Ubiquitin-specific protease 7 (USP7) regulates the stability and fate of many proteins, thereby influencing cellular processes such as cell cycle, chromatin remodeling, and protein synthesis. Elevated level of USP7 in cancer contributes to tumor progression by modulation of tumor microenvironment. USP7 has a variety of substrates, including MDM2 (Murine Double Minute 2) and the tumor suppressor p53 – well-established targets in cancer drug development. Here, we explored the USP7-MDM2-p53 pathway in the context of immunomodulation, rather than more studied direct cytotoxic effects on cancer cells.

The activity and selectivity of OAT- 4828, an inhibitor we designed, was assessed in enzymatic assays. After pharmacokinetic studies, the efficacy of OAT-4828 was evaluated in mouse in vivo models of melanoma (B16F10), colon cancer (CT-26), lymphoma (A20) and chronic lymphocytic leukemia (Eμ-TCL1). Tumor microenvironment was analyzed by flow cytometry. In ex vivo studies the influence of OAT-4828 was estimated in mouse and human T cells, as well as mouse bone marrow-derived macrophages by Western blotting, qPCR, ELISA tests and by a functional cell killing assay.

Oral administration of USP7i resulted in a significant reduction in tumor volume in syngeneic mouse models of colon, melanoma cancer, and lymphoma and leukemia. This reduction was associated with a substantial increase in T cell activation, as evidenced by higher levels of CD69 and CD44, as well as increased production of Granzyme B and IFN-γ. Antitumor activity of USP7i was improved when combined with anti-PD-1 antibodies, while the T cell depletion completely abrogated the therapy outcome. OAT-4828 caused increased production of IL-2 and upregulation of CD25 and CD69 in human T cells, which was associated with MDM2 downregulation and p53 stabilization. Similarly, USP7 inhibition led to MDM2 downregulation in macrophages, inhibiting their M2-like functions. These results indicate that the main mechanism of action of USP7i is based on the activation and improved cytotoxic functions of T cells and promoting anti-cancer activity relevant to the tumor microenvironment.

We conclude that USP7-MDM2-p53 is implicated in the antitumor function of immune cells and shapes the tumor microenvironment. Altogether, we confirm USP7 as an attractive target for cancer immunotherapy and present a strong rationale for the development of USP7 inhibitors for clinical use.