Discovery of indazole-carboxamide based USP8 inhibitor for immune activation and antitumor response in colorectal cancer.

BACKGROUND

Microsatellite-stable colorectal cancer (CRC), which accounts for most CRC cases, responds poorly to immune checkpoint inhibitors. Increasing evidence suggests that the deubiquitinase USP8 maintains PD-L1 protein expression by erasing its K48-linked ubiquitination, thereby preserving membrane PD-L1 and facilitating immune escape. How pharmacological inhibition of USP8 regulates PD-L1 stability and antitumor immunity in CRC, however, has not been fully clarified.

METHODS

Through structure-based optimization, we developed a novel indazole-carboxamide based compound, W-17, as a potent USP8 inhibitor. The effects of W-17 on PD-L1 stability, CD8⁺ T-cell cytotoxicity, and tumor growth were evaluated using enzymatic assays, cellular thermal shift assays (CETSA), Western blotting, T-cell killing assays as well as a syngeneic CT26 CRC mouse model.

RESULTS

W-17 inhibited USP8 enzymatic activity (IC50 = 83 nM), promoted PD-L1 polyubiquitination, and induced its proteasomal degradation. Pharmacological blockade of USP8 enhanced CD8⁺ T-cell mediated killing and increased the secretion of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in vitro. In an immunocompetent CT26 mouse model, W-17 significantly suppressed tumor growth, increased intratumoral CD8⁺ T-cell infiltration, and reduced PD-L1 expression, without overt systemic toxicity.

CONCLUSION

These findings identify USP8 as a post-translational regulator of PD-L1 stability in colorectal cancer and demonstrate that the novel USP8 inhibitor W-17 effectively promotes PD-L1 degradation and restores CD8⁺ T-cell-mediated antitumor immunity. Our study highlights W-17 as a promising lead compound for the development of USP8-targeted immunotherapeutic strategies for microsatellite-stable colorectal cancer.