title: Folylpolyglutamate synthetase splicing alterations in acute lymphoblastic leukemia are provoked by methotrexate and other chemotherapeutics and mediate chemoresistance.
abstract: Methotrexate (MTX), a folate antagonist which blocks de novo nucleotide biosynthesis and DNA replication, is an anchor drug in acute lymphoblastic leukemia (ALL) treatment. However, drug resistance is a primary hindrance to curative chemotherapy in leukemia and its molecular mechanisms remain poorly understood. We have recently shown that impaired folylpolyglutamate synthetase (FPGS) splicing possibly contributes to the loss of FPGS activity in MTX-resistant leukemia cell line models and adult leukemia patients. However, no information is available on the possible splicing alterations in FPGS in pediatric ALL. Here, using a comprehensive PCR-based screen we discovered and characterized a spectrum of FPGS splicing alterations including exon skipping and intron retention, all of which proved to frequently emerge in both pediatric and adult leukemia patient specimens. Furthermore, an FPGS activity assay revealed that these splicing alterations resulted in loss of FPGS function. Strikingly, pulse-exposure of leukemia cells to antifolates and other chemotherapeutics markedly enhanced the prevalence of several FPGS splicing alterations in antifolate-resistant cells, but not in their parental antifolate-sensitive counterparts. These novel findings suggest that an assortment of deleterious FPGS splicing alterations may constitute a mechanism of antifolate resistance in childhood ALL. Our findings have important implications for the rational overcoming of drug resistance in individual leukemia patients.
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.|Department Of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Anna
authors.initials: A
authors.lastname: Wojtuszkiewicz
authors.affiliation: Department Of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Shachar
authors.initials: S
authors.lastname: Raz
authors.affiliation: Department Of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Stark
authors.affiliation: Department Of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.affiliation: Department Of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.affiliation: Department Of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.affiliation: Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: Sonneveld
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Gertjan J L
authors.initials: GJ
authors.lastname: Kaspers
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.|Department Of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015153:Blotting, Western; D002648:Child; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000627:therapeutic use*; D010453:Peptide Synthases / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000201:enzymology*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000970:Antineoplastic Agents; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate
keywords: acute lymphoblastic leukemia; alternative splicing; drug resistance; folylpolyglutamate synthetase; methotrexate