DHX9 transcriptional upregulation and lactylation at K1024 fuels a glycolysis-histone lactylation loop in osteosarcoma.

BACKGROUND

Histone lactylation, a novel posttranslational modification driven by glycolytic lactate, has emerged as a key regulator of tumorigenesis. However, its role in osteosarcoma (OS) progression and the underlying metabolic‒epigenetic crosstalk remain poorly understood.

METHODS

Using immunohistochemistry, molecular biology, and functional assays in vitro and in vivo, we investigated lactylation levels, DHX9 regulation, Lactylation and glycolytic activity in osteosarcoma models. Techniques included CUT&Tag, LC-MS/MS, site-directed mutagenesis, and xenograft studies.

RESULTS

Here, we show global lactylation levels were significantly elevated in OS tissues compared with paracancerous controls. Glycolysis inhibition suppressed H3K9la and impeded OS malignancy. CUT&Tag identified H3K9la enrichment at the DHX9 promoter. DHX9 knockdown inhibited proliferation, migration, and invasion (in vitro) and tumor growth (in vivo), whereas DHX9 overexpression had the opposite effects. LC‒MS/MS revealed K1024 as a functional lactylation site on DHX9-K1024R mutation (lactylation-deficient) disrupted a feedforward loop: (1) reduced PKM2/LDHA expression → impaired glycolysis → decreased H3K9la; and (2) suppressed malignant phenotypes. Conversely, DHX9-K1024T (lactylation mimetic) partially rescued glycolytic enzyme expression and H3K9la levels.

CONCLUSION

We elucidated a self-amplifying H3K9la-DHX9-K1024la-glycolysis circuit that drives OS progression. DHX9 lactylation at K1024 serves as a critical metabolic‒epigenetic interface, suggesting that K1024 lactylation may represent a potential therapeutic target, warranting further investigation.