We have pursued our discovery and molecular pharmacology of novel topoisomerase I (Top1)-targeted anticancer agents to alleviate the limitations of camptothecins, which are yet routinely used to treat ovarian, colon and lung cancers as well as hematologic malignancies. We have pursued our studies with the two classes of novel non-camptothecin Top1-targeted drugs: the indenoisoquinolines and the Genzyme derivative, Genz-644282. The indenoisoquinolines have been discovered 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). We have continued to discover and characterize novel derivatives to optimize the indenoisoquinolines. As a result, two indenoisoquinolines (NSC 725776 and 743400) are in clinical trials at the NCI. This drug development is a collaboration between several groups: LMP (our group and Dr. Bonner for gamma-H2AX biomarker), 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 have also studied and characterized the other novel non-camptothecin and non-indenoisoquinoline topoisomerase inhibitors from Genzyme (Genz-644282), which is also in early clinical trial. We have characterized the molecular and cellular pharmacology of Genz-644282 and its metabolites. We have also shown the value of histone gamma-H2AX as a biomarker for Genz-644282. This research represents a strong translational component because it demonstrates our ability to discover and develop new drugs, to generate pharmacodynamic biomarkers and to work as a team both the NCI drug development teams and the pharmaceutical industry under clearly defined research agreements (referred to as CRADA). We have continued to study the importance of Top1-DNA complexes not only as the targets for Top1-targeted drugs, but also as potentially mutagenic lesions when Top1 processed abnormal DNA substrates. We recently reported in Science 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 ribonucleotide 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 also initiated a new set of studies that relate Top1 to transcription and published three manuscripts on these topics within the past year. First, we reveal 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. 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: mouse, rat, chicken, and zebra fish. However, 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 a 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. We have also generated Top1mt knockout mice and are presently studying their phenotype and their genotype. Our current studies are focused on the presence of other topoisomerases in mitochondria and on the phenotype of cells without Top1mt or with a toxic Top1mt.
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