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

Reviews on: DNA Repair Modulators as Anticancer Agents Repair of Topoisomerase I-mediated DNA damage Mechanism of action of topoisomerase I inhibitors Camptothecins and Topoisomerase I: A Foot in the Door. HIV-1 integrate as a target against HIV/AIDS HIV-1 Integrase Inhibitors: Update and Perspectives Chk2, ATM and DNA damage response: molecular interaction map p53-Mdm2: molecular interaction map Pharmacological and Therapeutics agents that Target DNA Replication

Chief, Laboratory of Molecular Pharmacology

pommier@nih.gov

Dr. Pommier received his M.D. and Ph.D. degrees from the University of Paris, France, and has been at the NIH since 1981. Dr. Pommier is a member of the Molecular Target steering committee at the NCI. He received an NIH Merit Award for his role in elucidating the function of topoisomerase enzymes as targets for anticancer drugs and Federal Technology Transfer Awards for studies on HIV-1 integrase and DNA topoisomerase inhibitors. Dr. Pommier is a program committee member of the American Association for Cancer Research, Senior Editor for Cancer Research, and associate editor for Cancer Research, Molecular Pharmacology, Leukemia, The Journal of Experimental Therapeutics and Oncology, The International Journal of Oncology, Drug Resistance Updates, and Current Medicinal Chemistry. Dr. Pommier serves as Chair for 2004-2005 Gordon conferences on the Molecular Therapeutics of Cancer. Dr. Pommier holds several patents for inhibitors of DNA topoisomerases I and II and HIV-1 integrase inhibitors.

DNA, Topoisomerases, DNA Damage Checkpoints, Molecular Interaction Maps, and HIV Integrase Molecular Pharmacology

DNA topoisomerases are essential for all DNA transactions because of the double helical structure of DNA. They change the topology of DNA by introducing reversible breaks in the DNA phosphodiester backbone. The broken DNA ends are held together by the topoisomerases in covalent complexes, which are referred to as cleavage complexes. We recently discovered a novel gene for a specific mitochondrial topoisomerase I, which demonstrate that human cells contain six topoisomerases genes: two type I topoisomerases (TOP1 and TOP1mt), two type II (TOP2-alpha and TOP2-beta) and two type III topoisomerases (TOP3-alpha and TOP3-beta). Mitochondrial Top1 (Top1mt) is present and restricted to all vertebrates whose genome has been sequenced to date, and the TOP1mt gene structure is remarkably conserved when compared to the nuclear TOP1 gene, suggesting that both genes derived from duplication of a common ancestor gene encoding both mitochondrial and nuclear type I enzymes.

 

Top1 and Top2 are the targets for potent anticancer drugs. Top2 inhibitors include the widely used anticancer agents: VP-16, adriamycin and mitoxantrone. Camptothecin is a specific Top1 inhibitor, and several camptothecin derivatives have recently been introduced in the clinic to treat solid tumors including colon, lung and ovarian carcinomas. Our aims regarding topoisomerase inhibitors are to discover novel inhibitors, and to study the molecular interactions between drugs, DNA and the enzyme-DNA complexes, as well as the cellular determinants of selectivity of cancers. We have shown that camptothecins 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. We refer to this concept as Interfacial inhibition, and we proposed interfacial inhibition as a Nature's paradigm for drug discovery. We have sequenced TOP1 point mutations that render the enzyme resistant to camptothecins, and structural studies are ongoing to further elucidate the structure of the Top1-DNA-camptothecin ternary complex. Such structures will be used for rational design of indenoisoquinolines as novel Top1 poisons.

 

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 lead to the formation of Top1 cleavage complexes. Top1 cleavage complexes also form during apoptosis induced by a broad range of inducers: arsenic trioxide, topoisomerase inhibitors, staurosporine (which we discovered as one of the most ubiquitous inducers of apoptosis), Fas, and Trail. Our working hypothesis is that these apoptotic Top1 cleavage complexes are trapped by oxidative DNA lesions induced by oxygen radicals (ROS) generated by the apoptotic cascade.

 

Topoisomerase inhibitors are only active on a fraction of patients, and therapeutic decisions remain largely empirical. We are investigating at the molecular level, why and how topoisomerase-mediated DNA damage selectively kills cancer cells. Elucidating the molecular determinant of sensitivity and resistance (i.e. identifying molecular markers of drug response) has the potential to: 1/ provide new ways to monitor and thereafter predict treatment outcome in clinical trials; 2/ rationalize therapeutic choices and drug combinations; and 3/ discover novel drug targets. We are investigating the DNA damage-induced repair and checkpoint pathways: (1) by characterizing the cellular lesions induced by Top1 cleavage complexes in cancer cells (replication-mediated DNA double-strand breaks and transcription complex interference); (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 (Bloom and Werner syndrome cells and cells deficient in PARP, beta-polymerase, XRCC1, etc.); and (4) by investigating the biochemical processing of Top1 cleavage complexes in vitro using oligonucleotides and purified repair factors (such as TDP and PNKP). To understand how the genetic makeup of human cells influences their cellular response to anticancer agents and the rationale underlying the selectivity of topoisomerase inhibitors toward cancer cells, we are studying cell lines from the NCI Anticancer Drug Screen and cell lines with selected gene disruptions. To comprehend the apparent complexity and redundancy of the repair and checkpoint pathways, we are developing molecular interaction maps.

 

Ecteinascidin 743 (Et743) is a natural product (from a Caribbean marine tunicate) remarkably active against sarcomas and presently in phase I/II clinical trials. Because of its unique activity profile, we are elucidating its mechanism of action. We first demonstrated that Et743 alkylates guanine N2 at selective sites in the DNA minor groove. This observation sets Et743 apart from the DNA alkylating agents currently in clinical use. We recently generated Et743-resistant cells, and found that these cells are deficient in nucleotide excision repair (NER). Additional studies led to the hypothesis that Et743 traps the transcription-coupled repair machinery as it attempts to remove the Et743-DNA adducts. Thus, Et743 is not just a Top1 and a transcription inhibitor, but its antiproliferative activity appears dependent upon TC-NER. To our knowledge, Et743 is the first drug with such a mechanism of action. This mechanism of action is opposite to cisplatin's, which is selectively toxic to TC-NER-deficient cells. Et743 might therefore represent another example of the "Interfacial Inhibition paradigm". In this case Et743 would trap the transcription-coupled repair complex. A translation program has been initiated to evaluate by proteomic analysis the relationship between TC-NER factors and therapeutic response to Et743 and cisplatin.

 

Our laboratory has pioneered the HIV integrase inhibitor research since 1993. 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. The rationale for searching HIV integrase inhibitors is that: (1) viruses with mutant integrase cannot replicate, and (2) integrase is one of the three retroviral enzymes (with reverse transcriptase and protease) with no cellular equivalent. 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. In collaboration with the Laboratory of Medicinal Chemistry, we have several families of inhibitors patented and available for development and licensing. Discovery and studies of HIV integrase inhibitors provides a new strategy for anti-AIDS therapy.

 

Selected recent publications:

1.         ________Zhang, H. et al. Human mitochondrial topoisomerase I. Proc Natl Acad Sci U S A 98, 10608-13. (2001).

2.         ________Furuta, T. et al. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA-double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J Biol Chem 278, 20303-20312 (2003).

3.         ________Khan, Q. A. et al. Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: Implications for interactions with intercalating anticancer agents. Proc Natl Acad Sci U S A 100, 12498-12503 (2003).

4.         ________Meng, L.-H., Liao, Z.-H. & Pommier, Y. Non-camptothecin DNA topoisomerase I inhibitors in cancer chemotherapy. Curr Topics Med Chem 3, 305-320 (2003).

5.         ________Pommier, Y. & Kohn, K. W. Cell cycle and checkpoints in oncology: new therapeutic targets. Med Sci (Paris) 19, 173-86 (2003).

6.         ________Pommier, Y. et al. Repair of and Checkpoint Response to Topoisomerase I-Mediated DNA Damage. Mutat Res 532, 173-203 (2003).

7.         ________Weinstein, J. N. & Pommier, Y. Transcriptomic analysis of the NCI-60 cancer cell lines. C R Biol 326, 909-20 (2003).

8.         ________Marchand, C. et al. Metal-Dependent Inhibition of HIV-1 Integrase by {beta}-Diketo Acids and Resistance of the Soluble Double-Mutant (F185K/C280S). Mol Pharmacol 64, 600-609 (2003).

9.         ________Chrencik, J. E. et al. Mechanisms of camptothecin resistance by human topoisomerase I mutations. J Mol Biol 339, 773-84 (2004).

10.        ______Johnson, A. A. et al. Position-specific suppression and enhancement of HIV-1 integrase reactions by minor groove benzo[a]pyrene diol epoxide deoxyguanine adducts: implications for molecular interactions between integrase and substrates. J Biol Chem 279, 7947-55 (2004).

11.        ______Pommier, Y., Sordet, O., Antony, S., Hayward, R. L. & Kohn, K. W. Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 23, 2934-49 (2004).

12.        ______Johnson, A. A., Marchand, C. & Pommier, Y. HIV-1 integrase inhibitors: a decade of research and two drugs in clinical trial. Curr Top Med Chem 4, 1059-77 (2004).

13.        ______Zhang, H., Meng, L. H., Zimonjic, D. B., Popescu, N. C. & Pommier, Y. Thirteen-exon-motif signature for vertebrate nuclear and mitochondrial type IB topoisomerases. Nucleic Acids Res 32, 2087-92 (2004).

14.        ______Sordet, O., Khan, Q. A. & Pommier, Y. Apoptotic Topoisomerase I-DNA Complexes Induced by Oxygen Radicals and Mitochondrial Dysfunction. Cell Cycle 3 (2004).

15.        ______Pommier, Y. & Cherfils, J. Interfacial protein inhibition: a nature's paradigm for drug discovery. Trends Pharmacol Sci In Press (2004).

16.        ______Kohn, K. W., Aladjem, M. I., Pasa, S., Parodi, S. & Pommier, Y. Cell cycle control: molecular interaction map. Nature Encyclopedia of the Human Genome 1, 457-474 (2004).

Last updated Mar. 2008