725776 and 743400) have begun clinical trial at the NCI with histone gamma-H2AX as biomarker in tumor and normal tissues. This translational drug development has been made possible by a close collaboration between the LMP (our group and Dr. Bonner for gamma-H2AX biomarker), the Clinical Oncology Branch (Dr. Doroshow and Shivaani Kummar for clinical trials), the NCI-DTP-DCTD (Dr. Hollingshead, Dr. Parchment and Dr. Kinders for mouse models and pharmacodynamic biomarkers), and Purdue University (Dr. Mark Cushman for drug synthesis). 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). We have also studied and characterized additional novel topoisomerase inhibitors. Those inhibitors belong to different chemical families: the homocamptothecins, camptothecins keto derivatives and aromathecins against Top1, and Dp44mT and epoxy-anthraquinones against Top2. We have also studied the molecular and cellular pharmacology of novel non-camptothecin Top1 inhibitors from Genzyme Co., which is just starting clinical trials. Using Top1-depleted human cells, we have shown the critical role of Top1 in genomic stability. Depletion of top1 produces replication damage. We have also shown for the first time that the trapping of Top1 (by drugs or potentially DNA damage) produces transcription-associated DNA double-strand breaks, as a result of R-loop formation. We have extended our studies on the repair of topoisomerase-associated DNA damage and focused on tyrosyl-DNA-phosphodiesterase (Tdp1). Our studies are focused on the cellular regulation of Tdp1 and the discovery of inhibitors that could be used in combination with camptothecins or indenoisoquinolines for the treatment of cancer. The first and still the only specific mitochondrial topoisomerase, Top1mt, was discovered in our laboratory. Top1mt is encoded by a nuclear gene present in all vertebrate genomes sequenced to date. The gene is absent in non-vertebrate including yeast and plants. We have proposed that Top1mt arose by duplication of a common ancestral TOP1 gene (found today in simple chordates) during evolution of vertebrates. The other TOP1 gene encodes the previously known Top1 devoted to the nuclear genome. We have generated specific antibodies for Top1mt, which enabled us to demonstrate that Top1mt is absent from nuclei and concentrated in mitochondria. We have also found that Top1mt can be trapped by camptothecin and used this finding to map the Top1mt binding sites in mitochondrial DNA (mtDNA). Mapping of Top1mt sites in the regulatory D-loop region of mtDNA in mitochondria revealed the presence of an asymmetric cluster of Top1mt sites confined to a 150-bp segment downstream from, and adjacent to, the site at which replication is prematurely terminated, generating an almost equal to 650-base (7S DNA) product that forms the mitochondrial D-loop. Moreover, we showed that inhibition of Top1mt by camptothecin reduces formation of the 7S DNA. Our results suggest novel roles for Top1mt in regulating mtDNA replication. In collaboration we Dr. Fritz Boege in Germany, we have shown that targeting (nuclear) Top1 is toxic, which demonstrate the different functions of Top1mt and Top1 in spite of their high degree of homology. We have also generated the Top1mt knockout mice and are studying their phenotype.
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