Leukemia (2001) 15, 875–890 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu REVIEW Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini1, JR Mackey2 and C Dumontet1,3 1Unite´ INSERM 453, Laboratoire de Cytologie Analytique, Faculte´ de Me´de´cine Rockefeller, Lyon, France; 2Medical and Experimental Oncology, Department of Oncology, University of Alberta, Edmonton, Alberta, Canada; and 3Service d’He´matologie, Centre Hospitalier Lyon Sud, France Nucleoside analogues (NA) are essential components of AML ter drugs have mostly been used in the treatment of low-grade induction therapy (cytosine arabinoside), effective treatments hematological malignancies.8,9 of lymphoproliferative disorders (fludarabine, cladribine) and are also used in the treatment of some solid tumors The importance of NA chemotherapeutic agents has (gemcitabine). These important compounds share some gen- recently increased as a result of the introduction of new com- eral common characteristics, namely in terms of requiring pounds into clinical use and the expansion of indications into transport by specific membrane transporters, metabolism and the field of solid tumors. During the same period, there has interaction with intracellular targets. However, these com- been rapid progress in the understanding of mechanisms of pounds differ in regard to the types of transporters that most drug resistance to NA. In this review, we highlight the current efficiently transport a given compound, and their preferential interaction with certain targets which may explain why some knowledge concerning the cellular mechanisms of resistance compounds are more effective against rapidly proliferating to NA and possible strategies that may be used to overcome tumors and others on neoplasia with a more protracted evol- such resistance. ution. In this review, we analyze the available data concerning mechanisms of action of and resistance to NA, with particular emphasis on recent advances in the characterization of nucleo- Normal nucleoside transport and phosphorylation side transporters and on the potential role of activating or inac- tivating enzymes in the induction of clinical resistance to these compounds. We performed an extensive search of published Because physiologic nucleosides are generally hydrophilic in vitro and clinical data in which the levels of expression of and do not readily permeate the plasma membrane, their nucleoside-activating or inactivating enzymes have been corre- cellular uptake occurs primarily via nucleoside-specific mem- lated with tumor response or patient outcome. Strategies aim- brane transport carriers (NT). Of the seven distinct NT activi- ing to increase the intracellular concentrations of active com- ties observed in human cells, only four have been defined pounds are presented. Leukemia (2001) 15, 875–890. 9 in molecular terms. These are classified into two categories: Keywords: nucleoside analogues; nucleoside transporters; 5 - 10,11 nucleotidase; drug resistance equilibrative (ENT) or concentrative (CNT). Human equil- ibrative NTs (hENTs) are found in virtually all studied cell types and have broad permeant selectivity, accepting both Introduction purine and pyrimidine nucleosides as substrates. The two cloned hENTs differ in their sensitivity to inhibition by nano- Nucleoside analogues (NA) constitute an important class of molar concentrations of nitrobenzylmercaptopurine ribonu- antimetabolites used in the treatment of hematological malig- cleoside (NBMPR): hENT1 has es activity (equilibrative and 1 NBMPR-sensitive) while hENT2 possesses ei (equilibrative and nancies and, more recently, in solid tumors. These thera- 12–14 peutic compounds mimic physiological nucleosides in terms NBMPR-insensitive) nucleoside transport activity. The of uptake and metabolism and are incorporated into newly human concentrative nucleoside transporters (hCNT) are synthesized DNA resulting in synthesis inhibition and chain inwardly-directed transporters that are capable of transporting nucleosides against a concentration gradient by utilizing the termination. Some of these drugs also inhibit key enzymes 15–17 involved in the generation of the purine and pyrimidine transmembrane sodium concentration gradient. The nucleotides and RNA synthesis, and directly activate the hCNT1 protein has greater affinity for pyrimidine nucleosides but also transports adenosine, while the hCNT2 protein trans- caspase cascade. All of these effects may lead to cell death. 18 The NA family includes various pyrimidine and purine ana- ports purine nucleosides and uridine. In all cases, human logues. Among the pyrimidine analogues cytosine arabinoside NTs accept only dephosphorylated compounds. (ara-C, cytarabine) is extensively used in the treatment of Deoxyribonucleotide triphosphate (dNTPs) pools present acute leukemia, while gemcitabine has more recently demon- within cells come from two sources, the de novo pathway strated activity in pancreatic, breast and lung cancer.2–4 The which is specifically activated in replicating cells, and the sal- vage pathway, which is the main source of nucleotides in two first purine analogues to have been described were the 19,20 thiopurines 6 mercaptopurine (6-MP) and 6-thioguanine (6- quiescent cells. The key step in the de novo pathway is TG). These compounds could more appropriately be desig- the conversion of ribonucleoside diphosphates into deoxyri- nated as ‘nucleobases’. Other new purine analogues are 2- bonucleoside diphosphates by ribonucleotide reductase (RR). chlorodeoxyadenosine (2-CdA) and fludarabine.5–7 These lat- Replicating hematopoietic cells are heavily dependent on the de novo pathway. Conversely in resting cells the salvage path- way, which recycles bases and nucleosides derived from DNA or RNA catabolism, is the unique provider of dNTPs. These Correspondence: CM Galmarini, Laboratoire de Cytologie Analytique, Faculte´ de Me´decine Rockefeller, 8 Avenue Rockefeller, 69373, Lyon compounds are first phosphorylated by nucleoside kinases Cedex 08, France; Fax: 33 4 78 95 35 05 such as deoxycytidine kinase (dCK), thymidine kinase 1 and Received 21 September 2000; accepted 1 February 2001 2, or deoxyguanosine kinase (dGK). This initial phosphoryl- Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 876 Table 1 Human nucleoside transporters (NT) mediating uptake of nucleoside analogues NT ara-Ca Gemcitabine Fludarabine 2-CdA Ref. hENT1 ++++ ++++ +++ ++++ 16, 96, 242, 95, 243 hENT2 ND ++ ++ ++ 11, 54, 81 hCNT1 + ++++ − − 17, 54, 81 hCNT2 ND −−− 18, 81 ND, not determined. aDegree of transport: (+), low transported; (++), moderate transported; (+++/++++), highly transported; (−), not transported. ation step constitutes the key step in the salvage pathway that to ‘self-potentiate’ their own cytotoxic effects. However each concludes with the formation of triphosphate or deoxytri- of these compounds also possesses specific properties in terms phosphate derivatives. There are thus two dNTP pools: one of drug–target interactions which may explain their differences derived from the de novo pathway that is predominantly in activity in various diseases. In particular the cytotoxic directed into replicating DNA and a second derived from effects of the purine analogues fludarabine and 2-CdA on salvage synthesis used for DNA repair.21,22 quiescent cells may be explained by interaction with targets involving DNA repair rather than replication, and direct or indirect effects on mitochondria. Mechanisms of action of nucleoside analogues and drug metabolism Ara-C All of the NA share common characteristics including active transport by membrane transporters (Table 1), activation by Ara-C (1-β-D-arabinofuranosylcytosine, cytosine arabinoside, kinases such as dCK allowing retention of the monophosphate cytarabine) is an structural analogue of deoxycytidine (dCyd) residues in the cell and the formation of the active triphos- (Figure 2) used for the treatment of acute leukemias and lym- phates metabolites, and dephosphorylation by 59-nucleotidase phomas. Ara-C differs from dCyd by the presence of a (59-NU) (Figure 1). Moreover, most of them have the ability hydroxyl group in the β-configuration at the 29-position of the sugar moiety. Intracellular penetration of ara-C depends on plasma ara-C concentrations.23–26 Standard-dose ara-C (SD ara-C; 100–200 mg/m2) achieves steady-state plasma levels of 0.5–1 µM.27,28 At these concentrations the expression of the hENT1 protein is the rate-limiting factor in ara-C uptake. In the presence of plasma concentrations greater than 50 µM, such as those reached with high-dose ara-C (HD ara-C; 2–3 g/m2), simple inward diffusion rates exceed those of pump- mediated transport.29 Once inside the cell, ara-C is phosphorylated by dCK and pyrimidine kinases to the active 59-triphosphate derivative ara- CTP.30,31 The catabolism of ara-C results from rapid deamin- ation by cytidine deaminase (CDD) to the non-toxic metab- olite arabinoside uridine while ara-CMP is dephosphorylated by the action of cytoplasmic 59-nucleotidase (59-NU).32 Ara- C cytotoxicity is believed to result from a combination of DNA polymerase inhibition and from incorporation of ara-CTP into Figure 1 Representation of the metabolism and drug target interac- tions of deoxynucleoside analogues in proliferating cells (NA). NA enters cells via specific nucleoside transporters. Once inside the cell, NA are phosphorylated by deoxycytidine kinase (DCK), NMPK and