Background Histone lactylation is a recently identified epigenetic modification derived from intracellular lactate, a key product of glycolysis. It has been implicated in regulating gene expression and cell fate in inflammatory and tumor contexts; however, its significance in hematologic malignancies remains largely unexplored. Given the critical role of glycolytic metabolism in multiple myeloma (MM), lactate-mediated epigenetic regulation may influence disease progression. LDHAL6B, a homolog of the glycolytic enzyme LDHA, was previously identified by our group as a novel prognostic marker highly expressed in MM, but its functional role remains undefined. This study aims to elucidate the biological role of LDHAL6B and its involvement in lactate production and histone lactylation in MM.

Methods Stable MM cell lines with LDHAL6B knockdown or overexpression were generated via lentiviral transduction. Intracellular lactate levels were measured using a colorimetric assay. Cell proliferation, migration, and invasion were assessed using CCK-8 and Transwell assays. Immunofluorescence staining of bone marrow biopsies from MM patients and healthy donors was performed to evaluate LDHAL6B expression and global lactylation, followed by correlation analysis. Western blotting was employed to quantify total protein lactylation and site-specific histone lactylation at lysine residues, including H3K9, H3K14, H3K18, H3K23, H3K27, H3K56, H4K5, H4K8, H4K12, and H4K16. To determine whether histones are direct substrates of lactylation, immunoprecipitation assays were conducted using a pan-lactylation antibody, followed by Western blotting with anti-H3 and anti-H4 antibodies.

Results LDHAL6B knockdown significantly reduced intracellular lactate production and suppressed MM cell proliferation, migration, and invasion, whereas LDHAL6B overexpression enhanced these malignant phenotypes. To determine whether lactate mediates these effects, MM cells were treated with glycolysis inhibitors or supplemented with exogenous lactate. Glycolytic inhibition mimicked the effects of LDHAL6B knockdown, while exogenous lactate promoted malignant behavior. Furthermore, lactate supplementation partially rescued the inhibitory effects of LDHAL6B knockdown, indicating that LDHAL6B exerts its effects, at least in part, through a lactate-dependent mechanism.

Immunofluorescence analysis revealed elevated global lactylation in MM bone marrow samples compared to healthy controls, with a strong positive correlation between LDHAL6B expression and lactylation intensity. Western blotting demonstrated a marked increase in global protein lactylation in MM cell lines relative to normal controls. Notably, lactylation signals were particularly enriched in the ~15 kDa region, corresponding to core histones. Immunoprecipitation further confirmed that histones H3 and H4 are targets of lactylation. Knockdown of LDHAL6B or inhibition of glycolysis significantly diminished lactylation intensity at this histone-enriched band. This reduction was partially reversed by exogenous lactate supplementation, supporting a lactate-dependent mechanism.To identify specific sites affected by LDHAL6B, multiple histone lysine residues were analyzed. Among these, H4K8 lactylation exhibited the most pronounced decrease following LDHAL6B knockdown or glycolytic inhibition. Further validation demonstrated that the enhanced H4K8 lactylation observed in LDHAL6B-overexpressing cells was abolished by glycolytic blockade, confirming that LDHAL6B regulates H4K8 lactylation via lactate production.

Conclusion This study identifies LDHAL6B as a novel regulator of glycolytic metabolism and histone lactylation in MM. By enhancing lactate production, LDHAL6B promotes H4K8 lactylation and facilitates malignant cellular behavior. Although the downstream effects of H4K8 lactylation require further investigation, these findings reveal a potential metabolic-epigenetic regulatory axis in MM and nominate LDHAL6B as a promising target for future mechanistic and therapeutic studies.

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2025
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