Targeted epigenetic modification has emerged as a critical strategy in cancer therapy, showing promising therapeutic potential in several tumor types. Recent studies have revealed that sialylation plays a pivotal role in tumor progression by regulating protein interactions, intracellular localization, vesicle trafficking, and signal transduction. This study aims to delve into the molecular mechanism by which the sialyltransferase ST6GALNAC4 drives the malignant progression of diffuse large B-cell lymphoma (DLBCL), clarify its role in glycolytic metabolic reprogramming, and provide new potential targets for DLBCL treatment.

Our preliminary studies found that the level of ɑ-2,6-sialylation modification was significantly upregulated in DLBCL patients compared with normal controls, and this was significantly associated with poor prognosis. Further research showed that the expression of the sialyltransferase ST6GALNAC4 was significantly elevated in DLBCL patients, which was also significantly correlated with poor prognosis. Functional experiments confirmed that ST6GALNAC4 could significantly promote DLBCL progression. To further explore the mechanism by which ST6GALNAC4 participates in DLBCL progression, we conducted a cross-analysis of sialylation-modified proteins in DLBCL cells and the top 100 binding proteins of ST6GALNAC4, identifying a total of 47 proteins potentially sialylated by ST6GALNAC4. Interestingly, a large number of key enzymes involved in glycolysis and fatty acid metabolism pathways were significantly enriched among them. Therefore, we selected key enzymes in glycolysis, fatty acid metabolism, and amino acid metabolism as candidate proteins. Preliminary experimental results showed that only the downregulation of LDHA could significantly inhibit the pro-cancer effect of ST6GALNAC4.

Through immunofluorescence co-localization experiments, we found that ST6GALNAC4 and LDHA co-localized in the cytoplasm, suggesting a specific binding between them in the cytoplasm. Lectin co-immunoprecipitation experiments further confirmed that LDHA undergoes significant sialylation modification. Knockdown of ST6GALNAC4 led to a decrease in the sialylation level of LDHA, indicating that the sialylation modification of LDHA is regulated by ST6GALNAC4. Meanwhile, protein molecular docking using PyMOL software revealed that LDHA and ST6GALNAC4 are highly embedded in their active pocket regions, which further supports a direct interaction between the two.

Given that sialylation modification can directly regulate the subcellular localization and functional activity of proteins, we performed mito-tracker labeling of mitochondria, immunofluorescence experiments, and enzyme activity assays. These studies found that knockout of ST6GALNAC4 significantly reduced both the co-localization of LDHA with mitochondria and the activity of LDHA, suggesting that sialylated LDHA is more inclined to localize to mitochondria. Detection of downstream products of LDHA showed that knockdown of ST6GALNAC4 significantly decreased ATP and lactate production, and reduced the NAD⁺/NADH ratio in DLBCL cells. Seahorse glycolytic stress tests indicated that cells overexpressing ST6GALNAC4 had significantly increased basal glycolytic rate, glycolytic capacity, and glycolytic reserve. Thus, we conclude that ST6GALNAC4 mediates the mitochondrial re-localization of LDHA and enhances its activity through sialylation modification, thereby promoting glycolytic metabolic reprogramming and lactate accumulation.

Studies have confirmed that lactate, a metabolic product of LDHA, plays an important role in tumor progression. Recent findings have shown that abnormal accumulation of lactate can further drive DLBCL progression through histone lactylation modification. We found that the global/pan-lysine lactylation level was elevated in DLBCL cell lines, with a distinct and consistent high-expression band at the histone position around 15 kDa. This suggests that lactate may participate in the regulation of downstream pro-cancer pathways through histone lactylation modification, forming a “metabolic-epigenetic regulation” vicious cycle, which ultimately promotes the malignant proliferation of DLBCL.

In summary, the results of our study clarify the critical role of the ST6GALNAC4-LDHA axis in metabolic reprogramming of DLBCL. Targeting ST6GALNAC4 and its regulated sialylation modification of LDHA is expected to become a new strategy for DLBCL treatment.

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2025
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