Lactate promotes DLBCL progression via NAT10/ldha axis and immune suppression

Abstract Introduction： Lactate, a metabolic byproduct, can induce protein lactylation that plays a key role in protein stability. However, the function of protein lactylation in diffuse large B-cell lymphoma (DLBCL) remains largely unknown. Tumor-derived lactate participates in multiple cellular processes and can be secreted into the tumor microenvironment, where it influences both tumor cells and immune cell function. Here, we investigated the functional importance and regulatory mechanisms of lactate-mediated lactylation in the tumorigenesis and immune suppression of DLBCL, aiming to identify novel therapeutic strategies. Methods： In vitro and in vivo experiments were conducted to evaluate the effects of lactate on lymphoma progression and the immune microenvironment. Mechanistically, quantitative proteomics and RNA-seq were used to identify lactylated proteins, which were further validated by western blot and RT-qPCR. RNA seq and acRIP-seq were performed to analyze dysregulated RNAs in DLBCL. Finally, a PEI/PC7A nanoparticle system targeting key genes involved in lactate production was developed for therapeutic intervention. Results：We first assessed the impact of lactate on lymphoma and found that lactate significantly enhanced lymphoma cell proliferation in vitro and promoted tumor progression in vivo. To determine whether lactate affects tumor-infiltrating immune cells, we performed multiple assays and observed a marked reduction in CD8+ T cell infiltration, accompanied by significantly decreased IFN-γ and GZMB levels. These findings suggest that lactate not only promotes lymphoma cell growth directly but also facilitates tumor progression by suppressing CD8+ T cell mediated antitumor immunity. Through quantitative proteomics and RNA-seq, we identified that lactate significantly upregulates NAT10 protein levels without affecting its mRNA expression, suggesting direct lactylation of NAT10. Lactylation omics identified residues Q8K224_K989 and Q8K224_K956 as NAT10 lactylation sites. Site-directed mutagenesis at these residues, followed by Western blot after lactate treatment, shows unchanged NAT10 protein levels and the absence of lactylation, confirming these residues as NAT10 lactylation sites. NAT10 overexpression promotes lymphoma cell proliferation, while the NAT10 inhibitor Remodelin and SiNAT10 suppress proliferation and induce apoptosis. In vivo, NAT10 accelerates lymphoma progression and reduces CD8+ T-cell infiltration, consistent with the effects of lactate. Further analysis of acRIP-seq demonstrates that ac4C peaks predominantly enriched in the CDS region of LDHA mRNA. SiNAT10 significantly reduces ac4C abundance, suggesting that NAT10-driven ac4C acetylation enhances LDHA mRNA stability and promotes its expression. In vivo, LDHA overexpression significantly increases tumor burden, reduces CD8+ T-cell infiltration, and decreases IFN-γ and GZMB levels. LDHA is a key enzyme that catalyzes the conversion of pyruvate to lactate. NAT10 and LDHA overexpressions enhance lactate production in both cell supernatants and tumor tissues, indicating a connection between NAT10 activity, LDHA expression, and lactate accumulation. The LDHA inhibitor GSK reduces tumor growth and increases CD8+ T-cell infiltration. Combined GSK and PD-1 blockade synergistically enhances CD8+ T-cell proportions and IFN-γ production, leading to robust tumor inhibition. Furthermore, PEI/PC7A nanoparticles were developed for LDHA-targeted delivery. Intra-tumoral PEI/PC7A/siLDHA nanoparticles suppress tumor growth while increase CD8+ T-cell infiltration and IFN-γ levels. These findings demonstrate that LDHA inhibition reduces lactate accumulation and enhances the antitumor efficacy of ICIs by improving tumor immune responses. Conclusions:This study provides preclinical evidence that tumor-intrinsic lactate plays a pivotal role in DLBCL progression and immunosuppression through protein lactylation. Mechanistically, lactate increases NAT10 protein levels via lactylation, and NAT10, in turn, promotes LDHA expression through mRNA acetylation. LDHA further enhances lactate production, forming a positive feedback loop that drives tumor progression and suppresses antitumor immunity. These findings identify lactate as a novel oncogenic factor in DLBCL and highlight its potential as a therapeutic target. Combining lactate inhibition with immunotherapy may offer a promising strategy for cancer treatment.