Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy.

OBJECTIVE:Myeloid-derived suppressor cells (MDSCs) contribute to tumour immunosuppressive microenvironment and immune-checkpoint blockade resistance. Emerging evidence highlights the pivotal functions of cyclin-dependent kinases (CDKs) in tumour immunity. Here we elucidated the role of tumour-intrinsic CDK20, or cell cycle-related kinase (CCRK) on immunosuppression in hepatocellular carcinoma (HCC). DESIGN:Immunosuppression of MDSCs derived from patients with HCC and relationship with CCRK were determined by flow cytometry, expression analyses and co-culture systems. Mechanistic studies were also conducted in liver-specific CCRK-inducible transgenic (TG) mice and Hepa1-6 orthotopic HCC models using CRISPR/Cas9-mediated Ccrk depletion and liver-targeted nanoparticles for interleukin (IL) 6 trapping. Tumorigenicity and immunophenotype were assessed on single or combined antiprogrammed death-1-ligand 1 (PD-L1) therapy. RESULTS:Tumour-infiltrating CD11b⁺CD33⁺HLA-DR^- MDSCs from patients with HCC potently inhibited autologous CD8⁺T cell proliferation. Concordant overexpression of CCRK and MDSC markers (CD11b/CD33) positively correlated with poorer survival rates. Hepatocellular CCRK stimulated immunosuppressive CD11b⁺CD33⁺HLA-DR^- MDSC expansion from human peripheral blood mononuclear cells through upregulating IL-6. Mechanistically, CCRK activated nuclear factor-κB (NF-κB) via enhancer of zeste homolog 2 (EZH2) and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. Hepatic CCRK induction in TG mice activated the EZH2/NF-κB/IL-6 cascade, leading to accumulation of polymorphonuclear MDSCs with potent T cell suppressive activity. In contrast, inhibiting tumorous Ccrk or hepatic IL-6 increased interferon γ⁺tumour necrosis factor-α⁺CD8⁺ T cell infiltration and impaired tumorigenicity, which was rescued by restoring Notably, tumorous Ccrk depletion upregulated PD-L1 expression and increased intratumorous CD8⁺ T cells, thus enhancing PD-L1 blockade efficacy to eradicate HCC. CONCLUSION:Our results delineate an immunosuppressive mechanism of the hepatoma-intrinsic CCRK signalling and highlight an overexpressed kinase target whose inhibition might empower HCC immunotherapy.

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