Yves Pommier, M.D., Ph.D.

DNA, Topoisomerases, DNA repair, Checkpoints, Pharmacogenomics and HIV Integrase Molecular Pharmacology

DNA topoisomerases are essential for all DNA transactions due to the double helical structure of DNA. They change the DNA topological structure by introducing reversible breaks in the DNA, which are referred to as cleavage complexes. Human cells contain six topoisomerases genes:  two for the type IB topoisomerases (TOP1 and TOP1mt), two for the type II (TOP2-alpha and TOP2-beta) and two for the type IA topoisomerases (TOP3-alpha and TOP3-beta).

Our laboratory discovered a nuclear-encoded topoisomerase gene specific for mitochondrial DNA (TOP1mt). Top1mt is present in all vertebrates, and the structure of the TOP1mt gene is remarkably conserved when compared to the nuclear TOP1 gene, suggesting that both genes derived from duplication of a common ancestor gene that may have encoded both mitochondrial and nuclear type IB topoisomerases. We are pursuing our studies on the functions of Top1mt and on the presence of other topoisomerases in mitochondria, and we are testing whether Top1mt could play a role in mitochondrial diseases.

Top1 and Top2 are targets for the most potent and widely used anticancer drugs. Top2 inhibitors include etoposide, doxorubicin (adriamycin) and mitoxantrone. Camptothecin is a specific Top1 inhibitor, and several camptothecin derivatives (topotecan, irinotecan and belotecan) are used to treat solid tumors including colon, lung and ovarian carcinomas, as well as hematopoietic and pediatric malignancies. Our aims regarding topoisomerase inhibitors are to discover novel inhibitors, and to rationalize the use and combination of these inhibitors with other anticancer agents. In collaboration with Dr. Mark Cushman (Purdue University, IN), we have discovered and optimized a novel family of Top1-targeted drugs, the indenoisoquinolines. Structural studies have elucidated the structure of the trapped Top1-DNA-camptothecin ternary complex. Such structures have been used for the rational design of the novel Top1-targeted indenoisoquinolines. Two derivatives are in clinical trial at the NIH. Histone gamma-H2AX (discovered by our coworker Dr. William Bonner, LMP, CCR, NCI) and Top1 levels are used as pharmacodynamic biomarkers to follow the effects of the drugs in the tumors and normal tissues.

We have shown that camptothecins and the indenoisoquinolines bind at the Top1-DNA interface. This mode of non-competitive inhibition (consisting of a trimer: the drug, the DNA, and Top1) represents a pharmacological paradigm, as the camptothecin molecule blocks a functional complex between the DNA and Top1 (by extension: two macromolecules) by inhibiting its dissociation rather than by blocking its formation.

It is noteworthy that recent studies have extended this paradigm to many natural products that target macromolecular protein complexes, such as microtubules (vinblastine, colchicine, taxol) or G-proteins (brefeldine A). Recently, interfacial mechanisms have been revealed by crystal structures for Top2 inhibitors (etoposide and quinolones) and HIV integrase strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir). Thus, interfacial inhibition is relatively common for medicinal drugs. And it is logical to propose screening for interfacial inhibitors as a novel avenue for drug discovery using strategies and assays based on stabilization of macromolecular complexes.

Top1-mediated DNA damage can be elicited by commonly occurring endogenous DNA modifications (mismatches, abasic sites, 8-oxoguanine, DNA breaks), as well as by carcinogenic polycyclic aromatic adducts (ethenoadenine, benzo[a]pyrene diol epoxide adducts). These observations suggest that frequently occurring DNA modifications can lead to the formation of Top1 cleavage complexes. In addition, we recently reported that Top1 can also convert misincorporated ribonucleotides into DNA breaks and generate small deletions and insertions. Together, these findings demonstrate the recombinogenic activity of Top1 and the importance of DNA repair for counteracting endogenous DNA damage induced by Top1.

To better use topoisomerase- and DNA-targeted drugs, we need to understand the determinants of response to those drugs. In other words, we need to understand at the molecular and pathway levels why cancer cells are, in many instances, more sensitive to topoisomerase-targeted drugs than normal cells. To reach this goal, we are investigating the mechanisms of damage and repair in several ways: (1) by characterizing the cellular lesions induced by Top1 cleavage complexes in cancer cells (replication-and transcription-mediated DNA double-strand breaks); (2) by elucidating the cellular responses/pathways elicited in response to such lesions (activation of DNA-PK, RPA phosphorylation, activation of histone phosphorylation [gamma-H2AX], transcriptional responses); (3) by analyzing the effects of camptothecins in mammalian cells with known genetic defects (Werner syndrome and cells deficient in PARP, beta-polymerase, XRCC1, etc.); and, (4) by investigating the biochemical processing of Top1 and Top2 cleavage complexes in vitro using oligonucleotides and purified repair factors (such as TDP1 and TDP2).

To understand how the genetic makeup of human cells influences their cellular response to anticancer agents and the rationale for the selectivity of topoisomerase inhibitors toward cancer cells, we are studying the cell lines from the NCI Anticancer Drug Screen and a set of chicken DT40 cell lines with single or double gene disruptions in selected DNA repair and checkpoint pathways.  The NCI-60 cell line panel is used by the NCI Developmental Therapeutics Program (DTP) to screen for and characterize novel anticancer drugs. The cell lines encompass 9 tissues of origin including breast, central nervous system, colon, lung, prostate and renal cancers as well as leukemia and melanomas. Hundreds of thousands of drugs have been tested over the years and our laboratory maintains a database for approximately 16,000 drugs, including the FDA-approved anticancer drugs. Over the years, our laboratory has characterized the genome of these 60 cell lines [by multiple gene expression microarray platforms, spectral karyotypic analyses (SKY), comparative genomic hybridization (aCGH) and microRNA expression microarrays]. In collaboration with the NCI Division of Cancer Treatment and Diagnosis (DCTD) and the Genetics Branch (NCI-CCR), we are currently sequencing all the genes for the entire set of cell lines. This exome sequencing effort will be made publicly available in the near future and will enable large-scale system biology studies related to oncogenes, tumor suppressor genes and pharmacogenomics.

Anti-HIV integrase (IN) small molecules are being actively pursued following the FDA approval of raltegravir (RAL; Isentress; MK-0518; Merck & Co.) and the promising activity of several IN inhibitors in clinical trials: elvitegravir, dolutegravir and BMS-707035. These drugs are highly targeted to the IN active site and are often referred to as INSTIs (integrase strand transfer inhibitors). INSTIs act by binding two metal ions at the interface of the enzyme and the viral DNA, thereby inhibiting integration by blocking the binding of target/chromosomal DNA (see interfacial inhibition paragraph above). As a consequence of the high affinity targeting of IN, point mutations of IN are emerging as a major mechanism of resistance to raltegravir and elvitegravir.

We are currently focusing on the characterization of these resistance mutations and developing recombinant IN proteins to test their biochemical properties and molecular pharmacology. We investigate the molecular interactions of drugs with retroviral integrases using recombinant integrases in biochemical assays and by exploring different steps of the integration reaction. Our goals are to discover new antiviral agents, evaluate which steps of the integration reactions are affected by drugs, and determine the drug binding site in the HIV-1 integrase-DNA complex.