H3K9me3 regulates T cell effector function and colon tumor liver metastasis

Patients with the microsatellite stable (MSS) subset of colorectal cancer, which accounts for 85-90% of all human colorectal cancer cases, do not respond to PD-(L)1 immune checkpoint inhibitor immunotherapy. Metastasis accounts for over 90% of human colorectal cancer mortality and liver metastasis accounts for 90% of human colorectal cancer metastasis. The mechanism underlying colorectal tumor cell immune evasion and the resultant liver metastasis is incompletely understood. The SUV39H1-H3K9me3 epigenetic pathway has emerged as a key regulator of CTL persistence and effector function in anti-tumor immunity. We therefore aimed at testing the hypothesis that H3K9me3 induces CTL dysfunction to promote colorectal tumor liver metastasis. Using a mouse MSS colon tumor experimental liver metastasis model, we observed that PD-1 blockade immunotherapy does not induce CTL activation in the liver metastases and exhibits no significant efficacy in suppression of liver metastasis. CTL adaptive transfer increased accumulation of the tumor-specific CTLs in liver metastases but also exhibited no significant efficacy in suppression of liver metastasis. However, pharmacological inhibition of H3K9me3-specific histone methyltransferases with the small molecule F5446 significantly suppressed liver metastasis in mice. scRNA-seq analysis of liver metastases-infiltrating immune cells revealed that F5446 therapy does not increased total level of T cell tumor infiltration. Instead, inhibition of H3K9me3 increased Tex and decreased Tcm in liver metastases. Analysis of human colon tumor scRNA-Seq datasets identified Tex, Tcm and Tem-like clusters in both primary and liver metastasis with higher level of Tex subset of T cells in liver metastases than primary tumor. Our findings determine that colon tumor cells induce H3K9me3 to regulate T cell differentiation and effector function in liver metastases microenvironment and targeting H3K9me3 is a promising approach for suppression of CRC liver metastasis. CT26 cells were injected to spleens of BALB/c mice to form liver metastases. The tumor-bearing mice were treated with vehicle control of F5446 (10 mg/kg body weight) 4 days after tumor cell injection every 2 days for 5 times. Livers were harvested and underwent single cell suspension. CD45 cells were isolated from the samples using mouse CD45 positive selection beads before sequencing.