TP53 mutation promotes diffuse large B-cell lymphoma progression via mediating lactylation of ATP5E at Lys44 Free

Lactate has recently been shown to play an important role in remodeling the tumor microenvironment and regulating energy metabolism through lysine lactylation (Kla). Mutant TP53 (MUT-TP53) can mediate tumor metabolic reprogramming through gain-of-function (GOF) mutations, but its specific molecular mechanism in diffuse large B-cell lymphoma (DLBCL) remains to be fully elucidated. This study focuses on the regulatory mechanisms of TP53 mutations on DLBCL lactate metabolism and lactylation modification.

According to the IARC TP53 database, TP53 mutations in DLBCL predominantly occur in Exon 5. We detected the TP53 protein expression in DLBCL cells with different TP53 mutation statuses, compared to naive B-cells. Analysis using the TCGA database showed significant activation of oxidative phosphorylation, mitochondrial respiratory chain complexes, and ATP synthase activity pathways in TP53-mutated DLBCL patients. RNA-seq analysis demonstrated that pathways involved in metabolism, cell cycle regulation, and DNA replication were significantly activated in DLBCL.

The above findings prompted us to investigate the biological function of the TP53 mutation in DLBCL. Knockdown of MUT-TP53 resulted in no significant changes in cell viability. In contrast, knockdown of WT-TP53 promoted cell proliferation and shortened the G0/G1 phase, and significantly decreased LDHA expression and intracellular lactate levels, whereas knockdown of WT-TP53 had no notable effect. Moreover, knockdown of MUT-TP53 reduced cellular sensitivity to APR-246, a small-molecule inhibitor targeting MUT-TP53. APR-246 treatment significantly suppressed lactate production and induced cell cycle arrest.

To elucidate the regulatory effects of TP53 mutations on the lactate metabolism and lactylation modification in DLBCL, we measured significantly elevated serum lactate concentrations in DLBCL patients compared to healthy controls. We revealed markedly increased lactylation modification levels in DLBCL tissues, with high lactylation levels correlating significantly with poorer progression-free survival (PFS) and overall survival (OS). The widespread lactylation modifications were confirmed in DLBCL cell lines. Exogenous supplementation with sodium lactate (LNa) increased lactylation levels, whereas inhibition of glycolysis or LDH activity using 2-DG or Galloflavin significantly reduced lactylation levels. Through proteomic analysis, 3156 lactylated proteins were identified in DLBCL cells. Functional enrichment analysis demonstrated that these proteins were predominantly involved in biological processes such as cell cycle regulation, mitochondrial function, transcriptional control, and signal transduction. Notably, the K44 site on ATP5E was identified as a highly conserved lactylation modification site.

We further elucidated the molecular mechanism of ATP5E K44la in DLBCL. ATP5E was highly expressed in DLBCL cells, and LNa treatment further enhanced its expression. K44la extended the ATP5E protein half-life and increased its expression. In vitro and in vivo experiments verified that overexpression of WT ATP5E significantly promoted cell proliferation, whereas the K44R mutation reversed this effect. Differentially expressed genes (DEGs) in overexpression of ATP5E WT and the K44R mutant were enriched in pathways related to DNA replication, mitochondrial function, cell cycle regulation, and OXPHOS. Cells in the WT group exhibited reduced mtDNA copy number, elevated mitochondrial membrane potential, decreased mtROS levels, and increased intracellular ATP levels, whereas the K44R mutant reversed these changes. Furthermore, mass spectrometry identified that TP53 interacted with ATP5E. Knockdown of MUT-TP53, but not WT-TP53, significantly decreased cellular lactylation levels. mIHC confirmed the co-localization of TP53 and Kla in DLBCL tissues. Overexpression of ATP5E enhanced its interaction with TP53, while mutation at the lactylation site (K44) reduced this interaction. Functional analyses indicated that MUT-TP53 knockdown increased mtROS levels and decreased mitochondrial membrane potential. Taken together, these findings suggest that DLBCL tissues and cells exhibit widespread elevated lactylation modification levels mediated by TP53 mutation.In conclusion, our results revealed that TP53 mutations recruit and enhance ATP5E K44la, stabilizing its expression and modulating mitochondrial functions, thus providing a novel mechanism for metabolic reprogramming in DLBCL.