G. Jansen

MRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of β-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR × FPGH)/(RFC × FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Impaired bortezomib binding to mutant beta 5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells.

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the β5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant β5 subunit of the proteasome. These newly identified β5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the β5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant β5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNFα

Objective: To determine whether overexpression of cell membrane associated drug efflux pumps belonging to the family of ATP binding cassette (ABC) proteins contributes to a diminished efficacy of sulfasalazine (SSZ) after prolonged cellular exposure to this disease modifying antirheumatic drug (DMARD). Methods: A model system of human T cells (CEM) was used to expose cells in vitro to increasing concentrations of SSZ for a period of six months. Cells were then characterised for the expression of drug efflux pumps: P-glycoprotein (Pgp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Results: Prolonged exposure of CEM cells to SSZ provoked resistance to SSZ as manifested by a 6.4-fold diminished antiproliferative effect of SSZ compared with parental CEM cells. CEM cells resistant to SSZ (CEM/SSZ) showed a marked induction of ABCG2/BCRP, Pgp expression was not detectable, while MRP1 expression was even down regulated. A functional role of ABCG2 in SSZ resistance was demonstrated by 60% reversal of SSZ resistance by the ABCG2 blocker Ko143. Release of the proinflammatory cytokine tumour necrosis factor α (TNFα) was threefold higher in CEM/SSZ cells than in CEM cells. Moreover, twofold higher concentrations of SSZ were required to inhibit TNFα release from CEM/SSZ cells compared with CEM cells. Conclusion: Collectively, ABCG2 induction, augmented TNFα release, and less efficient inhibition of TNFα production by SSZ may contribute to diminished efficacy after prolonged exposure to SSZ. These results warrant further clinical studies to verify whether drug efflux pumps, originally identified for their roles in cytostatic drug resistance, can also be induced by SSZ or other DMARDs.

Classification Of Ex Vivo Methotrexate Resistance In Acute Lymphoblastic and Myeloid Leukaemia

The introduction of antifolates in the late 1940s started the era of modern chemotherapy and the antifolate methotrexate (MTX) is still an important component in the treatment of acute lymphoblastic leukaemia (ALL) (Bertino, 1993). Combination chemotherapy nowadays cures 80% of the children diagnosed with precursor B ALL, but treatment is not effective in the remaining 20% of these children (Pui & Evans, 1998). Treatment failure is at least partially due to cellular drug resistance (Pieters et al, 1997). Drug resistance is also frequently found in the T-lineage phenotype (T-ALL) (Pieters et al, 1998), which is generally classified as a highrisk feature (Pui & Evans, 1998). Intensification of therapy, including higher-dose MTX, results in equal survival rates for children with T-ALL compared with precursor B ALL (Uckun et al, 1998). This improved survival rate suggests that cellular drug resistance can be partially overcome by dose intensification. High cure rates do not apply for childhood acute myeloid leukaemia (AML), for which event-free survival rates of 50±60% have been reported (Pui, 1995; Stevens, 1998). A remarkable difference between the treatment schedules for AML and ALL is the absence of MTX in the treatment of AML. The exclusion of MTX from AML therapy was based on disappointing results in early clinical trials (Bender, 1975), leading to the assumption that AML cells are intrinsically MTX resistant. This review first summarizes the recent developments in MTX research in experimental models and subsequently describes the clinically relevant mechanisms of MTX resistance, as observed in samples of acute leukaemia patients. Suggestions are made for approaches to circumvent MTX resistance.

A possible role for methotrexate in the treatment of childhood acute myeloid leukaemia, in particular for acute monocytic leukaemia

Acute myeloid leukaemia (AML) is thought to be methotrexate (MTX)-resistant. However, a small study suggested that acute monocytic leukemia (AML-M5) is sensitive to MTX. We measured MTX accumulation/polyglutamylation in 20 AML-nonM5, 37 AML-M5 and 83 common/preB-acute lymphoblastic leukaemia (c/preB-ALL) samples. Membrane transport was determined in 11 childhood AMLs (including 3 AML-M5) and in 25 c/preB-ALL samples. MTX sensitivity was determined in 23 AML-nonM5, 15 AML-M5 and 63 common/preB-ALL samples using the thymidylate synthase (TS) inhibition assay. MTX transport was higher in AML samples compared with c/preB-ALL precluding a transport defect in AML. Accumulation of long-chain polyglutamates MTX-Glu(4-6) was 3-fold lower for AML-nonM5 compared with c/preB-ALL cells (median 268 versus 889 pmol MTX-Glu(4-6)/10(9) cells; P < or = 0.001); for AML-M5 samples, median accumulation of MTX-Glu(4-6) was 0 pmol/10(9) cells (P < or = 0.001). After short-term MTX exposure, AML-nonM5 was 6-fold more resistant to MTX compared with c/preB-ALL cells (2.16 versus 0.39 microM; P < 0.001), while AML-M5 was 2-fold more resistant (P = 0.02). In both AML-nonM5 and AML-M5 cells, MTX resistance was circumvented by continuous MTX exposure (median TSI(50) values: 0.052 and 0.041 microM, respectively) compared with a c/preB-ALL value of 0.066 microM. In conclusion, AML-M5 is relatively sensitive to MTX compared with other AML-subtypes even though polyglutamylation of MTX is poor. Using continuous exposure, AML-nonM5 and AML-M5 cells were at least as sensitive to MTX as c/preB-ALL cells. This report suggests that MTX might be an overlooked drug in the treatment of childhood AML.

Continuous Exposure to Methotrexate (MTX) Overcomes Resistance in Childhood Acute Myeloid Leukemia (AML) in Vitro

Continuous Exposure to Methotrexate (MTX) Overcomes Resistance in Childhood Acute Myeloid Leukemia (AML) in Vitro

Lack of Cross-Resistance Between Prednisolone and Methotrexate in Childhood Acute Lymphoblastic Leukemia?

We studied the cross-resistance between prednisolone (PRD) and methotrexate (MTX) in children with newly diagnosed acute lymphoblastic leukemia (ALL). This was done because of a previous observation that such patients could show a good clinical response to systemic PRD monotherapy plus intrathecal MTX, despite in vitro PRD resistant ALL cells (as determined with the MTT assay). This suggests an antileukemic effect of MTX, and thus the lack of cross-resistance between PRD and MTX. A systemic antileukemic effect of intrathecally administered MTX has been reported in the literature. Clinical good responders with PRD resistant ALL cells (n = 15) did not show unfavorable MTX-polyglutamylation nor unfavorable low inhibition of thymidylate synthase by MTX, as compared to a heterogeneous group of newly diagnosed ALL children (n = 47). In addition, we did not find a significant correlation between these two parameters and in vitro PRD resistance within the clinical good responders with PRD resistant ALL cells. In conclusion, we did not find a significant cross-resistance between PRD and MTX in vitro. It therefore may be that the good clinical response to systemic PRD plus intrathecal MTX in patients with in vitro PRD resistant ALL cells was caused by a systemic antileukemic activity of the intrathecally administered MTX.