ABSTRACT
BACKGROUND: Recent advances in immune checkpoint blockade (ICB) have improved patient prognosis in mismatch repair-deficient and microsatellite instability-high colorectal cancer (dMMR/MSI-H CRC); however, PD-1 blockade has faced a challenge in early progressive disease. We aimed to understand the early event in ICB resistance using an in vivo model. METHODS: We subcutaneously transplanted the MC38 colon cancer cells into C57BL/6 mice, intraperitoneally injected anti-PD-1 antibody and then isolated ICB-resistant subclones from the recurrent tumors. RESULTS: Comparative gene expression analysis discovered seven genes significantly downregulated in the ICB-resistant cells. Tumorigenicity assay of the MC38 cells knocked out each of the seven candidate genes into C57BL/6 mice treated with anti-PD-1 antibody and bioinformatics analysis of the relationship between the expression of the seven candidate genes and the outcome of cancer patients receiving immunotherapy identified Rtp4, an interferon-stimulated gene and a chaperon protein of G protein-coupled receptors, as a gene involved in ICB resistance. Immunohistochemical analysis of transplanted tumor tissues demonstrated that anti-PD-1 antibody failed to recruit T lymphocytes in the Rtp4-KO MC38 cells. Mouse and human RTP4 expression could be silenced via histone H3 lysine 9 (H3K9) trimethylation, and public transcriptome data indicated the high expression level of RTP4 in most but not all of dMMR/MSI-H CRC. CONCLUSIONS: We clarified that RTP4 could be silenced by histone H3K9 methylation as the early event of ICB resistance. RTP4 expression could be a promising biomarker for predicting ICB response, and the combination of epigenetic drugs and immune checkpoint inhibitors might exhibit synergistic effects on dMMR/MSI-H CRC.