Metabolome Analysis Reveals Excessive Glycolysis via PI3K/AKT/mTOR and RAS/MAPK Signaling in Methotrexate-Resistant Primary CNS Lymphoma-Derived Cells

Purpose: Metabolome analysis is an emerging method that provides insight into intracellular and physiologic responses. Methotrexate (MTX) is an antifolate that suppresses DNA syntheses by inhibiting dihydrofolate reductase. High-dose methotrexate treatment with deferred radiotherapy is a standard protocol in primary central nervous system lymphoma (PCNSL) treatments. However, most cases come to relapse-acquired resistance, in which the role of metabolic pathways is largely unknown. Experimental Design: Metabolome analysis in methotrexate-resistant PCNSL-derived cells (designated as TK-MTX and HKBML-MTX) was performed to detect alternative metabolites and pathways. Results: The metabolomic analyses using capillary electrophoresis-time-of-flight mass spectrometry detected 188 and 169 peaks in TK- and HKBML-derived cells, respectively, including suppression of central carbon metabolism, lipid metabolism, nucleic acid metabolism, urea cycle, branched chain and aromatic amino acids, and coenzyme metabolism. Particularly, whole suppressive metabolic pathways were demonstrated in TK-MTX, whereas HKBML-MTX indicated partially enhanced pathways of the urea cycle, amino acid metabolism, and coenzyme metabolism. Reciprocally detected metabolites for glycolysis, including induced glucose and reduced glycogen, and induced lactate and reduced pyruvate, in addition to increased lactate dehydrogenase activity, which is involved in Warburg effect. Thereby, ATP was increased in both methotrexate-resistant PCNSL-derived cells. Furthermore, we specifically found that PI3K/AKT/mTOR and RAS/MAPK signaling pathways were activated in TK-MTX but not in HKBML-MTX by growth rate with inhibitors and gene expression analysis, suggestive of cell type-specific methotrexate-resistant metabolic pathways. Conclusions: These results can help us understand targeted therapies with selective anticancer drugs in recurrent CNS lymphoma-acquired resistance against methotrexate.