BACKGROUND Interpatient differences in the accumulation of methotrexate’s active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODS To identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTS We documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10–5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10–3 to 3.7 × 10–8). A multivariable model including the ALL subtype (P = 1.1 × 10–14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10–4), the MTX infusion time (P = 1.5 × 10–3), FPGS mRNA expression (P = 2.1 × 10–3), and MTX systemic clearance (P = 4.4 × 10–2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10–38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONS These findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDING THE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).
Elixabet Lopez-Lopez, Robert J. Autry, Colton Smith, Wenjian Yang, Steven W. Paugh, John C. Panetta, Kristine R. Crews, Erik J. Bonten, Brandon Smart, Deqing Pei, J. Robert McCorkle, Barthelemy Diouf, Kathryn G. Roberts, Lei Shi, Stanley Pounds, Cheng Cheng, Charles G. Mullighan, Ching-Hon Pui, Mary V. Relling, William E. Evans