Differences in Folylpolyglutamate Synthetase and Dihydrofolate Reductase Expression in Human B-Lineage versus T-Lineage Leukemic Lymphoblasts: Mechanisms for Lineage Differences in Methotrexate Polyglutamylation and Cytotoxicity

Abstract

SUMMARYCellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B-lineage ALL (129 children) compared with those with T-lineage ALL (20 children) ( p Ͻ 0.01) and was characterized by a saturable (E max ) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 M), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (E max ) in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM6 cells, and v) 4 -6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC 50 ) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.

Histone deacetylase inhibitors induce FPGS mRNA expression and intracellular accumulation of long-chain methotrexate polyglutamates in childhood acute lymphoblastic leukemia: implications for combination therapy

“…14 This is consistent with the higher IC 50 for MTX in CCRF-CEM (T-ALL) as compared with in NALM6 and other Bp-ALL cells in our study and other reports. 14 The data presented here, demonstrate that upregulation of FPGS mRNA expression by SAHA results in a net pharmacological increase of 30% in intracellular of long-chain MTX-PGs accumulation in ALL cell lines as compared with that in untreated control. Further, long-chain MTX-PGs inhibit not only DHFR but also other important folate pathway enzymes such as TS and aminoimidazolecarboxamide ribonucleotide formyltransferase.…”

“…12,13,72 Our laboratory and others have previously demonstrated that Bp-ALL cells express 2-to 3-fold higher levels of FPGS mRNA, protein, enzyme activity, and accumulate higher intracellular concentrations of MTX-PGs as compared with T-ALL. 10,14 The ability of cells to accumulate higher intracellular concentrations of MTX-PGs correlates with their in vitro and in vivo sensitivity to MTX. 14 This is consistent with the higher IC 50 for MTX in CCRF-CEM (T-ALL) as compared with in NALM6 and other Bp-ALL cells in our study and other reports.…”

“…[10][11][12][13] Our laboratory and others have demonstrated using real-time quantitative reverse transcription-PCR (qRT-PCR), enzymatic activity assays and gene microarrays that FPGS expression (mRNA and enzyme activity) is lineage-specific, with higher level in B-precursor (Bp)-ALL when compared with that in T-lineage (T)-ALL, in both cell line models and clinical samples from patients with ALL. 10,14,15 In normal and malignant lymphohematopoietic cells, the FPGS gene is controlled by at least two mechanisms: one tissue-specific (lineage-specific) and a second proliferation-dependent. 10,16 FPGS activity is distributed to both cytosolic and mitochondrial compartments of mammalian cells.…”

Resistance to antifolates

“…The lower levels of MTX polylgutamates (with lower chain lengths) in T-lineage ALL, as compared to B-lineage ALL, is associated with both a decrease in transport and FPGS activity. Lower polyglutamate levels may be the basis, in part, for the poor response of T-cell ALL to chemotherapies that include MTX (Barredo et al, 1994;Galpin et al, 1997).…”

Long-term results of Total Therapy studies 11, 12 and 13A for childhood acute lymphoblastic leukemia at St Jude Children's Research Hospital

“…In this regard, several studies have shown that T-lineage blasts accumulate methotrexate and its active metabolites (methotrexate polyglutamates) less avidly than do B-lineage blasts and that higher doses could result in greater accumulation of the drug. [7][8][9] Based on the premise that early intensification of systemic chemotherapy can forestall the emergence of drug-resistant blast cells, we reasoned that the same approach applied to intrathecal chemotherapy should further reduce the central nervous system (CNS) relapse hazard, leading to an improved outcome overall. Indeed, in study 13A, this strategy reduced the early CNS relapse hazard to near zero, boosting the overall effectiveness of ALL treatment.…”