Osteopetrosis-associated transmembrane protein1 modulates CD8⁺ T-cell responses during immunotherapy in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is the third leading cause of cancer-related deaths worldwide. Although atezolizumab plus bevacizumab (Atezo+Beva) has shown some efficacy in the treatment of advanced HCC, its therapeutic benefit remains limited. Systematically identifying these regulatory factors is crucial for recognizing predictive biomarkers and therapeutic targets in HCC. Methods: An in vivo genome-wide CRISPR/Cas9 knockout screen was performed in a murine HCC model treated with anti-programmed death ligand 1 (anti-PD-L1) and anti-vascular endothelial growth factor a (anti-VEGFa) antibodies to identify key regulators of immunotherapy resistance. Osteopetrosis-associated transmembrane protein 1 (OSTM1), an uncharacterised oncogene, was identified as a leading candidate. It was subsequently validated through tumour-cell-specific knockout in both orthotopic and hydrodynamic tail-vein injection (HDTVi) mouse models. Bulk RNA sequencing was performed on tumour tissues from OSTM1-knockout and control groups. Results: The screening nominated OSTM1 as a key mediator of resistance to anti-PD-L1 and anti-VEGFa. Knockout of OSTM1 in tumour cells improved response to therapy and reduced tumour growth in both orthotopic and HDTVi mice models. Clinically, elevated OSTM1 levels have been demonstrated to be associated with diminished survival rates in patients with HCC who have been treated with Atezo+Beva. Mechanistically, OSTM1 fostered an immunosuppressive microenvironment by impairing CD8⁺ T-cell infiltration and multiple effector functions, including Interferon gamma (IFN-gamma) production, and by reducing markers of proliferation and cytotoxicity such as kiel 67 (KI67), granzyme b (GZMB), and Perforin. Moreover, we merged differentially expressed genes from both the orthotopic and HDTVi OSTM1-knockout models consistently revealed PRELI domain containing 2 (PRELID2) as a downstream gene suppressed by OSTM1. Given that PRELID2 is involved in mitochondrial regulation and there is a new link between mitochondrial function and immune cell activity, this finding suggests that OSTM1 may indirectly influence immune cell infiltration and function in the tumor microenvironment through a potential axis. Conclusion: OSTM1 contributes to poor immunotherapy response in HCC, suggesting its utility as a biomarker and candidate therapeutic target during Atezo+Beva therapy. Future research will investigate how targeting OSTM1 may enhance CD8⁺ T-cell infiltration and function in the tumour microenvironment.