10th Conference on DNA Topoisomerases in therapy

PERSPECTIVE 10th Conference on DNA Topoisomerases in therapy

Table 1 Major clinically approved anticancer drugs that target DNA topoisomerases Drug

Chemical class

Topoisomerase

Giuseppe Giaccone,1 Giovanni Capranico2

Doxorubicin Daunorubicin Epirubicin Idarubicin Mitoxantrone Etoposide (VP-16) Teniposide (VM-26) Irinotecan Topotecan

Anthracycline Anthracycline Anthracycline Anthracycline Anthraquinone Epipodophyllotoxin Epipodophyllotoxin Camptothecin Camptothecin

IIA IIA IIA IIA IIA IIA IIA IB IB

1

Medical Oncology,Academic Hospital Vrije Universiteit,Amsterdam,The

Netherlands, 2Moruzzi Department of Biochemistry, Bologna University, Bologna, Italy

Abstract The 10th Conference on DNA Topoisomerases in Therapy 6–8 October 1999 in Amsterdam,The Netherlands) covered basic research on DNA topoisomerases and aspects of topoisomerase-directed therapy.The understanding of basic aspects of enzyme functions and structures was discussed throughout the meeting, as this knowledge is fundamental to further developments of new and more effective therapeutic approaches. Several new crystal structures were presented, and implications for function and interaction with DNA and drugs were discussed. Knock-out mice for various topoisomerase genes have been produced and genes have been shown to differ in importance for development and survival.The interaction of topoisomerases with other proteins involved in DNA metabolism, chromosome stability and physiology were discussed.The main focus for cancer therapy was on camptothecins (CPT) and related compounds stabilizing covalent DNA-intermediates of topoisomerase I. Reports on recent clinical trials of first-generation, water-soluble CPTanalogs (topotecan and irinotecan) confirmed earlier findings of activity in several solid tumors and hematological malignancies. Improvements in efficacy and toxicity profiles are being sought in orally absorbable compounds and other drug formulations (e.g. in liposomes). Several new CPT-analogs at preclinical stages of development might also provide a greater stability of the lactone ring, higher DNA-binding affinity, and improved water solubility. New drugs have also been developed from a number of new non-CPT compounds, which inhibit the activity of DNA-topoisomerases but do not stabilize the DNAlinked form of the enzymes.Another focus of the meeting was on therapeutic targeting of microbial DNA topoisomerases. The antibiotic potential of the quinolones has been extended to gram-positive pathogens, particularly Streptococcus pneumoniae.The cloning and biochemical characterization of the DNA-topoisomerases of eukaryotic parasites such as Plasmodium falciparum or Candida albicans have been completed and the search for specific inhibitors targeting these enzymes is under way. © 1999 Harcourt Publishers Ltd Key words: DNA topoisomerase I, camptothecins, DNA topoisomerase II, meeting

doxorubicin and etoposide, have curative potential for several hematological malignancies and solid tumors. However, significant toxicities limit their clinical use and dose escalation.Thus, the development of anti-topoisomerase II agents with reduced toxic side-effects is highly desirable. In addition, two topoisomerase I-directed camptothecins, irinotecan and topotecan (Table 1), have recently been introduced in the treatment of refractory colon and ovarian cancers, respectively, while several other analogs are in earlier phases of clinical development. Current knowledge indicates that, apart from the sideeffects, other obstacles that limit the use of anti-topoisomerase I and anti-topoisomerase II drugs are: (a) slow growing cancers, which may have reduced enzyme levels; (b) membrane pumps such as P-glycoprotein (PgP), which can decrease drug uptake and thus activity; (c) topoisomerase mutations, which can affect drug binding and efficacy of enzyme poisoning by drugs; and (d) alterations of cell processing of drug-stimulated DNA lesions, which can increase cancer cell survival following drug treatments.The last two topics constituted a major focus at the meeting held at the Amsterdam Free University. At the same time, the understanding of basic aspects of enzyme functions and structures was discussed throughout the 3 days of the meeting, as this knowledge is fundamental to further developments of new and more effective therapeutic approaches. The meeting was organized by the European Cancer Centre (Amsterdam,The Netherlands) under the direction of Giuseppe Giaccone (Oncology Department, Free University, Amsterdam, The Netherlands). It followed the 9th Symposium of this series, held in New York last year, and was attended by over 150 participants from academia and pharmaceutical industry. STRUCTURES AND FUNCTIONS OF TOPOISOMERASES

INTRODUCTION

C

linical and preclinical research has been very active in the topoisomerase field in the last two decades, and at least six anticancer agents that target topoisomerase II are currently approved for general clinical use by most countries (Table 1). Some of these drugs, particularly

The structure of human topoisomerase I was recently published.1 At the conference, J. Champoux (Seattle, USA) discussed the resolution of novel crystals of the enzyme bound to different DNA oligomers. In particular, Lys532 may form hydrogen bonds with different oxygens of the DNA phosphate backbone. DNA and protein domains are oriented in a slightly different way in two structures, thus suggesting that  1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 347–350 DOI: 10.1054/drup.1999.0109, available online at http://www.idealibrary.com on

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Giaccone and Capranico the protein ‘cap’ and ‘linker’ regions are quite flexible.These observations are both compatible with the proposed controlled rotation model for top1 DNA relaxation.1,2 J.C. Wang (Harvard, USA) reported on topoisomerase gene knockout mice. While homozygous top3b mice are apparently viable, disruption of all other topo genes are lethal during mouse development. Interestingly, absence of top2b leads to neural and neuromuscular abnormalities suggesting that the b form of top2 may have crucial roles during neural development. Topoisomerase interactions with other proteins is a hot issue in the field. For the eukaryotic enzymes, the effect of the RNA-splicing factor PSF/p54nrb on topo 1 was discussed by F. Boege (Wuerzburg, Germany). It has been previously shown that the splicing factor was able to increase topo 1 DNA relaxation activity. Using a suicide DNA substrate, it has now been demonstrated that the splicing factor increases the DNA dissociation rate of topo 1 thus switching the enzyme from a processive to a more distributive mode of operation. Interactions of eukaryotic top3 with RecQ helicases are relevant for several cancer-prone syndromes in humans. Yeast genetics have been invaluable in dissecting functions of top3 and helicases that appear to be essential for viability and for suppressing DNA recombination. The Bloom’s syndrome protein, a human RecQ helicase, has been shown to interact with two independent domains of top3a (I. Hickson, Oxford, UK). Deletion of top3 gene in yeast causes a cell phenotype hypersensitive to DNA damaging agents such as hydroxyurea and methanesulfonate. Interestingly, human lymphoblastoid cells from patients with Werner syndrome (which have a gene defect related to Bloom’s protein) are more sensitive to camptothecin cell killing effects.3 DNA damage recognition often elicits a cell cycle checkpoint. In mutant studies with yeast, R. Rothstein (New York, USA) has identified SML1 gene that allows cell viability in the absence of checkpoint genes such as MEC1 and RAD53. Further data suggested that Sml1 protein inhibits dNTP synthesis by binding directly to Rnr1 (the large subunit of ribonucleotide reductase) and that Mec1 and rad53 are required to relieve this inhibition at S phase and after DNA damage. Topoisomerase 1 interaction with other proteins can control or modulate enzyme activity in nuclear chromatin.The fusion of human top1 to a heterologous specific DNA-binding domain (Gal4) has been presented by G. Capranico (Bologna, Italy). The fusion enzyme was targeted at a site recognized by Gal4 under controlled conditions.4 If this is achieved in living cells as well, it may provide new means to further define topoisomerase functions and to better understand the cell killing mechanisms of topoisomerase poisons. DNA TOPOISOMERASES IN CHROMATIN DYNAMICS The modulation of chromatin and chromosome structures by DNA topoisomerases was also discussed at the Conference. Topoisomerase II is one of the most abundant non-histone proteins in chromosomes. A new type II topo, topo VI, has been found in Saccharomyces cerevisiae, called Spo11, which has been shown to have a crucial role during meiosis since it produces a double-stranded DNA cut (DSB) that initiates meiotic recombination.The distribution of DSB 348

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along a chromosome is not uniform and centromere sequences suppress meiotic recombination. New evidence now show that this suppression operates at the initiation step by repressing formation of DSB (M. Lichten, Bethesda, USA). Moreover, M. Lichten presented data suggesting that DNA replication is required to target DSB-forming protein complexes to DNA sites known to be hot spots meiotic recombination. The functional interactions of topo II with cohesins and condensins in Xenopus has been presented by T. Hirano (Cold Spring Harbor, USA). These proteins are responsible for chromosome condensation at mitosis and sister chromatid cohesion established during S phase. Their interactions with topoisomerases are essential to fold and organize properly chromatin fibers. Polycomb (PcG) and trithorax (trxG) group genes encode components of multiprotein complexes involved in chromatin remodeling implicated in the maintenance of cellular identity at the level of chromatin structure. Chromatin binding sites of PcG, topo II as well SARs have been mapped in Drosophila cells (V. Orlando, Milan, Italy). Seven topo II binding sites were found at the bithorax complex locus, three of them matching SARs and the others Pc/trx protein binding sites.All SARs and topo II sites were confined to the repressed domain of the bithorax locus whereas the active region was without them. The results suggest a model in which processes of chromosome condensation are driven by SARs, topo II and PcG proteins thus cooperating in the maintenance of the repressed state of homeotic genes. MECHANISMS OF SENSITIVITY AND RESISTANCE Camptothecin reversibly stabilizes topoisomerase I-linked DNA single-strand breaks, which then interact with replication forks giving rise to irreversible DNA double-strand breaks. These are considered the primary cytotoxic lesions induced by this drug and alterations of the molecular mechanisms that process the lesion inside the nucleus can affect for drug activity.5 Y. Pommier (Bethesda, MD, USA) presented elegant data showing that these double-strand breaks are only formed on the leading strand, whereas on the lagging strand a blockage of replication fork progression occurs. M.A. Bjornsti (Memphis, TN, USA) showed that cellular coping with such lesions induced by covalently DNA-linked topoisomerase I at the replication fork involves proteins required for replication initiation (CDC45 and DBP11), suggesting that polymerase switching may be a compensatory mechanism.6 DNA-tyrosine phosphodiesterase (DTP1), an enzyme able to cleave the phosphodiester linkage between the active site tyrosine of topoisomerase I and DNA, might constitute another compensatory mechanism and can affect cell sensitivity. H. Nash (Bethesda, MD, USA), who discovered the enzyme 3 years ago, presented information on the primary structure and genetics of DTP1.The enzyme cannot be assigned to any known protein family and is highly conserved in all organisms expressing topoisomerase I. DTP1knockouts are hypersensitive to camptothecin. Thus DTP1 seems to constitute a major physiological modulator of camptothecin activity. Another cellular mechanism that could confer cellular resistance to topoisomerase I-poisons appears to be the degradation of DNA-linked topoisomerase

DNA topoisomerases I, which is mediated by modifications induced by ubiquitin and also by small ubiquitin-like modulator proteins (SUMO). Several topoisomerase mutations were presented. Enzyme mutations affecting drug activity may indicate the binding site of the drugs. However several mutations of topoisomerase II that affect chemically distinct poisons map in the ATP-binding domain of the enzyme. In general, the data indicate that the multidrug resistance/sensitivity of these mutant enzymes can be explained by specific defects in ATP utilization during enzyme catalysis.7 CLINICAL EXPERIENCE WITH CPT-11 AND TPT The clinical use of topoisomerase inhibitors has preceeded the discovery of their cellular target by several years. After the demonstration of covalently DNA-linked catalytic intermediates of DNA-topoisomerase as a crucial mechanism in cytotoxic chemotherapy, much effort has been invested into the discovery and development of new substances targeting these enzymes in bacteria, parasites, and tumor cells. While topoisomerase II-directed therapeutics were more clearly the focus of previous meetings, this year’s Conference was almost completely devoted to topoisomerase I inhibitors. The reason for this change is due to the recent introduction of water-soluble camptothecin derivatives, such as irinotecan and topotecan (Table 1), into routine anticancer therapy. Both drugs are in fact now registered in several countries. Irinotecan (CPT-11) is now registered worldwide for second-line systemic treatment of metastatic colorectal cancer, based on the increase in response and survival over 5-FU chemotherapy8 or best supportive care.9 The combination of CPT-11 with 5-FU in first-line treatment of colorectal cancers has also produced improved results in comparison with 5-FU based regimens, and synergistic effects with various agents have been observed in the first-line treatment of several rather chemoresistant tumors, such as gastric cancer, cervical cancer, and non-small cell lung cancer. Ongoing developments of systemic therapy with CPT-11 aim at the formulation of oral CPT-11, which might have the advantage of continuous administration and ease for the patient. Topotecan (TPT) is currently registered for second-line treatment of ovarian cancer and also in second-line treatment of small cell lung cancer in some countries (Table 1). There is a major schedule dependency, as far as toxicity is concerned, when the drug is combined with other agents, in particular cisplatin, which limits considerably the possibility of combining this agent with other agents which have predominant myelosuppression as side-effect. Randomized studies in second-line treatment of ovarian cancer and small cell lung cancer have indicated that the oral formulation of the drug yields similar results as the intravenous formulation but is less toxic. Both formulations have been studied in combinations with various other agents. Most notably, the combination with ara-C appears to be synergistic. This may be partially due to the fact that ara-C in itself possesses some activity as a topoisomerase I poison. A number of novel camptothecin analogs are at preclinical or early clinical stages of development. All these drugs appear to have a stronger inhibitory activity than camptothecin or SN38, the active metabolite of CPT-11.

Two different approaches were followed to improve on the first generation of camptothecins. In one approach, in order to make the lactone ring of water-soluble compounds more stable, a methylene spacer has been inserted between the alcohol and carboxyl functions of the E-ring in homocamptothecins. Moreover, decomposition to the inactive open ring form is irreversible, which enables a more predictable relationship between dosage and toxicity, because reformation of the active compound at low pH is avoided.The inhibitory activity of homocamptothecins was further enhanced by fluorination of the A-ring at several positions. The most potent compound of this class (BN 80915) has been selected for further development. Lurtotecan (NX211), a watersoluble camptothecin, showed favorable properties in phase II clinical trials, and a liposomal formulation of lurtotecan has been developed, which exhibits a markedly enhanced plasma stability of the lactone ring, prolonged plasma circulation, and a marked increase in cellular drug delivery. In xenograft tumor models the compound was most active in ovarian and small cell lung cancers. In an attempt to minimize the decomposition of the lactone and find a drug suitable for oral application, karenitecin (BNP 1350), a semisynthetic highly lipophilic CPT-derivative was developed. It has been engineered (computer-assisted drug design) for superior oral bioavailability, greater lactone stability, increased potency, and is not a substrate of drug transporters such as PgP or MRP. Moreover, BNP1350 shows a significantly increased DNA-binding affinity, which may also explain its greater stability. It was effective in eight out of nine experimental human cancers tested and oral administration was as effective as the i.p. route. It has been suggested that BNP1350 would be a suitable compound for the oral treatment of cancer and it was recently introduced into phase I clinical trials in the US. Rubitecan (9-nitrocamptothecin, RFS2000) is another insoluble CPT-derivative, which is in advanced development as an oral cancer therapy.A large randomized study has been initiated in untreated pancreatic cancer and several other studies are investigating combinations of rubitecan with other antitumor agents in several tumor types. CATALYTIC INHIBITORS OF HUMAN DNA TOPOISOMERASES A number of compounds structurally unrelated to CPT have been discovered in the last 2 or 3 years.They inhibit earlier steps of the catalytic cycle (DNA-binding and/or DNA-cleavage) and consequently have been termed catalytic topoisomerase I inhibitors.10 A number of substances with such an activity profile were presented. These include 1-n-alkylcarbamoyl-5-fluorouracil, bis-netropins, tyrphostins, rebeccamycin analogues, and pyrazolo (1,5-a) indole derivatives. F 11782, a fluorinated lipophylic epipodophylloid and a series of anthraquinone-peptide conjugates were presented as catalytic inhibitors of both topoisomerase I and II. Most of these substances exhibit rather good antitumor activity, although it is still unclear why catalytic inhibition of topoisomerase I should kill tumor cells. Another interesting area of application of catalytic topoisomerase I-inhibitors might be the treatment of viral infections, such as HIV, where the virus  1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 347–350

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Giaccone and Capranico needs host topoisomerases for replication. Data were presented showing replication of murine HIV and infection of murine cell lines by the virus to be reduced up to 90% after treatment with tyrphostins. However, it remains to be demonstrated that topoisomerases are indeed the crucial cellular targets of these substances.

Received 12 November 1999; Revised 1 December 1999; Accepted 10 December 1999 Correspondence to: Giuseppe Giaccone, Department of Medical Oncology, Academic Hospital,Vrije Universiteit De Boelelaan, 1117 1081 HV Amsterdam,The Netherlands. Fax: +31 20 444 4079;Tel: +31 20 444 4300; E-mail: [email protected]

NEW TARGETS AMONG MICROBIAL DNA TOPOISOMERASES Among known bacterial topoisomerases, the type II enzymes DNA gyrase and topoisomerase IV have been exploited by nature and the pharmaceutical industry as antibacterial targets.11 Natural products that inhibit one or both of these topoisomerases include the coumarins, cyclothialidines, flavones, and terpenoid derivatives. Interestingly, plasmidencoded peptides, micron B17 and CcdB, inhibit DNA gyrase as well. However, only the quinolones, a synthetic class of bacterial type II topoisomerase poisons, have a broad clinical use. Studies of an expanding set of resistant mutant enzymes and the crystal structure of the homologous enzyme in yeast have contributed to our understanding of interactions of these drugs with topoisomerase-DNA complexes and the ways in which mutations affect resistance. Renewed interest in the fluoroquinolones stems from the recent development of drugs active against gram-positive bacterial pathogens, particularly Streptococcus pneumoniae. Several agents including sparfloxacin, grepafloxacin, and gemifloxacin display enhanced antipneumococcal activity over that of ciprofloxacin and are either approved or in clinical trials. It seems that in S. pneumoniae these new quinolones target preferentially DNA-gyrase, whereas ciprofloxacin acts through topoisomerase IV, which may explain the altered spectrum of sensitivity and the lack of cross resistance between these two drugs. New approaches to the treatment of malaria may be derived in the future from the cloning and characterization of the DNA-topoisomerases of Plasmodium falciparum. So far, two topoisomerases have been cloned, which are homologs of eukaryotic topoisomerase I and II.A third topoisomerase is possibly present in P. falciparum, which might be a homolog of E. coli gyrase, but which has not been completely identified. Extensive structural differences between P. falciparum topoisomerases I and II and their human homologs have been detected, suggesting that specific inhibitors can be developed, which will not target the human enzymes. Moreover, expression of P. falciparum topoisomerases I and II raises as the parasite progresses to the trophozoite and schizont stages of development.Thus it seems feasible that the cycle of infection could be effectively disrupted by specific inhibitors of these enzymes.

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References 1 Redinbo MR, Champoux JJ, Hol WG. Structural insights into the function of type IB topoisomerases. Curr Opin Struct Biol 1999; 9: 29–36. 2 Redinbo MR, Stewart L, Champoux JJ, Hol WG. Structural flexibility in human topoisomerase I revealed in multiple non-isomorphous crystal structures. J Mol Biol 1999; 292: 685–696. 3 Poot M, Gollahon KA, Rabinovitch PS.Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase. Hum Genet 1999; 104: 10–14. 4 Beretta GL, Binaschi M, Zagni E, Capuani L, Capranico G.Tethering a type IB topoisomerase to a DNA site by enzyme fusion to a heterologous site-selective DNA-binding protein domain. Cancer Res 1999; 59: 3689–3697. 5 Shao RG, Cao CX, Zhang H, Kohn KW,Wold MS, Pommier Y. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA: DNA-PK complexes. EMBO J 1999; 18: 1397–1406. 6 Reid RJ, Fiorani P, Sugawara M, Bjornsti MA. CDC45 and DPB11 are required for processive DNA replication and resistance to DNA topoisomerase I-mediated DNA damage. Proc Natl Acad Sci U S A 1999; 96: 11440–11445. 7 Mao Y,Yu C, Hsieh TS et al. Mutations of human topoisomerase II alpha affecting multidrug resistance and senstivity. Biochemistry 1999 38: 10793–10800. 8 Rougier P,Van Cutsem E, Bajetta E et al. Randomised trial of irinotecan versus fluorouracil by continuous infusion after fluorouracil failure in patients with metastatic colorectal cancer: Lancet 1998; 352: 1407–1412. 9 Cunningham D, Pyrhonen S, James RD et al. Randomised trial of irinotecan plus supportive care versus supportive care alone after fluorouracil failure for patients with metastatic colorectal cancer. Lancet 1998; 352: 1413–1418. 10 Capranico G, Giaccone G, D’Incalci M. DNA topoisomerase II poisons and inhibitors. In: Pinedo HM, Longo DL, Chabner BA, eds. Cancer chemotherapy and biological response modifiers Annual vol. 18. Elsevier Science Publs, 1999; 126–144. 11 Hooper DC. Bacterial topoisomerases, anti-topoisomerases, and anti-topoisomerase resistance. Clin Infect Dis 1998; 27 (Suppl 1): S54–S63.