In Vivo CRISPR Screens Identify E3 Ligase Cop1 as a Modulator of Macrophage Infiltration and Cancer Immunotherapy Target

SUMMARY Despite remarkable clinical efficacies of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits in triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that inhibition of the E3 ubiquitin ligase Cop1 in cancer cells decreases the secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, and shows synergy in anti-tumor immunity with ICB. Transcriptomics, epigenomics, and proteomics analyses revealed Cop1 functions through proteasomal degradation of the C/ebpδ protein. Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. Cop1 inhibition stabilizes C/ebpδ to suppress the expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy by regulating chemokine secretion and macrophage levels in the TNBC tumor microenvironment. Highlights Large-scale in vivo CRISPR screens identify new immune targets regulating the tumor microenvironment Cop1 knockout in cancer cells enhances anti-tumor immunity Cop1 modulates chemokine secretion and macrophage infiltration into tumors Cop1 targets C/ebpδ degradation via Trib2 and influences ICB response