Modeling Mechanisms of In Vivo Variability in Methotrexate Accumulation and Folate Pathway Inhibition in Acute Lymphoblastic Leukemia Cells

John C Panetta,Mary V Relling,Alex Sparreboom,Ching-Hon Pui,William E Evans,Jason A Papin

doi:10.1371/journal.pcbi.1001019

Abstract

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.

Highlights

Methotrexate (MTX) is one of the primary anticancer agents used for the treatment of acute lymphoblastic leukemia (ALL) [1,2,3]
Gene Expression and Polymorphisms We investigated how the methotrexate polyglutamates (MTXPG) model parameters related to gene expression in ALL cells and germline polymorphisms in or flanking genes related to MTX transport and metabolism
Because MTXPG accumulation was significantly lower in Tlineage ALL compared to B-lineage hyperdiploid ALL, we investigated how this differential accumulation affected the inhibition of de novo purine synthesis (DNPS) by comparing the simulated baseline DNPS activity to its activity over a 72 hr post MTX treatment interval

Summary

Introduction

Methotrexate (MTX) is one of the primary anticancer agents used for the treatment of acute lymphoblastic leukemia (ALL) [1,2,3]. The ability of cells to accumulate intracellular polyglutamate metabolites of MTX (MTXPG) is an important factor in its antileukemic effects [4]. MTXPG inhibits the folate pathway by competitively inhibiting several important enzymes including: dihydrofolate reductase (DHFR), thymidylate synthase (TS), glycinamide ribonucleotide transformylase (GART), and aminoimidazole carboxamide ribonucleotide transformylase (AICART). This inhibition leads to reduced or blocked TS and de novo purine synthesis (DNPS), which are needed for DNA synthesis. Developing a better understanding of the underlying mechanisms responsible for these differences in cellular disposition of MTX is important for understanding the basis for inter-patient differences in MTX’s antileukemic effects and to identify strategies to circumvent mechanisms of resistance

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