Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

Leukemia (2002) 16, 2379–2387  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells AJ Gifford1, M Haber1, TL Witt2, JR Whetstine2, JW Taub2, LH Matherly2 and MD Norris1 1 Children’s Cancer Institute Australia for Medical Research, Sydney, Australia; and 2Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transportdefective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, Kis for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease. Leukemia (2002) 16, 2379–2387. doi:10.1038/sj.leu.2402655 Keywords: acute lymphoblastic leukemia; reduced folate carrier gene; drug resistance; trimetrexate Introduction The antifolate methotrexate (MTX) is a critical component of the therapy of pediatric acute lymphoblastic leukemia (ALL), the most common childhood cancer.1,2 Current combination chemotherapy cures approximately 70% of children diagnosed with ALL,3 however, the remaining 30% of patients typically succumb to the disease due to the emergence of drug-resistant cell populations. Resistance to MTX is an important cause of treatment failure in these children.4 Mechanisms of MTX resistance include reduced drug transport; increased activity of the target enzyme, dihydrofolate reductase; structural alterations in dihydrofolate reductase, and decreased MTX polyglutamylation.5 Defective MTX transport has been described commonly in ALL.6–8 Cloning of the reduced folate carrier (RFC) gene,9–13 encoding the major membrane transport protein for MTX, has permitted the characterization of molecular changes leading to defective MTX transport in resistant cells. The RFC is an integral membrane protein with 12 putative transmembrane Correspondence: Dr MD Norris, Children’s Cancer Institute Australia for Medical Research, PO Box 81 (High Street), Randwick, Sydney, NSW 2031, Australia; Fax: (612) 9382 1850 Received 13 November 2001; accepted 15 May 2002 domains.9,11,14 A large number of RFC gene mutations leading to an antifolate-resistant phenotype have been described in rodent and human cell lines.15–21 Although mutations have been identified throughout the RFC coding region, they appear to be disproportionately clustered within the first putative transmembrane domain (eg G44E, E45K, S46N, I48F).16– 20,22 Of particular interest is E45K, originally identified in MTX-resistant murine cells19 and, subsequently, in separate MTX-resistant CCRF-CEM human T-cell ALL sublines from our own23 and another laboratory.16 Upon transfection into MTXresistant L1210 cells, the murine E45K RFC results in the markedly increased uptake of reduced folates and folic acid compared to MTX.19 Although there have been no reports of transfection of this mutation into human cells, qualitatively similar results were reported for the E45K human RFC (hRFC)-overexpressing CCRF-CEM subline (designated CEM/MTX-LF), selected in subphysiologic levels of reduced folates.16 However, there were notable discrepancies between the transport parameters obtained with the murine RFC transfectants19 and CEM/MTX-LF cells.16 This may reflect species differences or, perhaps, the presence of additional gene alterations resulting from the low folate selection. In this report, we stably transfected MTX-resistant CCRFCEM cells (CEM/MTX R0) lacking a functional hRFC with wild-type and E45K hRFC constructs, in order to characterize the functional and metabolic consequences of E45K hRFC expression in an isogenic human leukemia cell background. In addition, we have assessed the possible clinical relevance of mutations in the first transmembrane domain of the hRFC, including the E45K mutation, in leukemic blasts isolated from bone marrow aspirates from children diagnosed with ALL. Materials and methods Cell lines and patient samples All cell lines were maintained as suspension cultures in RPMI 1640 containing 2.3 ␮M folic acid, supplemented with 10% fetal calf serum (FCS) (Life Technologies, Grand Island, NY, USA) and 2 mM L-glutamine at 37ºC in a humidified atmosphere of 5% CO2. Both CEM/MTX R24 and CEM/MTX R0 sublines were independently derived from CCRF-CEM cells by stepwise exposure to increasing doses of MTX and were selected in final concentrations of 0.4 ␮M MTX and 5 ␮M MTX, respectively. For polyglutamylation, growth requirement and 3 H folic acid transport assays, cells were depleted of endogenous folates by growth in folate-free RPMI 1640 containing adenosine (60 ␮M) and thymidine (10 ␮M) supplemented with dialysed FCS (Life Technologies) and L-glutamine, as above. Routine screening for mycoplasma indicated that cell lines were free of contamination. Bone marrow samples were obtained after informed consent from pediatric ALL patients presenting at Sydney Chil- E45K-reduced folate carrier gene mutation AJ Gifford et al 2380 dren’s Hospital (Sydney, Australia) or from the Children’s Hospital of Michigan and the Pediatric Oncology Group. Australian children were uniformly treated using the Australia and New Zealand Children’s Cancer Study Group ALL Study V or Study VI protocol.25 Patients presenting at American institutions were registered on the POG#8600 leukemia classification protocol and treated with the POG#8602 antimetabolite-based treatment protocol.26 The diagnosis ALL specimens were those used in our previous studies of MTX resp (...truncated)