FPGS relapse-specific mutations in relapsed childhood acute lymphoblastic leukemia

Abstract

Although the cure rate for childhood acute lymphoblastic leukemia (ALL) has exceeded 80% with contemporary therapy, relapsed ALL remains a leading cause of cancer-related death in children. Relapse-specific mutations can be identified by comprehensive genome sequencing and might have clinical significance. Applying whole-exome sequencing to eight triplicate samples, we identified in one patient relapse-specific mutations in the folylpolyglutamate synthetase (FPGS) gene, whose product catalyzes the addition of multiple glutamate residues (polyglutamation) to methotrexate upon their entry into the cells. To determine the prevalence of mutations of the FPGS mutations, and those of two important genes in the thiopurine pathway, NT5C2 and PRPS1, we studied 299 diagnostic and 73 relapsed samples in 372 patients. Three more FPGS mutants were identified in two patients, NT5C2 mutations in six patients, and PRPS1 mutants in two patients. One patient had both NT5C2 and PRPS1 mutants. None of these alterations were detected at diagnosis with a sequencing depth of 1000X, suggesting that treatment pressure led to increased prevalence of mutations during therapy. Functional characterization of the FPGS mutants showed that they directly resulted in decreased enzymatic activity, leading to significant reduction in methotrexate polyglutamation, and therefore likely contributed to drug resistance and relapse in these cases. Thus, besides genomic alterations in thiopurine metabolizing enzymes, the relapse-specific mutations of FPGS represent another critical mechanism of acquired antimetabolite drug resistance in relapsed childhood ALL.

Figure 1

Droplet digital PCR for the follow-up of the patient with FPGS R419W. The FPGS R419W clone was undetected after stem cell transplantation; however, the clone recurred, and the patient had second relapse.

Figure 2

FPGS mutations in relapsed pediatric ALL. (a) Schematic representation of the structure of the FPGS protein. Tetrahydrofolylpolyglutamate synthase, Mur ligase family, and glutamate ligase domains are indicated. FPGS mutations identified in relapsed pediatric samples are shown. Filled circles represent heterozygous mutations. (b) DNA sequencing chromatograms of paired diagnosis and relapse genomic ALL DNA samples showing representative examples of relapse-specific heterozygous FPGS mutations, with the mutant allele sequence highlighted in red.

Figure 3

Effects of FPGS mutation on polyglutamation enzymatic activity using methotrexate as the substrate. (a) FPGS activity was measured by quantifying ATP consumption with a phosphate sensor kit. Wild-type or mutant FPGS proteins were expressed and purified to homogeneity. Mutant FPGS proteins exhibited 30% to 66% reduction in activity under the same experimental conditions. (b) FPGS proteins 2.5 ng (c) FPGS proteins 5.0 ng.