HDAC6 inhibition attenuates RIPK1/RIPK3/MLKL signalling and improves anti-tumor immune response in oral cancer

RIPK1/RIPK3/MLKL signalling and its activation can lead to a regulated form of inflammatory cell death characterised by the release of intracellular contents into the extracellular space, contributing to an immunosuppressive tumor microenvironment (TME). In head and neck squamous cell carcinoma (HNSCC), approximately half of cell death involves necrosis. Targeting RIPK1/RIPK3/MLKL signalling represents a promising therapeutic strategy for oral squamous cell carcinoma (OSCC). In this study, we evaluated the impact of histone deacetylase-6 (HDAC6) inhibition on this pathway and TME using Tubastatin A (TSA), a highly selective HDAC6 inhibitor. Our findings revealed that HDAC6 expression was significantly upregulated during necrotic cell death in MOC2-OSCC cells. Treatment with TSA suppressed the translocation of phosphorylated MLKL (p-MLKL) to the cell membrane, thereby effectively inhibiting cell death. Using an MOC2-induced syngeneic OSCC mouse model, we observed that administration of TSA significantly reduced tumor volume and weight. TSA also decreased RIPK1/RIPK3/MLKL expression in tumor-bearing mice and modulated the immunosuppressive TME by altering immune cell populations. Specifically, TSA increased CD45⁺ immune cells and M1 macrophages while reducing myeloid-derived suppressor cells (MDSCs). Additionally, TSA enhanced T-cell-mediated anti-tumor immunity, as indicated by lower levels of CD8a⁺PD1⁺ and CD4⁺PD1⁺ regulatory T cells and higher levels of CD8a⁺Granzyme⁺, CD4⁺Granzyme⁺, CD8a⁺IFNγ⁺, and CD4⁺IFNγ⁺ cells in the spleen and tumor. In conclusion, TSA attenuates RIPK1/RIPK3/MLKL-signalling and reprograms the immunosuppressive TME, inhibiting OSCC progression and highlighting its potential as a therapeutic agent in clinical conditions.