FPGS (folylpolyglutamate synthase) catalyzes the ATP-dependent conjugation of glutamate residues to folate cofactors, enabling their intracellular accumulation and retention. The enzyme preferentially acts on reduced folates and also converts methotrexate to polyglutamate derivatives, which enhance drug retention and efficacy. FPGS localizes to both the cytoplasm and mitochondrial matrix, where it maintains distinct folate pools critical for one-carbon transfer reactions supporting purine, pyrimidine, and amino acid synthesis. FPGS inactivation emerges as a key mechanism of drug resistance in relapsed acute lymphoblastic leukemia (ALL). In early and late relapses, FPGS mutations occur as part of convergent evolution in approximately 65% of cases, acquired through therapy-induced mutagenesis 1. Recent evidence indicates that FPGS loss in relapsed ALL involves decreased gene expression, focal deletion, impaired catalytic activity, and increased protein degradation, collectively reducing folate polyglutamylation capacity and inducing methotrexate resistance 2. Alternative splicing dysregulation of FPGS also contributes to altered drug uptake and metabolism in cancer 3. Germline FPGS polymorphisms, particularly rs1544105 and rs10106, associate with both MTX toxicity and treatment response in hematologic malignancies and rheumatoid arthritis. The FPGS rs1544105 T allele correlates with increased gastrointestinal, hepatic, and hematologic toxicity 4, while the C allele associates with remission achievement 4. In relapsed ALL with FPGS inactivation, polyglutamylation-independent lipophilic antifolates such as trimetrexate represent an alternative therapeutic approach to overcome chemoresistance 2.