CBP-mediated H3K18 lactylation drives GLI2 transcription to promote progression and EGFR-targeted therapy resistance in head and neck squamous cell carcinoma

Protein lactylation plays critical roles in cancer, but the participation of histone H3 lysine 18 lactylation (H3K18la) in the progression and EGFR-targeted therapy resistance in head and neck squamous cell carcinoma (HNSCC) remains largely unclear. Through a combination of RNA-seq and CUT&Tag, we identified the H3K18la-mediated genes such as GLI2. CCK-8, colony formation, ChIP-qPCR, qRT-PCR, Co-IP, and Western blotting were applied to evaluate tumor malignant phenotypes and molecular mechanisms. CAL27 cell-derived xenograft mouse models were developed for analyzing the effects of 2DG, and SGC-CBP30 in combination with gefitinib, respectively. Gefitinib-resistant HNSCC cell lines were established to evaluate the regulatory roles of H3K18la in gefitinib resistance. In HNSCC, hypoxia increased H3K18la and EGFR protein levels. Single-cell RNA-seq analysis revealed a high-lactylation state in HNSCC. Inhibition of protein lactylation by 2DG or oxamate attenuated HNSCC cell proliferation and colony formation, while lactate supplementation reversed these effects. The integrated RNA-seq and CUT&Tag analysis revealed that H3K18 lactylation was enriched at the GLI2 locus, and GLI2 was transcriptionally activated. Knockdown or inhibition of GLI2 suppressed cell proliferation and colony formation, whereas overexpressing it promoted these phenotypes. The lactyltransferase CBP promoted H3K18la, and molecular docking and Co-IP confirmed the binding between CBP and H3K18la. Knocking down CBP or treating with SGC-CBP30 or C646 reduced H3K18la levels and GLI2 expression, thereby inhibiting HNSCC cell proliferation and colony formation. EGFR knockdown or gefitinib/cetuximab treatment suppressed the levels of global protein lactylation, H3K18la and GLI2, and decreased cellular lactate concentration and inhibited glucose uptake. EGFR bound to and stabilized GLUT3. Inhibitors of the H3K18la-GLI2 axis sensitized HNSCC cells to gefitinib and cetuximab, and importantly, SGC-CBP30 greatly improved the anti-tumor effects of gefitinib in vivo. The gefitinib-resistant HNSCC cell lines were established, and the H3K18la-GLI2 axis was activated in these gefitinib-resistant cells. Notably, blocking this pathway overcame gefitinib resistance in HNSCC cells. CBP-mediated H3K18 lactylation drives GLI2 transcription to promote progression and EGFR-targeted therapy resistance in head and neck squamous cell carcinoma, providing a promising target for HNSCC therapy.