Targeting NEK9 synergises with immunotherapy in hepatocellular carcinoma by remodelling the immunosuppressive microenvironment

Background Immune checkpoint inhibitors (ICIs) demonstrate limited efficacy in hepatocellular carcinoma (HCC), largely attributable to a profoundly immunosuppressive tumour microenvironment (TME). Objective To investigate the kinase never-in-mitosis A-related kinase 9 (NEK9) as a potential tumour-intrinsic driver of immune evasion and therapeutic target. Design NEK9 expression and its clinical relevance were analysed in HCC cohorts. Functional investigations employed genetic and specific pharmacological approaches in HCC cell lines and orthotopic mouse models. The TME was comprehensively profiled using single-cell RNA sequencing, flow cytometry and multiplex immunohistochemistry. Mechanistic insights were gained through co-immunoprecipitation, phosphoproteomic analysis and kinase assays. Synergy between NEK9 inhibition and programmed death-ligand 1 (PD-L1) blockade was quantitatively assessed using zero interaction potency (ZIP) reference models. Results NEK9 was significantly upregulated in HCC and correlated with poor survival, diminished intratumoral CD8 + T cell infiltration and increased myeloid-derived suppressor cells (MDSCs). Mechanistically, NEK9 directly phosphorylated TRIM28 and USP46, stabilising nuclear factor-κB2 (NF-κB2) and driving PD-L1 and CXCL1 transcription, thereby promoting CD8 + T cell dysfunction and CXCR2-dependent recruitment of MDSCs. Pharmacological NEK9 inhibition destabilised NF-κB2 and reversed the immunosuppressive TME. Importantly, two novel small-molecule NEK9 inhibitors (MIPO, FPTP) were identified, which synergised strongly with anti-PD-L1 therapy, enhancing CD8 + T cell effector function and tumour suppression in vivo. Conclusions NEK9 is a druggable driver of immune evasion in HCC. Targeting NEK9 remodels the immunosuppressive TME and synergises with PD-L1 blockade, offering a promising strategy to overcome ICI resistance.