Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia

Abstract

BACKGROUND Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with combination chemotherapy containing methotrexate (MTX), drug resistance contributes to treatment failure for a substantial fraction of patients. The primary transporter for folates and MTX is the reduced folate carrier (RFC). Impaired drug transport is a documented mechanism of MTX resistance in patients with ALL; however, to the authors' knowledge it is not known whether inactivating RFC mutations are a contributing factor. METHODS The authors devised a genomic polymerase chain reaction‐single strand conformational polymorphism assay followed by sequencing and screened the entire RFC coding region for sequence alterations in DNA from 246 leukemia specimens from patients with diverse ethnic variation, 24 at the time of recurrence and the rest at the time of diagnosis. This cohort was comprised of 203 B‐precursor ALL specimens (82.5%), 32 T‐lineage ALL specimens (13%), and 11 acute myeloblastic leukemia specimens (4.5%). RESULTS Of 246 DNA samples, only 3 diagnosis B‐precursor ALL specimens (1.2%) were found to harbor alterations in the RFC gene, including heterozygous single nucleotide changes resulting in D56H and D522N substitutions in the first extracellular loop and the C‐terminus of this transporter, respectively. The third sample had a sequence alteration in exon 3 that could not be identified because of the lack of availability of DNA. CONCLUSIONS Whereas inactivating RFC mutations are a frequent mechanism of MTX resistance in human leukemia cell lines and in patients with osteosarcoma, they are not common and do not appear to play any significant role in intrinsic or acquired resistance to MTX in childhood leukemia. This is the first study of RFC mutations in multiple pediatric leukemia specimens. Cancer 2004;100:773–82. © 2003 American Cancer Society.

Relationship between RFC gene expression and intracellular drug concentration in methotrexate-resistant osteosarcoma cells

“…Our results showed a decreased expression of RFC mRNA in drug-induced cells. In primary cells, RFC mRNA is significantly higher than that of the inducible cell line, and this is consistent with the literature (Guo et al, 1999;Hattinger et al, 2003, Ifergan et al, 2003Kaufman et al, 2004).…”

“…Conversely, Kaufman et al (2004) found that in a child with acute lymphoblastic tumor, the mutations of genes are not a potential cause of MTX resistance. Guo et al (1999) analyzed RFC and DHFR mRNA in 42 cases (primary and metastatic osteosarcoma specimens) by semi-quantitative RT-PCR and found the mechanism of osteosarcoma chemotherapy resistance.…”

The role of folate receptor α in cancer development, progression and treatment: Cause, consequence or innocent bystander?

“…21,22 The remaining discussion is restricted to FRa, the most widely studied FR isoform. The transport kinetics and affinity for folates and antifolates differ significantly between systems (Table I), in part, because FRa does not bind and carry folate into the cell as does RFC, but binds and internalizes folate via endocytosis.…”

“…22 It is a ubiquitous resident of most cells, and mediates the uptake of reduced folates and methotrexate with relatively high affinity and at higher, micromolar concentrations (K m : 1-10 lM); it has relatively poor affinity for oxidized folic acid (K m : 200-400 lM). 21,27,28 The potential binding capacity of the RFC for reduced folates is several hundred times greater than the amount of folate in serum.…”

Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier

“…Consistently, alternative phosphorylations also have been reported to render Sp1 a transcriptional repressor. 29,30 An important mechanism of antifolate resistance in ALL 31 and other malignancies 32 is impaired antifolate transport due to loss of RFC function, 2,33 resulting from mutational inactivation 34 and/or transcriptional silencing. [9][10][11] This loss of function, as we have recently shown in multiple antifolate-resistant human leukemia cell lines with defective antifolate uptake, is due either to mutations in the RFC gene 34 or decreased RFC mRNA levels caused by a methylation-independent transcriptional silencing of the RFC gene.…”