PKCα/ZFP64/CSF1 axis resets the tumor microenvironment and fuels anti-PD1 resistance in hepatocellular carcinoma

BACKGROUND & AIMS: Despite remarkable advances in treatment, most patients with hepatocellular carcinoma (HCC) respond poorly to anti-programmed cell death 1 (anti-PD1) therapy. A deeper insight into the tolerance mechanism of HCC against this therapy is urgently needed. METHODS: We performed next-generation sequencing, multiplex immunofluorescence, and dual-color immunohistochemistry and constructed an orthotopic HCC xenograft tumor model to identify the key gene associated with anti-PD1 tolerance. A spontaneously tumorigenic transgenic mouse model, an in vitro coculture system, mass cytometry, and multiplex immunofluorescence were used to explore the biological function of zinc finger protein 64 (ZFP64) on tumor progression and immune escape. Molecular and biochemical strategies like RNA-sequencing, chromatin immunoprecipitation-sequencing and mass spectrometry were used to gain insight into the underlying mechanisms of ZFP64. RESULTS: We showed that ZFP64 is frequently upregulated in tumor tissues from patients with anti-PD1-resistant HCC. Elevated ZFP64 drives anti-PD1 resistance by shifting macrophage polarization toward an alternative activation phenotype (M2) and fostering an inhibitory tumor microenvironment. Mechanistically, we primarily demonstrated that protein kinase C alpha (PKCα) directly phosphorylates ZFP64 at S226, leading to its nuclear translocation and the transcriptional activation of macrophage colony-stimulating factor (CSF1). HCC-derived CSF1 transforms macrophages to the M2 phenotype to drive immune escape and anti-PD1 tolerance. Notably, Gö6976, a protein kinase inhibitor, and lenvatinib, a multi-kinase inhibitor, reset the tumor microenvironment and restore sensitivity to anti-PD1 by blocking the PKCα/ZFP64/CSF1 axis. CONCLUSIONS: We propose that the PKCα/ZFP64/CSF1 axis is critical for triggering immune evasion and anti-PD1 tolerance. Inhibiting this axis with Gö6976 or lenvatinib overcomes anti-PD1 resistance in HCC. LAY SUMMARY: Despite remarkable treatment progress, most patients with hepatocellular carcinoma respond poorly to anti-PD1 therapy (a type of immunotherapy). A deeper insight into the tolerance mechanisms to this therapy is urgently needed. Herein, we unravel a previously unexplored mechanism linking tumor progression, macrophage polarization, and anti-PD1 resistance, and offer an attractive novel target for anti-PD1 combination therapy, which may benefit patients with hepatocellular carcinoma.