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

Abstract • 194

In the treatment of childhood precursor B- acute lymphoblastic leukemia (pre-B ALL) MTX is considered a clinically effective and important drug, whereas childhood AML is regarded to be intrinsically resistant. This is based on data from the literature in which children with AML showed less response to relatively low-dose MTX (mostly by iv push) in comparison with patients with pre-B ALL. We adapted an MTX sensitivity assay based upon the MTX-induced inhibition of the thymidylate synthase (TS)-catalyzed conversion of ³H-dUMP to dTMP and ³H₂O. Blasts from children with pre-B ALL and AML were also analyzed for ³H-MTX accumulation and -polyglutamylation as well as for levels of folylpolyglutamate synthetase (FPGS), which catalyses the formation of polyglutamates and of folylpolyglutamate hydrolase (FPGH), which breaks down the polyglutamates. Results: we analyzed AML cells from 22 children and compared them with untreated ALL-cells from 43 pre-B ALL patients. AML cells were 6- fold more resistant to MTX after a 3 hour MTX-exposure followed by a drug-free period (IC₅₀ values 2.2 µM and 0.38 µM, resp.; p<0.001), but no difference in resistance was seen after a 21 hours continuous MTX exposure. Accumulation of the pharmacologically important long chain polyglutamates (MTX-Glu_4-6), after a 24 hour exposure to 1 µM ³H-MTX, was 4-fold higher for 40 pre-B ALL patients compared to 14 AML patients (906 and 225 pmol/10⁹cells, resp.; p<0.001). The activity of FPGS was 2-fold lower in 19 AML samples compared to 39 pre-B ALL samples (6.9 vs. 14.6 pmol of MTX-[³H]Glu₂ formed/hr/10⁶ cells, resp.; p=0.004). FPGH was 3-fold higher in 33 AML samples compared to 94 pre-B ALL samples (1.15 vs. 0.36 nmol MTX-Glu₂ hydrolysed/hr/10⁶ cells, resp.; p<0.001). Conclusions: After short term exposure polyglutamylation defects may explain the observed MTX-resistance in childhood AML. However, resistance could be overcome by continuous MTX exposure for 21 hrs. This warrants a clinical trial with continuous infusion of MTX in pediatric AML.