FPGS (folylpolyglutamate synthase) is a crucial enzyme in folate metabolism that catalyzes the conversion of folates to polyglutamate derivatives, enabling cellular concentration and intracellular retention of folate cofactors essential for one-carbon transfer reactions in purine, pyrimidine, and amino acid synthesis 1. The enzyme preferentially uses unsubstituted reduced folates as substrates and also metabolizes methotrexate (MTX) to polyglutamates, which is critical for MTX efficacy in cancer treatment 2. FPGS functions in both cytoplasm and mitochondria, playing a central role in antifolate drug retention and activity. In cancer, particularly acute lymphoblastic leukemia (ALL), FPGS dysfunction represents a major resistance mechanism. Relapsed ALL frequently exhibits decreased FPGS network activity, inactivating mutations, focal gene deletions, and increased protein degradation, leading to MTX resistance 23. These alterations are enriched in early relapses and can arise through therapy-induced mutagenesis 3. FPGS inactivation creates a therapeutic vulnerability, as cells become sensitive to non-polyglutamatable antifolates like trimetrexate 2. Additionally, FPGS polymorphisms influence MTX treatment response and toxicity across various conditions, with variants like rs1544105 associated with adverse events in rheumatoid arthritis and increased ALL susceptibility 456. Novel regulatory mechanisms include Smad4/Ets-1 complex binding to exon12, which can suppress FPGS expression in resistant leukemia cells 1.