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.Despite the high cure rates that are currently achieved, approximately 20% of pediatric acute lymphoblastic leukemia (ALL) patients experience a relapse and face poor prognosis. 1,2 Drug resistance in leukemia continues to be a major obstacle to targeted combination therapy. Although various molecular mechanisms have been classified in model systems, the major mechanisms for clinical drug resistance remain largely unknown. 3 One of the key components of contemporary treatment of ALL is the folate antagonist methotrexate (MTX). Folates play a crucial role as one-carbon donors in multiple biosynthetic pathways including de novo synthesis of purines and thymidylate, amino acid metabolism, mitochondrial protein synthesis and DNA methylation. 4,5 Antifolates including MTX are potent inhibitors of several folate-dependent enzymes engaged in nucleotide biosynthesis, thereby inhibiting DNA replication and leading to cell death. 6 The cytotoxic activity of various antifolates is largely dependent on the activity of folylpolyglutamate synthetase (FPGS). The latter enzyme catalyzes the addition of multiple glutamate residues (i.e., polyglutamylation) to both folates and antifolates upon their entry into the cell. 7 This unique metabolic conversion dramatically enhances the intracellular retention of antifolates including MTX as the polyglutamate forms of antifolates are no longer substrates of various efflux transporters. 8 Polyglutamylation also decreases the Ki of MTX and other antifolates like pemetrexed to their target enzymes in...

The association of aberrant folylpolyglutamate synthetase splicing with ex vivo methotrexate resistance and clinical outcome in childhood acute lymphoblastic leukemia

“…The ratio of this splice variant over the wild type transcript markedly increased in response to treatment with antifolates and other chemotherapeutic agents in antifolate-resistant cells, but not in their parental antifolate sensitive counterparts. 10 Moreover, FPGS intron 8 PR was predicted to cause a premature stop codon insertion which resulted in dysfunctional FPGS protein as indicated by in vitro FPGS catalytic activity analysis.…”

“…7 The identity of the obtained PCR fragments was previously confirmed by sequencing to represent FPGS splice variants. 10 For more details on patient characteristics, MTX related variables and splice variant analysis see Online Supplementary Materials and Methods and Online Supplementary Table S1. Of note, since not all variables characterising MTX sensitivity could be measured in all the patients in our cohort (due to limited number of leukemic blasts available as well as logistic reasons), the number of patients included in the various analyses varied.…”

“…10 These observations imply that intron 8 PR presumably reflects a broader splicing defect resulting in resistance to multiple chemotherapeutics; most likely affecting other genes as well. These genes have not yet been identified, but could be involved either in drug metabolism or regulation of apoptosis.…”

The translational expression of ABCA2 and ABCA3 is a strong prognostic biomarker for multidrug resistance in pediatric acute lymphoblastic leukemia

“…Despite the successes achieved in the treatment of childhood ALL in the past 4 decades, relapse was observed in ~20% of cases 4,5. The most important cause of relapse is resistance to chemotherapy medications,6,7 which is often attributed to genes with MDR. MDR is generated by mechanisms such as the increased efflux of a wide range of chemotherapeutics from the cells 7–9.…”

Could drugs inhibiting the mevalonate pathway also target cancer stem cells?

“…New antifolates targeting various enzymes are being introduced into clinical use (Gonen and Assaraf, 2012). An intracellular metabolism of these drugs and enzymes involved in it are better understood (Assaraf, 2007;Wojtuszkiewicz et al, 2016). Cancer cells can develop various mechanisms of drug resistance, like fast efflux of anticancer drug mediated by multidrug resistance proteins, drug-accumulating lysosomes and highly acidic microenvironment of tumors (Taylor et al, 2015;Zhitomirsky and Assaraf, 2016).…”