Folylpoly-gamma-gluatamate
synthetase (FPGS) catalyzes the polyglutamylation and thus intracellular retention of folates and
antifolates (eg,
methotrexate; MTX) through the addition of multiple
glutamate equivalents to their gamma-carboxyl residue. Since polyglutamylation of
antifolates is crucial for their pharmacological activity in
leukemia, loss of FPGS function results in decreased cellular levels of polyglutamylation-dependent
antifolates and consequent drug resistance. Whereas resistance to pulse exposure to
antifolates is frequently associated with loss of FPGS activity, the underlying molecular mechanism remains elusive. Here we explored the molecular basis of
antifolate resistance in human MTX-resistant
leukemia cell lines displaying marked loss of FPGS activity. We demonstrate that these MTX-resistant cells exhibit impaired splicing of FPGS
mRNA based on intron retention and/or exon skipping, thereby resulting in loss of FPGS function due to premature translation termination. Furthermore, analysis of FPGS transcripts in blood or bone marrow specimens from patients with
acute lymphoblastic leukemia revealed exon 12 skipping, both at diagnosis and at relapse, the latter of which occurs after high-dose MTX-containing
chemotherapy. These results constitute the first demonstration of the loss of FPGS function via aberrant
mRNA splicing, thereby resulting in loss of
antifolate retention and drug resistance. The clinical ramifications of these novel findings are discussed.