Reversing KDM6A phosphorylation-driven epigenetic suppression restores pyroptosis and synergizes with anti-PD-1 therapy in HNSCC

Abstract Immune checkpoint blockade (ICB) has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment. Yet, a major barrier to response is that tumor cells are epigenetically reprogrammed to resist immunogenic cell death, particularly pyroptosis. To elucidate how tumor cells are pre-conFigured for pyroptosis resistance, we investigated the role of post-translational modifications on epigenetic regulators, focusing on the phosphorylation-dependent control of histone demethylase KDM6A. Through integrated phosphoproteomics and functional genomics in HNSCC models, we identified phosphorylation of KDM6A at Ser829 as a master regulatory switch. Mechanistically, phosphorylation at Ser829 subverts KDM6A function, leading to a global accumulation of repressive H3K27me3 marks. This establishes an epigenetic program that locks key pyroptosis effector genes ( GSDMD , NLRP3 ) in a transcriptionally refractory, heterochromatic state. To pharmacologically dismantle this barrier, we developed a novel lipid nanoparticle (LNP) system encapsulating mRNA encoding a phosphorylation-defective KDM6A S829A variant. This strategy functionally reverses the oncogenic epigenetic state, restoring endogenous demethylase activity specifically at pyroptosis loci and reigniting the cell death pathway. In vivo, a single administration of KDM6A S829A mRNA inhibited tumor growth by 39% ( p < 0.01). Strikingly, when combined with anti-PD-1, this strategy achieved a synergistic 79% tumor reduction ( p < 0.0001), characterized by robust CD8 + T cell infiltration. Our work identifies KDM6A phosphorylation as a druggable epigenetic-immune checkpoint and establishes mRNA-based modulation of enzyme post-translational status as a transformative paradigm to potentiate ICB in HNSCC.