Abstract 2173: Targeting USP7 with MEK1/2 inhibition synergizes to induce pyroptosis and enhance immunotherapy in <i>NRAS</i> mutant melanoma

Introduction: There is an urgent need for targeted therapies in metastatic melanoma patients with NRAS mutations, who comprise more than 25% of this population. This study investigates the therapeutic potential of targeting Ubiquitin-Specific Peptidase 7 (USP7) with a highly selective inhibitor, FT671, in NRAS mutant melanoma. In addition, the synergistic effects of the combination of FT671 with MEK1/2 inhibitor (MEKi) on pyroptosis induction, tumor suppression and immune activation were evaluated. Moreover, the potency of triple combination of FT671, MEKi and anti-PD-1 antibody was assessed. Experimental Procedures: We employed a combination of genetic knockout models and pharmacological inhibition to validate the selectivity of FT671 in vitro. Cell proliferation assays were used to determine IC50s and the synergistic effects. Flow cytometry assays were performed to monitor cell cycle arrest and cell death and analyze intratumoral immune cells. Western blot assays were used to examine if the combinations markedly induced the cleavage of GSDME. A CRISPR based screen was performed to explore the mechanism of action of FT671. We also utilize isograft melanoma models in immunocompetent mice to assess tumor growth, immune cell infiltration, and therapeutic outcomes. Results: As a validated USP7 selective inhibitor (USP7i), FT671 treatment effectively induced cell cycle arrest, reduced cell proliferation in vitro, and delayed tumor growth in NRAS mutant melanoma models in vivo. Knockout of TP53BP1, TP53 or CDKN1A conferred resistance to FT671, indicating activation of a functional p53 pathway is a major mechanism of action for the efficacy of USP7i in melanoma. The combination of FT671 with MEKi resulted in synergistic effects, and induced pyroptosis in vitro. In vivo, the combined treatment significantly suppressed tumor growth, prolonged survival, and enhanced anti-tumor immunity, evidenced by increased infiltration of CD8+ T cells and mature dendritic cells in tumors. Notably, the triple therapy with FT671, MEKi, and anti-PD-1 antibody led to tumor regression and durable responses in an isograft melanoma model. Conclusions: These findings highlight the potential of USP7 inhibition, combined with MEKi, as a promising strategy for treating NRAS mutant melanoma, an 'undruggable' target. Moreover, this combinatorial approach is able to enhance the efficacy of anti-PD-1 antibody, providing a compelling rationale for its clinical application as an adjuvant for immune checkpoint inhibitors. Citation Format: Liya Su,Dinghao Wang,Timothy J. Purwin,Sophia Ran,Qi Yang,Qingrun Zhang,Weijia Cai. Targeting USP7 with MEK1/2 inhibition synergizes to induce pyroptosis and enhance immunotherapy in NRAS mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2173.