Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia.
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.
Stark
Michal
M
The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Rambam Medical Center, and Rappaport Facultyof Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Wichman
Chen
C
Avivi
Irit
I
Assaraf
Yehuda G
YG
D000328:Adult; D017398:Alternative Splicing; D015152:Blotting, Northern; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm; D005091:Exons / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007438:Introns / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology*; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution
D005493:Folic Acid Antagonists; D012333:RNA, Messenger; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate