REVIEW Nucleoside Analogues: Mechanisms of Drug Resistance and Reversal Strategies

Home , Cytarabine, Deoxycytidine, Deoxyribonucleoside, Nucleoside, Nucleoside analogue, Purine analogue

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 inactivating 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 equilibrative 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 salvage 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 pathway, 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 deamination by cytidine deaminase (CDD) to the non-toxic metabolite 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 interactions 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 NDPK to the active 59-triphosphate derivatives. NA catabolism can result from rapid deamination by cytidine deaminase to non-toxic metabolites. Cytoplasmic 59-nucleotidase (59Nucl) activity opposes that of DCK by dephosphorylating 59-monophosphate derivatives, thereby preventing the production of the active form. NA exerts their action by incorporation into newly synthesized DNA resulting in chain termination and cell death. Also, some NA indirectly block DNA replication by inhibiting ribonucleotide reductase (RR) enzyme, that in turn inhibit reduction of ribonucleotides diphosphate (NDPs) to deoxyribonucleotides diphosphate (dNDPs). The decrease of deoxyribonucleotides triphosphate (dNTPs) pools favors incorporation of NA active 59-triphosphate derivatives into DNA. Figure 2 Chemical structure of pyrimidine analogues.

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 877 DNA, in competition with deoxycytidine triphosphate (dCTP). This incorporation causes chain termination, resulting in a block of DNA synthesis.33–35 Sustained high cellular concentrations of ara-CTP relative to that of dCTP are thought to favor drug incorporation into replicating DNA, thereby initiating the leukemic cell death associated with therapeutic response.36 Low incorporation of ara-CTP into the DNA of blast cells in vitro has been shown to be predictive of an adverse outcome in AML patients receiving ara-C-based therapy.37,38

Gemcitabine

Gemcitabine (difluorodeoxycytidine, dFdC) (Figure 2) is a new pyrimidine analogue with promising activity in solid tumors.39,40 This compound is a dCyd analogue with two flu- orine substituted for the two hydrogen atoms in the 29 position of the deoxyribose sugar.41 After the initial phosphorylation into dFd-CMP by dCK,42 gemcitabine is subsequently phosphorylated by pyrimidine kinases to the active 59-diphosphate (dFd-CDP) and triphosphate (dFd-CTP) derivatives.43 Inacti- Figure 3 Chemical structure of purine analogues. vation of gemcitabine can occur by deamination into its inactive metabolite dFd-U by CDD or dephosphorylation of dFd- CMP by 59-NU.44,45 malignancies, in particular hairy-cell leukemia.51–53 2-CdA dFd-CTP is incorporated into DNA by replication synthesis enters cells via NT54 and is phosphorylated to 2-CdATP by in the C sites of the growing DNA strand.46 Once incorporated dCK, AMP kinase, and nucleoside diphosphate kinase. Mito- into the DNA strand, an additional natural nucleoside is chondrial dGK has also been identified as a 2-CdA phos- added, masking gemcitabine and preventing DNA repair by phorylating enzyme.55 2-CdAMP can be dephosphorylated by base pair excision. Thereafter, the DNA polymerases are 59-NU. The net accumulation of the triphosphate derivative unable to proceed,47 a process designated as ‘masked DNA has been shown to depend on the relative concentrations of chain termination’. The active diphosphate metabolite of gem- dCK and 59-NU.56 citabine also inhibits DNA synthesis indirectly through inhi- 2-CdA is cytotoxic both to dividing and resting cells.57,58 bition of RR.47 This effect blocks the de novo DNA synthesis In dividing cells, this compound inhibits DNA synthesis by pathway and self-potentiates gemcitabine activity by decreas- incorporation of its triphosphate metabolite into DNA.59,60 ing intracellular concentrations of normal dNTPs (particularly Once incorporated 2-CdATP is capable of terminating chain dCTP). Reduction in cellular dCTP results in increased gemcit- elongation mediated by DNA polymerases, inducing an S abine nucleotide incorporation into DNA and increased for- phase-specific apoptosis.61 2-CdA also inhibits DNA repli- mation of active gemcitabine di- and triphosphates, since dCK cation indirectly through its inhibitory action on RR,62 causing activity is down-regulated by high cellular dCTP levels. Other a subsequent reduction of the dNTPs pool required for DNA self-potentiating mechanisms of gemcitabine include a synthesis.59,63 The reduction of intracellular dATP results in decreased elimination of gemcitabine nucleotides by direct self-potentiation of 2-CdA as it enhances the incorporation of inhibition of CDD.48 Gemcitabine is not only incorporated triphosphate metabolites into DNA. Moreover, the decrease into DNA, but also into RNA.49 in dCTP could result in an increased rate of phosphorylation The spectrum of activity of gemcitabine in solid tumors may of 2-CdA and other NA, since dCK activity is regulated by be due to different characteristics. Compared with ara-C, gem- cellular dCTP levels. An original mechanism of 2-CdA cyto- citabine serves as a better transport substrate for membrane toxicity is related to the inhibition of ADA and S-adenosylho- pumps, is phosphorylated more efficiently, and is eliminated mocysteine hydrolase (SAHH).64 Inhibition of ADA could lead more slowly, favoring a longer retention time of the active to increased levels of Ado and dAdo, thereby causing the metabolite in tumor cells.48,50 Moreover, gemcitabine cytotox- inactivation of SAHH and reduced homocysteine formation. icity is enhanced by a number of unique self-potentiating This might result in altered methylation reactions and mechanisms that contribute to maintaining high intracellular indirectly inhibit nucleotide synthesis and cellular prolifer- concentrations of the active metabolites. This prolonged pres- ation.65,66 ence of active gemcitabine derivatives may explain part of its Different mechanisms have been proposed to explain why effect on slowly dividing tumor cells. These differences, 2-CdA is toxic to a cell that is not undergoing replicative DNA together with greater lipophilicity, masked chain termination, synthesis. First, 2-CdA-induced killing in non-dividing cells RNA incorporation and RR inhibition, which are not observed occurs by inhibition of cellular DNA repair processes. Incor- with ara-C, may explain why gemcitabine is, and ara-C is not, poration of 2-CdATP into DNA by the repair machinery ter- active in solid tumors. minates the nucleotide excision repair process leading to the progressive accumulation of DNA single-strand breaks which eventually initiate apoptosis by p53-dependent or p53-inde- 2-CdA pendent pathways.67,68 p53 activation subsequently regulates the levels of expression of p53-dependent proteins such as 2-CdA (Figure 3) is an analogue of deoxyadenosine (dAdo) Bcl-2 and Bax leading to the activation of the caspase path- resistant to deamination by adenosine deaminase (ADA). 2- way.69 Second, incorporation of 2-CdA into DNA may alter CdA is commonly used in the treatment of indolent lymphoid gene transcription with a consequent depletion of proteins

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 878 required for cell survival. It has been demonstrated that when sine triphosphate (dATP) for incorporation into the A sites of 2-CdA metabolites were present in one or both DNA strands, elongating DNA by DNA polymerases.84 Once incorporated it the yield of full-length transcripts was reduced.70 Moreover, is capable of terminating chain elongation mediated by DNA RNA polymerase did not bind as well to 2-CdATP-containing polymerases α (F-ara-ATP).85 RR inhibition causes a sub- promoters. In addition, on binding to the substituted promoter, sequent reduction of dNTPs pools.63,86 Similarly to 2-CdA, the RNA polymerase had an altered conformation that led to decrease of cellular dATP and dCTP results in a self-potenti- enhanced proteolytic clipping by endoproteinase Glu-C.70 ation of the drug and other NA. Other enzyme targets of F- Alternatively, it has been shown that 2-CdA cytotoxicity ara-ATP in dividing cells include DNA primase, DNA poly- may involve alterations of mitochondrial function or integrity. merase α, DNA ligase and topoisomerase II.84 Thus, the mech- 2-CdA has been shown to disrupt the integrity of mitochondria anism of action of F-ara-A in proliferating cells is mainly cell in CLL cells.69 The nucleoside-induced damage leads to the cycle-specific, and incorporation of F-ara-A into DNA during release of the pro-apoptotic mitochondrial protein cyto- S phase is required for the induction of apoptosis.87 chrome c, thereby initiating the caspase proteolytic cascade. In non-dividing cells, the inhibition of cellular DNA repair Moreover, 2-CdATP can cooperate with cytochrome c and processes appears to be the major mechanism of cytotoxicity Apaf-1 to activate caspase-3 and trigger a caspase path- of F-ara-A. Incorporation of F-ara-AMP into DNA by the repair way.71,72 2-CdA also interferes either directly or indirectly machinery terminates the polymerization step of the nucleo- with mitochondrial transcription that will eventually reduce tide excision repair process causing irreversible damage the levels of mitochondrial proteins necessary for electron recognized by the cells, which would signal for p53-mediated transport and oxidative phosphorylation. In vitro studies have or PARP-mediated apoptosis.20 As occurs after exposure to 2- demonstrated that 2-CdATP is incorporated into mitochon- CdA, fludarabine may induce PARP activation resulting in drial DNA by mitochondrial DNA polymerase.62 This incor- consumption of NAD and depletion of total adenine nucleo- poration may decrease transcription by mitochondrial RNA tides with subsequent cell death.88 polymerase. On the other hand, high intracellular 2-CdATP Other mechanisms of action of F-ara-A may explain its cyto- levels could indirectly repress transcription of mitochondrial toxicity in non-cycling cells.82 First, F-ara-AMP and F-ara-ATP genes by inhibiting reduction of ADP to dADP.73 This leads incorporation into RNA results in premature termination of the to high ADP concentrations that cause a net influx of adenine RNA transcript, impairing its function as a template for protein nucleotides into mitochondria and an increase in the intrami- synthesis.89 F-ara-ATP also inhibits RNA synthesis by sup- tochondrial ATP concentration.59,74 High intramitochondrial pressing the activity of RNA polymerase II.89 Inhibition of RNA ATP levels in turn inhibit mitochondrial RNA polymerase transcription correlates with the cytotoxic action of fludarab- activity.74 ine in CLL cells.90 Secondly, F-ara-ATP is the most potent PARP activation has been suggested to be involved in p53- nucleotide activator of APAF-1, with the consequent acti- independent mechanisms of cell killing by 2-CdA.57 In vation of the caspase-9 and caspase-3 pathways.71 Moreover, addition to being a target for proteolytic cleavage during F-ara-A has been reported to downregulate Bcl-2 mRNA apoptosis, PARP is thought to play a role in DNA repair by expression, thereby favoring apoptosis.91 Finally, F-ara-A signaling genomic damage.75 PARP binds to DNA breaks, affects mitochondria but only as a late event, suggesting an becomes activated and, using NAD as a substrate, adds ADP- indirect mechanism related to the p53-apoptotic pathway.69 ribose polymers to a range of nuclear proteins including itself, The different effects of F-ara-A and 2-CdA on mitochondria histones and DNA repair enzymes.76,77 These and other find- integrity may be related to the fact that 2-CdATP may accumu- ings have led to a proposed model of 2-CdA action involving late more rapidly than F-ara-ATP in the mitochondria because the following sequence of events: inhibition of ongoing DNA it is converted with higher efficiency by mitochondrial dGK.92 repair by 2-CdA with accumulation of DNA breaks, PARP It has also been stated that 2-chloro derivatives are slightly activation with resultant consumption of NAD, and depletion more hydrophobic than the corresponding 2-fluoro derivatives of total adenine nucleotides.57,76–78 Given that the cell mem- and therefore may be able to insert more easily into the mito- brane requires ATP for many of its functions, it seems likely chondrial membrane, thereby affecting their integrity.69 In that PARP-mediated ATP depletion might contribute to the summary inhibition of DNA repair, termination of mRNA tran- cell membrane disruption that occurs during apoptosis.79 scription and the consequent depletion of proteins required for cell survival, as well as the capacity of F-ara-ATP to activate the apoptosome pathway appear to contribute to the Fludarabine cytotoxicity of fludarabine in non-dividing cells.

β 9- -D-arabinosyl-2-fluoroadenine (F-ara-A) (Figure 3) is an Mechanisms of resistance to nucleoside analogues analogue of adenosine (Ado) which is resistant to deamination by adenosine deaminase (ADA).80 F-ara-A is administered as Three general mechanisms of resistance to NA have been the 59-monophosphate form (fludarabine). The phosphate described in cell lines and in clinical samples. A primary group on the 59- position of the arabinose has the sole func- mechanism of resistance to NA arise from an insufficient intra- tion of making the clinical formulation water-soluble. Before cellular concentration of NA triphosphates, which may result entering cells, fludarabine is rapidly dephosphorylated by from inefficient cellular uptake, reduced levels of activating membrane CD73 into F-ara-A which is transported inside the enzymes, increased NA degradation by increased 59-NU or cell via hNTs.81 Like other NA, it requires phosphorylation to CDD activity, or expansion of the dNTP pools.93 A second its triphosphate form, F-ara-ATP for cytotoxic activity.82,83 The type of resistance mechanism may be due to the inability to initial step in this activation process is performed by dCK. achieve sufficient alterations in DNA strands or dNTP pools, Like 2-CdA, F-ara-A is cytotoxic both against dividing and either by altered interaction with DNA polymerases, by lack resting cells. In dividing cells, this compound inhibits DNA of inhibition of RR, or because of inadequate p53 exonuclease synthesis and RR.82 DNA synthesis inhibition is mediated by activity. Finally, drug resistance may be a consequence of a competition of the triphosphate metabolite with deoxyadeno- defective induction of apoptosis.

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 879 Membrane transporters decrease in the activity level of this enzyme as one of the mechanisms responsible for clinical resistance to ara-C, flud- In vitro studies have demonstrated that NT-deficient cells are arabine or 2-CdA. Conversely, other authors reported no sig- highly resistant to NA.94 Several cell lines exhibit cross-resist- nificant relationship between dCK activity and clinical out- ance to a spectrum of NA because of reduced uptake.11,54,95 come to NA in these diseases.120,121 In isolated leukemic blasts, sensitivity to ara-C, fludarabine Structural analyses of the dCK gene have show inactivating and 2-CdA correlated with the cellular abundance of mutations and deletions as a cause of dCK deficiency in vitro. hENT196,97 determined by cellular binding of NBMPR. In Owens and co-workers122 claimed that to acquire a drug- these cells, the number of such binding sites ranged from 500 resistant phenotype, cells required two independent mutations to 27600 sites/cell.11,26 As initial rates of cellular drug uptake that markedly disrupt the catalytic activity of the enzyme. are, in general, proportional to hENT1 abundance, this wide However, these cDNA mutations rarely occur in vivo and variation suggests that transport-deficient cells would display therefore may not constitute a major mechanism of clinical a drug-resistant phenotype. In AML patients, Wiley et al98 NA resistance.115,116,123 Moreover, it seems that most of the observed that low transport rates correlated with poor clinical aminoacid replacements did not occur in a functionally rel- responsiveness to ara-C therapy. Although the measurement evant region of the enzyme.124 More recently alternatively of hENT1 expression by a quantitative flow cytometric assay spliced forms of dCK mRNA were detected in leukemic blasts with the hENT1 ligand, SAENTA fluorescein, shows promise from patients with clinically resistant AML but not in leukemic in identifying transport-deficient leukemic cells, the clinical blasts from patients with sensitive AML.125 These alternatively significance of NT deficiency to in vivo resistance is not yet spliced forms of dCK mRNA were shown to be inactive in an well established. This is due to the difficulty of assessing drug in vitro dCK activity assay. These data indicate that the pres- transport in clinical samples, the contribution of transporters ence of dCK splice products may contribute to the occurrence other than hENT1, and the technical difficulties of quantifying of clinical ara-C resistance. NTs in neoplastic clones admixed with normal cells.11 Structural alterations of the dCK gene complex other than mutations may play a role in drug resistance to NA in vivo.126 Downregulation of dCK activity in cytarabine-resistant cells Deoxycytidine kinase has been shown to correlate with hypermethylation of the promoter region of the gene.127,128 Decreased levels of dCK Deoxycytidine kinase is the rate-limiting enzyme that cata- mRNA has been shown to be induced under various con- lyzes the initial step of the 59-phosphorylation of dCyd to ditions in vitro.127,129 Finally dCK activity may be regulated dCMP and of many NA to their corresponding monophos- post-translationally, since this enzyme is feedback-inhibited phates.99–101 The dCK activity is high in quiescent cells in by dNTPs (particularly dCTP) and by some phosphorylated which it phosphorylates nucleosides necessary for DNA repair analogues such as ara-CTP.130 and, depending on the cell type, may increase several-fold when the cells enter the S-phase of the cell cycle.102–105 Deoxycytidine kinase activity has been shown to be Deoxyguanosine kinase decreased or absent in different cell lines resistant to NA.106–109 Transfection of the dCK gene in dCK-deficient tumor cell lines Deoxyguanosine kinase (dGK) is another enzyme responsible restores in vitro sensitivity to ara-C.110,111 Moreover, in vitro for the phosphorylation of purine deoxynucleosides, in mam- models have shown crossresistance between 2-CdA, gemcita- malian cells. This enzyme showed a broad substrate speci- bine, fludarabine and ara-C with reduced dCK activity as the ficity towards natural purine deoxynucleosides, as well as underlying determinant of resistance.109,112 It is noteworthy towards nucleoside analogues such as ara-G and 2- that in vitro only significant reductions in the levels of dCK CdA.55,73,92 dGK is located predominantly in the mitochon- activity (10% or less) are associated with a resistant pheno- dria.131 However, Petrakis et al132 described a novel amino- type, suggesting that under baseline conditions dCK is present terminally truncated isoform that corresponds to about 14% of in excess. the total dGK mRNA population in mouse spleen. This isoform In the clinical setting, it is important to note that bone mar- cannot translocate into the mitochondria and thus may rep- row and lymphoid tissues exhibit the highest activities of dCK resent a cytoplasmic enzyme. resulting in the clinical success observed when treating these Mitochondrial dGK is responsible for phosphorylation of target tissues with NA.113,114 However, the relevance of dCK purine deoxyribonucleosides and their analogues in the mito- deficiency in clinical resistance to NA is controversial (Table chondrial matrix providing the dNTPs necessary for mitochon- 2). In childhood acute lymphoblastic leukemias (ALL),115,116 drial DNA synthesis.133–136 Cytoplasmic dGK may also con- AML117,118 and lymphoproliferative disorders119 some authors tribute to the supply of purine deoxynucleotides for nuclear observed decreased expression of the dCK gene or significant DNA replication and repair.132 In this way, nucleoside ana-

Table 2 Correlation between low levels of expression of dCK gene or low dCK activity and drug resistance in hematological malignancies

Author Disease n Drug Technique Resistance Ref.

Tattersal AML 30 ara-C EA assay Yes 117 Stammler ALL 107 ara-C rt-PCR Yes 116 Albertioni CLL 17 2-CdA EA assay No 120 Kawasaki HCL/CLL 7/25 2-CdA IA Yes 119 Leiby NHL 25 Fludarabine EA assay No 121

N, number of patients; EA, enzyme activity; IA, immunoassay; rt-PCR, reverse transcriptase polymerase chain reaction.

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 880 Table 3 Human 59-nucleotidase (59-NU) activities involved in NA resistance

9 Enzyme activity Cytosolic low Km Cytosolic high Km Cytosolic Ecto-5 -NU 59-NU 59-NU 59(39)-nucleotidase (CD73)

Ref. 244, 245 131, 140, 244 244 131, 246 NMM (kDa) 143 210 45 120–160 SMM (kDa) 76 53 22 60–80 Preferred substrates CMP.dUMP.G DIMP.dGMP dIMP.dUMP.dT AMP MP.AMP MP.dGMP Optimum pH 7.4–9 6.5 6.0–6.5 7.0–8.0 Stimulation dATP, GTP, 2, 3 DPG ATP, ADP Inhibition ADP, ATP(dADP, dATP, PI) Pi, orthophosphate (Pi)

NMM, native molecular mass; SMM, subunit molecular mass.

logues phosphorylated by dGK may exert their cytotoxic patients with no expression.144 However, due to the multi- effects by interference with both mitochondrial and nuclear plicity of 59-NU activities, and the limited ability of classical DNA synthesis. Whether inactivation of dGK is involved in biochemical methodology to differentiate between these resistance to NA is not presently known. activities, the particular 59-NU enzyme(s) with the largest impact on clinical resistance to NA have not yet been convincingly identified.145 59-Nucleotidase Cytidine deaminase 59-NU dephosphorylates nucleoside monophosphates and cytotoxic mononucleotides through its hydrolysis of the ester- Cytidine deaminase catalyses the inactivation of cytidine and bond between the 59-carbon and the phosphate group. 59-NU dCyd to uridine and deoxyuridine, respectively. CDD also activity therefore opposes that of dCK. 59-NUs comprise a deaminates ara-C and gemcitabine.146 Several lines of evi- large and complex group of enzyme activities differing in dence have indicated a role for increased levels of CDD in cellular localization, pH sensitivity, and inhibition or stimu- the development of resistance to ara-C.147–149 Different studies lation by ATP (Table 3). Two broad classes of 59-NU are impli- have revealed that transfection of the human CDD gene into cated in drug resistance to NA: cytosolic and membrane- murine fibroblasts and hematopoietic cells confers drug bound 59-NUs. Membrane-bound or ‘ecto-59-NU’ is also resistance to ara-C and gemcitabine.44,150,151 known as CD73, and is frequently expressed in acute leuke- However, the in vivo correlation between CDD activity and mias.137,138 The physiological function of CD73 is the salvage ara-C resistance remains controversial and the relative contri- of extracellular nucleotides into nucleosides capable of cellu- bution of CDD to drug resistance has not yet been fully eluci- lar entry by means of nucleoside transport proteins. In con- dated (Table 4). The clinical relevance of a high level of CDD trast, several intracellular 59-NU activities have been ident- activity as a major cause of ara-C resistance in AML patients ified and cloned to date.139–141 The ‘low-Michaelis-Menten has been emphasized by several investigators.118,152–154 Con- 9 117 constant’ (Km)5-NU is specific for pyrimidine monophos- versely, Tattersal et al did not find a relationship between phates at low micromolar Km values and is inhibited by ATP. increased CDD activity and resistance to therapy in AML 9 The other, high Km 5 -NU is purine selective at high micromo- patients. 142,143 lar Km values, and it is activated by ATP. In vitro, Structural analyses of the CDD gene correlated polymor- increased 59-NU activity has been consistently associated with phism at codon 27 to substantially different deamination rates nucleoside drug resistance.45,58,109 of ara-C in vitro.155 This structural aberration did not seem to In the clinic, levels of cyto-59-NU activity have been corre- represent a major cause of the observed differences of CDD lated to clinical response in CLL, HCL and AML (Table 4). In activities between ara-C-sensitive and ara-C-resistant patients. CLL and HCL, Kawasaki et al119 demonstrated that pretreat- Schro¨der and co-workers154 demonstrated a significant corre- 9 ment levels of high Km 5 -NU allowed prediction of 2-CdA lation of the amount of CDD mRNA with CDD enzyme activi- responsiveness, since 59-NU levels were significantly lower in ties in AML blasts suggesting that variations in CDD activity responders than in non-responding patients. In AML, we have result from differences in gene expression. It therefore appears 9 reported that patients whose blasts expressed high Km 5 -NU that CDD activity in vivo is correlated with transcriptional mRNA had shorter time-to-relapse and overall survival than regulation rather than with CDD gene aberrations.

Table 4 59-NU and CDD enzyme activity measurements in hematological malignancies

Author Disease Drug Enzyme Results Ref.

Galmarini AML ara-C 59-NU High mRNA levels correlates with DR 144 Kawasaki HCL/CLL 2-CdA 59-NU Increased EA correlates with DR 119 Colly AML ara-C CDD Increased EA correlates with DR 118 Jahns AML ara-C CDD Increased EA correlates with DR 153 Schro¨der AML ara-C CDD Increased EA correlates with DR 154 Tattersal AML ara-C CDD No relationship to DR 117

Tech, technique; EA, enzyme activity; DR, drug resistance.

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 881 Levels of dCTP resistance to NA.68,188 p53 appears to have a speciﬁc role in resistance to NA since it possesses exonuclease activity, and Various cell lines and AML blasts containing high levels of may therefore be able to excise illegally DNA-incorporated dCTP have been found to be resistant to ara-C.126,156,157 dCTP NA. Recently, Feng et al reported that although the 39-59 levels regulate ara-C metabolism at three levels: by feedback exonuclease of wild-type p53 (wt-p53) protein was able to inhibition of dCK activity with the consequent decrease in ara- bind and excise gemcitabine residues from DNA in vitro, C phosphorylation;117 by allosteric activation of the catabolic removal of the drug molecules from cellular DNA was slow enzyme, CDD;126 and by competing with ara-CTP for incor- in sensitive cells containing wt-p53, and undetectable in drug- poration into DNA.158,159 Moreover, Ohno and coworkers160 resistant mut-p53 cells. Collectively, these data imply that have shown that dCTP in sufﬁciently high concentrations can molecular regulators of apoptosis should be taken into con- overcome ara-C DNA strand termination so that strand elong- sideration when analyzing factors associated with resistance ation continues past the ara-C incorporation site. dFd-CTP to NA. also compete with dCTP for DNA polymerase and for incorporation into DNA.46 Thus, elevated levels of dCTP will favor the survival of cells exposed to these NA. Reversal strategies

It is now widely accepted that the antileukemic effect of NA Ribonucleotide reductase and DNA polymerases requires sufficiently high intracellular concentrations of triphosphate analogues in tumor cells.38,82,189 One approach has RR consists of two non-identical proteins called M1 and M2, been to develop compounds which are insensitive to degra- both of which are required for activity. Protein M1 contains dation. Fludarabine and cladribine are resistant to the effect the substrate and effector binding sites and is present through- of ADA. Another approach could consist in the use of specific out the cell cycle in proliferating cells. Protein M2 is a dimer inhibitors of degradative enzymes, such as 59-NU. Combi- that contains stoichiometric amounts of non-heme iron and a nations of a 59-NU inhibitor with a NA could be expected to unique tyrosyl free radical essential for reductase activity. Its potentiate the cytotoxicity of the NA. However, there is still expression is specific of S phase and in quiescent cells M1 uncertainty regarding the relevant 59-NU enzyme(s) that and M2 mRNA and proteins are not detectable.161 Clinically mediate NA resistance, and there are currently no clinically useful NA target RR, altering dNTPs concentrations.62,162 available inhibitors of 59-NU activity. Because many potential Recently, Goan et al163 reported a human KB cancer cell line inhibitors of 59-NU are themselves nucleoside analogues, resistant to gemcitabine with overexpression of the M2 sub- competition with cytotoxic NA for membrane transporter sites unit as the sole mechanism explaining this resistance.163 may be problematic. Regulation of dCK activity could also be Most of the NA inhibit DNA polymerase activity by compet- a target for modulation. It has been shown that dCK activity ing with dNTPs,164 thereby contributing to the inhibition of is stimulated after treatment with 2-CdA, as well as with flud- DNA synthesis.165 The degree of inhibition of DNA poly- arabine and ara-C in normal human lymphocytes and various merases is not the same for all the NA. For example, DNA leukemic cell lines.190,191 The increase of dCK activity polymerase α has been shown to be more sensitive to inhi- induced by NA was considered to result from post- bition by ara-CTP than DNA polymerase β.166 F-ara-ATP is translational modifications of dCK during inhibition of DNA not a potent inhibitor of DNA polymerases β and γ or DNA synthesis.192,193 primase.84,162 In vitro, overexpression of DNA polymerases The importance of the ratio of nucleoside analogues to nor- may be a significant factor in the development of drug resist- mal nucleosides has been recognized more recently. Reduc- ance.167 Tanaka et al168 found that DNA polymerase α sensi- ing the intracellular pools of normal nucleotides therefore tivity to ara-CTP and F-ara-ATP was lower in blast cells appears to be a therapeutic alternative to increasing the con- obtained from ALL patients than in cells obtained from AML centrations of cytotoxic NA triphosphates. In practice, given patients, and concluded that DNA polymerase α from ALL the feedback inhibitory effects of dNTPs on NA activation, blast cells has a decreased affinity for NA.168 these two goals may be attained simultaneously. Various biochemical modulation strategies, based on NA combinations, have been suggested to increase the concentration of NA Defective cell death pathways and p53 exonuclease triphosphate in leukemic cells. activity

DNA damage caused by NA induces expression of p53, lead- Inhibition of ribonucleotide reductase ing to the induction of pro-apototic molecules such as Bax and to down-regulation of anti-apoptotic proteins, such as Large amounts of dNTPs generated by RR are directed into Bcl-2.169–171 These events lead to induction of apoptosis.172,173 replicating DNA, a process that effectively excludes NA from Various investigators have shown that B-CLL and AML replicating DNA synthesis.194 Furthermore intracellular dNTP patients with mutations in the p53 gene displayed resistance pools inhibit dCK activity, thereby reducing NA activation. to fludarabine, 2-CdA or ara-C and/or poorer response and Thus, drugs capable of depleting or reducing normal dNTPs shorter remission and/or overall survival174–177 (Table 5). Con- by inhibiting RR might be expected to potentiate cytotoxicity versely, other authors failed to find any relationship between of different NA. Sequential incubation of K562 human leuke- p53 abnormalities and resistance to NA in these dis- mia cells with F-ara-A followed by ara-C enhanced ara-CTP eases.178,179 Other genes regulating apoptosis, such as Bcl-2, accumulation, thereby suggesting possible therapeutic syner- Bcl-X/L, Mcl-1 or Bag-1, have been suggested to influence gism between ara-C and fludarabine.86 Subsequent clinical sensitivity to NA, although the results remain contro- studies have demonstrated the effectiveness of this strat- versial.180–187 egy.195,196 Lymphocytes isolated from patients with CLL and Alterations in p53 function have been related to in vitro incubated with ara-C after in vitro F-ara-A incubation or a

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 882 Table 5 Involvement of p53 protein in drug resistance to NA

Author Disease Drug Tech Results Ref.

El-Rouby CLL Flud SSCP DR in patients with mutations 174 Wattel CLL/AML Flud/ara-C SSCP DR in patients with mutations 175 Johnston CLL Flud/2-CdA SSCP, IB DR in patients with mutations 176 Lens CLL Flud SB, ICC No correlation with DR 178 Dohner CLL Flud FISH, SSCP DR in patients with mutations 177 Kasimir AML ara-C SSCP No correlation with DR 179

Tech, technique; Flud, ﬂudarabine; DR, drug resistance; IB, immunoblot; SB, Southern blot; ICC, immunocytochemistry.

therapeutic infusion of fludarabine (25 or 30 mg/m2) displayed higher concentrations of ara-CTP, greater ratios of analogue an increase in ara-CTP accumulation.197 The same phenom- triphosphates to normal dNTPs and tandem incorporation of enon was observed in leukemic lymphocytes and AML 2-Cl-dATP and ara-CTP in growing DNA strands. blasts.198,199 This increase was caused by a higher rate of Although potentially interesting, the usefulness of NA com- anabolism rather than by a slower rate of catabolism of ara- binations in the clinic remains to be determined. In particular CTP. More recently, Seymour et al200 have shown that F-ara- the toxicity to normal cells due to the increase of NA accumu- ATP concentrations (30–50 µM, achieved with usual clinical lation appears to be significant, casting doubt on an improve- doses of fludarabine), enhances both intracellular ara-CTP ment of the therapeutic index in comparison to single agent accumulation and incorporation into cellular DNA at concen- NA therapy. A potential consequence related to NA modu- trations of ara-C which are clinically achievable. Such drug lation therapy may be the increased risk of secondary malig- combinations have induced responses in untreated and nancies in a group of patients whose disease already places refractory patients with acute leukemias.201,202 them at a great risk of second cancers, and dose-related effects This modulatory effect of fludarabine can be extended to like severe neurotoxicity.210,211 Apparently, the frequency and other NA such as 9-β-D-arabinofuranosylguanine (ara-G).203 severity of neurotoxicity may be greater with the combination In vitro studies using human leukemia cell lines and primary therapy than with either drug used alone. leukemia cells obtained from patients established that the effectiveness of ara-G is due to intracellular accumulation of ara-GTP. The rate-limiting step of ara-GTP accumulation is the Modulation of dCK and 59-NU activitity with initial phosphorylation performed both by dGK and dCK. In bryostatin vivo, Gandhi and coworkers demonstrated in a phase I clinical trial that patients who responded to therapy with the Bryostatin, a macrocyclic lactone and protein kinase C acti- water-soluble ara-G prodrug GW506U achieved a signifi- vator, has been shown to increase the ratio of dCK/59-NU cantly greater concentration of ara-GTP in their circulating activity and thereby increase sensitivity to fludarabine and cla- cells than patients who did not respond.204,205 In this setting, dribine in CLL cells, both in vitro and in vivo.212,213 Phase I the combination with fludarabine could increase the intra- studies of single agent bryostatin have been completed and cellular accumulation of ara-GTP in target cells and improve this compound is now in clinical trials in combination with response rates in patients.205 NA.214 Other inhibitors of RR can be used for the modulation of ara-C metabolism. Iwasaki and coworkers194 showed that inhibition of RR with gemcitabine and the consequent Use of hematopoietic growth factors decrease in cellular dCTP pools favored ara-C incorporation into replicating DNA. Colly et al206 and Bhalla et al207 tested Different in vitro studies have shown that pretreatment of AML the ability of the RR inhibitor hydroxyurea to enhance ara-C blasts by growth factors (GF) enhances ara-C mediated cyto- metabolism and cytotoxicity in drug-sensitive and -resistant toxicity against leukemic cells, possibly through their prolifer- cell lines. This combination significantly reduced dCTP pools, ation-inducing effects.215,216 GF may also enhance ara-C cyto- increased DNA incorporation of ara-C and enhanced drug toxicity by increasing hENT1 expression. High cellular cytotoxicity, even in dCK deficient ara-C-resistant cell lines. proliferation rates are associated with high levels of hENT1- Similarly, as 2-CdA inhibits the reduction of CDP and ADP, mediated activity that increase the nucleoside drug a decrease in the dCTP and dATP pools is expected when uptake.217,218 Wiley et al219 determined that in vitro treatment cells are exposed to this compound. The decrease of these of isolated human leukemic cells with GM-CSF, resulted in an pools would increase the ratios of 2-CdATP to dATP and ara- increase in hENT1 expression. Reuter et al220 established that CTP to dCTP. In vitro incubation of AML blasts with 2-CdA blasts from AML patients treated with GM-CSF displayed an followed by ara-C produced a higher rate of ara-CTP accumu- enhanced drug uptake after low or SDAC doses. In this study, lation than did ara-C alone.208 Gandhi and coworkers209 stud- GM-CSF also increased intracellular ara-CTP/dCTP pool ratios ied 17 patients with refractory AML receiving combination and enhanced ara-C incorporation into DNA by activation of chemotherapy with ara-C and 2-CdA. Seven of nine patients DNA polymerases. In vivo, GM-CSF priming of leukemic studied during therapy had an increased rate of ara-CTP for- blasts prior to induction therapy translates into a higher anti- mation and peak ara-CTP concentrations in leukemic blasts. leukemic activity as indicated by a prolonged survival of This modulation strategy increased the effective dose intensity treated mice with advanced AML and a high rate of complete of the active metabolite of ara-C by 40% in the tumor cells. remission in patients with AML.221,222 However, various ran- The co-administration of 2-CdA and ara-C resulted in domized clinical trials failed to demonstrate the effectiveness maximum inhibition of DNA synthesis, accumulation of of the GF-priming strategy and concluded that administration

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 883 of GF during and after induction chemotherapy does not improve the clinical outcome of ara-C-treated AML patients.223,224

DNA repair induction

Another way of enhancing incorporation of NA into DNA consists of using the DNA repair pathway after combined exposure of tumor cells to NA and DNA damaging agents.225 The recognition and repair of the damaged strand caused by the DNA damaging agent allow resynthesis using the opposite strand as a template. This creates an opportunity for the inser- Figure 4 Chemical structure of troxacitabine. tion of the NA instead of its normal counterpart into the DNA repair patch. The incorporated NA is relatively resistant to repair excision and causes irreversible damage recognized by 4) is a stereochemically synthetic nucleoside analogue that the cell. Alternatively inhibition of DNA repair by NA may has potent antitumor activity in preclinical models.235–237 It is increase the accumulation and slow the removal of DNA phosphorylated in vitro by deoxycytidine kinase (dCK) with a lesions induced by DNA damaging agents, thereby potentiat- Km similar to its natural substrate, dCyd, but unlike gemcitab- ing the cytotoxicity of these latter compounds. In vitro, combi- ine and ara-C, it is not a substrate of CDD. Troxacitabine tri- nation of gemcitabine and fludarabine with cisplatin gener- phosphate can be incorporated into cellular DNA,235 which ated synergistic cytotoxicity.225 Van Den Neste et al226 causes rapid chain termination. However, neither ribonucleo- recently reported the in vitro synergistic cytotoxicity of 2-CdA tide reductase nor mitochondrial DNA synthesis are inhibited and cyclophosphamide in B-CLL cells isolated from patients by troxacitabine in cell culture. Because of its broad preclini- pretreated with alkylating agents. cal spectrum with cytotoxicity both against leukemic and epi- In vivo, the combination of a DNA damaging agent and a thelial malignancies, and promising phase I activity demon- NA may prove to be particularly useful to destroy cancer cells strated in acute leukemia,238 phase II testing is now being without significant DNA replication activity such as CLL and carried out in several diseases. indolent lymphomas. The combination of fludarabine with doxorubicin and prednisone is clinically active against CLL.227 Fludarabine and ara-C with or without cisplatin, have been 2-amino-9-β-D-arabinosyl-6-methoxy-9H-guanine administered to patients with CLL with cytoreductive activity.228 Results of clinical studies using mitoxantrone In 1983, Cohen et al239reported that the deoxyguanosine (which induces protein-associated DNA strand breaks), fluda- derivative ara-G (9-β-D-arabinofuranosylguanine) (Figure 5) rabine, and dexamethasone demonstrated a high response was resistant to cleavage by purine nucleoside phosphorylase rate, especially in follicular lymphomas, with a number of (PNP) and was toxic to T-lymphocytes. The devolopment of patients achieving complete remission.229,230 A combination this drug was limited by water insolubility. However, 2-amino- of mitoxantrone and ara-C for the treatment of acute leukemia 9-β-D-arabinosyl-6-methoxy-9H-guanine (GW506U78) is 10- has been encouraging.231,232 Moreover, clinical trials evaluat- fold more soluble, and is rapidly converted to ara-G by ing NA in combination with DNA damaging agents have plasma adenosine deaminase activity. shown effectiveness against solid tumors with low growth In phase I clinical testing, this agent has shown particular fractions.233,234 promise in the therapy of T cell malignancies,240 where 54% of patients achieved partial or complete remissions after one to two cycles of drug treatment. Of particular interest, neuro- New nucleoside analogues toxicity was dose limiting, with little clinical myelosuppres- sion.241 This raises the possibility of successful combination The broadening of the spectrum of nucleoside analog activity therapy with other active agents, including other hematolog- to solid tumors has triggered renewed interest in this class of ically active NA. Although no formal studies have been perfor- antimetabolites. Many new nucleoside drugs have been syn- med to demonstrate mechanisms of ara-G resistance, accumu- thesized and studied over the past decade, with several that lation of ara-GTP in leukemic blasts correlated with the are in early phase clinical trials, and others that have become standardly prescribed agents (gemcitabine and capecitabine). We will review the most promising of these agents: troxacitabine, and the GW506U78, an ara-G prodrug. We have excluded from this discussion 5-FU prodrugs such as capecitabine since their mechanisms of action and resistance mechanisms differ substantially from those of other anticancer nucleosides.

Troxacitabine

Troxacitabine (BCH-4556; (−)-2-(S)-hydroxymethyl-4-(s)- (cytosin-19-yl)-1,3-dioxolane; β-L-dioxalane cytidine) (Figure Figure 5 Chemical structure of ara-G.

Leukemia Nucleoside analogues: mechanisms of drug resistance and reversal strategies CM Galmarini et al 884 cytotoxic activity against malignant cells.241 This suggests that amic and pharmacokinetic properties and therapeutic potential in the early steps of uptake and metabolism of ara-G may be haematological malignancies. Drugs 1993; 46: 872–894. major determinants of cellular drug sensitivity, as for other 6 Robertson LE, Huh YO, Butler JJ, Pugh WC, Hirsch-Ginsberg C, Stass S, Kantarjian H, Keating MJ. Response assessment in chronic NA. lymphocytic leukemia after fludarabine plus prednisone: clinical, pathologic, immunophenotypic, and molecular analysis. Blood 1992; 80: 29–36. Conclusions 7 Ross SR, McTavish D, Faulds D. Fludarabine. A review of its pharmacological properties and therapeutic potential in malignancy. During the past decade there has been dramatic progress in Drugs 1993; 45: 737–759. 8 Juliusson G, Liliemark J. Long-term survival following cladribine the understanding of the individual mechanisms of resistance (2-chlorodeoxyadenosine) therapy in previously treated patients to nucleoside analogues, including the role of cellular drug with chronic lymphocytic leukemia. Ann Oncol 1996; 7: 373– uptake, drug metabolism, interaction with cellar targets, and 379. the resulting apoptotic cascade. However, most studies have 9 Sorensen JM, Vena DA, Fallavollita A, Chun HG, Cheson BD. focused on a single mechanism of resistance, thereby failing Treatment of refractory chronic lymphocytic leukemia with fluda- to provide the relative importance of the different known rabine phosphate via the group C protocol mechanism of the mechanisms of resistance. It is clear that malignancies are het- National Cancer Institute: five-year follow-up report. J Clin Oncol 1997; 15: 458–465. erogeneous in terms of mechanisms of resistance, and that the 10 Boleti H, Coe IR, Baldwin SA, Young JD, Cass CE. Molecular clinical benefit of resistance-reversal strategies will only be identification of the equilibrative NBMPR-sensitive (es) nucleoside demonstrated when targeted to the appropriate subgroup of transporter and demonstration of an equilibrative NBMPR-insensi- patients. tive (ei) transport activity in human erythroleukemia (K562) cells. The ultimate goal of research into anticancer therapy Neuropharmacology 1997; 36: 1167–1179. remains improving patient survival. However, in diseases such 11 Baldwin SA, Mackey JR, Cass CE, Young JD. Nucleoside transporters: molecular biology and implications for therapeutic devel- as CLL or indolent lymphoma in which several treatments are opment. Mol Med Today 1999; 5: 216–224. effective but not curative, a realistic goal would be the identi- 12 Crawford CR, Patel DH, Naeve C, Belt JA. Cloning of the human fication of tumor cell traits which could allow tailored or tar- equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)- getted therapy. In such patients, a predictive assay to deter- insensitive nucleoside transporter ei by functional expression in a mine a phenotype of probable resistance to certain NA would transport-deficient cell line. J Biol Chem 1998; 273: 5288–5293. be of great value in the choice of optimal therapy, and the 13 Griffiths M, Beaumont N, Yao SY, Sundaram M, Boumah CE, Dav- avoidance of ineffective but toxic treatments. ies A, Kwong FY, Coe I, Cass CE, Young JD, Baldwin SA. Cloning of a human nucleoside transporter implicated in the cellular In diseases such as AML in which curative drug combi- uptake of adenosine and chemotherapeutic drugs. Nat Med 1997; nations exist, the objective is to optimize the efficacy of cur- 3: 89–93. rently available NA and to develop newer, more effective ana- 14 Griffiths M, Yao SY, Abidi F, Phillips SE, Cass CE, Young JD, Bald- logues that are less susceptible to the resistance mechanisms win SA. 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