Methionine promotes glycolysis and tumor progression in multiple myeloma by enhancing POU2AF1 expression via m⁶A modification

Abstract INTRODUCTION: Multiple myeloma (MM), the second most prevalent hematologic malignancy, is characterized by the abnormal proliferation of clonal plasma cells in the bone marrow. Methionine, an essential amino acid, plays a critical role in fundamental physiological processes. Tumors frequently exhibit heightened dependence on exogenous methionine, making methionine restriction a potential therapeutic strategy. However, whether MM displays methionine dependence and how methionine specifically contributes to MM progression remain unclear. This study aims to validate methionine dependence in MM, elucidate the underlying mechanisms driving MM progression, and provide novel insights for future therapeutic interventions. METHODS: To compare methionine concentrations in the bone marrow microenvironment of MM patients and healthy controls, we quantified methionine levels in clinical samples using fluorometric assays. In vitro, we examined the effects of methionine supplementation, restriction, and its metabolites [S-adenosylmethionine (SAM) and homocysteine] on MM cell proliferation, apoptosis, migration, and invasion. We established a C57BL/KaLwRij mouse model to assess the impact of methionine on MM progression in vivo. N6-methyladenosine (m6A) dot blot and colorimetric assays were employed to analyze m6A methylation changes following methionine intervention. RNA sequencing was performed on methionine-treated MM cells to identify key regulatory factors and pathways, with subsequent validation by RT-qPCR and Western blotting. Glycolytic activity was assessed using Seahorse assays, glucose uptake, and lactate production measurements. Potential regulators of methionine-driven glycolysis in MM were screened using the ChIP-Atlas database. MeRIP-qPCR was employed to verify m6A modification changes in POU2AF1, and an actinomycin D assay was conducted to evaluate its impact on the stability of POU2AF1 mRNA. The functional role of POU2AF1 in MM glycolysis and malignant progression was further investigated using shRNA-mediated knockdown. RESULTS: We observed that methionine levels in the bone marrow supernatant of MM patients were significantly higher than those in healthy controls. In vitro, MM cell proliferation was dependent on methionine and SAM but not on homocysteine. Supplementation with methionine and SAM significantly inhibited apoptosis while enhancing the migration and invasion capabilities of MM cells. In contrast, methionine restriction produced the opposite effects. SAM served as the principal methyl donor for methylation modifications. We found that methionine, as a precursor for SAM, increased global m6A methylation levels in MM cells. Transcriptomic profiling of methionine-treated MM cells revealed significant alterations in the expression of solute carrier (SLC) transporter genes. Validation experiments confirmed that MM cells upregulate the surface transporters SLC6A18 and SLC38A5 to facilitate enhanced methionine uptake. Further KEGG enrichment analyses revealed significant enrichment of differentially expressed genes (DEGs) involved in glycolysis and one-carbon metabolism pathways. We subsequently found that methionine supplementation enhanced glycolytic flux and upregulated enzymes associated with the glycolysis pathway. The ChIP-Atlas database predicted POU2AF1 as a key regulator of methionine-driven glycolysis in MM. Analysis of publicly available MM datasets revealed that POU2AF1 expression was elevated in MM patients, with higher levels significantly associated with advanced ISS stage and poorer clinical outcomes. Mechanistically, methionine promoted m6A methylation of POU2AF1, which increased its expression by enhancing mRNA stability. Further studies confirmed that POU2AF1 knockdown and methionine restriction markedly suppressed glycolytic activity and malignant phenotypes in MM cells, including reduced proliferation, migration, and invasion capabilities. CONCLUSION: MM exhibits a pronounced dependence on methionine, with enhanced methionine uptake through the upregulation of the transporters SLC6A18 and SLC38A5. Methionine promotes MM glycolysis and tumor progression by facilitating the m6A modification of POU2AF1, which increases its stability and expression. These findings suggest that targeting methionine metabolism represents a promising therapeutic strategy for MM.