Discovery of o-aminodiphenyl-based USP7 inhibitor for immune activation and antitumor response in colorectal cancer.

[BACKGROUND] Colorectal cancer is a leading cause of cancer mortality, with microsatellite-stable tumors exhibiting poor response to immune checkpoint inhibitors. Ubiquitin-specific protease 7 (USP7) stabilizes programmed death-ligand 1 (PD-L1), promoting immune evasion.

[METHODS] Through screening, synthesis, and optimization, we identified an o-aminodiphenyl derivative, U-20, as a potent USP7 inhibitor (IC₅₀ = 83 nM). USP7 inhibition, PD-L1 modulation, and immune activation were evaluated using in vitro enzymatic and cellular assays, T-cell killing assays, and an immunocompetent CT26 colorectal cancer mouse model.

[RESULTS] U-20 selectively bound USP7, destabilized PD-L1 via proteasome-mediated degradation, and increased PD-L1 polyubiquitination without affecting transcription. In colorectal cancer cell-T-cell co-cultures, U-20 enhanced CD8 T-cell cytotoxicity and cytokine secretion (IFN-γ, TNF-α), effects abolished by ubiquitination-resistant PD-L1 mutants. In vivo, U-20 significantly inhibited tumor growth, increased intratumoral CD8 T-cell infiltration, and elevated effector cytokine levels, without apparent toxicity.

[CONCLUSION] U-20 effectively reprograms the colorectal cancer immune microenvironment by blocking USP7-mediated PD-L1 stabilization, thereby restoring antitumor immunity. These findings provide a preclinical rationale for USP7 inhibition as a novel immunotherapeutic strategy for colorectal cancer, particularly in microsatellite-stable disease.