Abstract 4040: Hypoxia-induced HIF-1α/USP3 pathway promoting immune escape in lung squamous cell carcinoma

Abstract Background: Immune escape poses a significant mechanism for tumor progress as well as enormous challenge for the treatment of lung squamous cell carcinoma (LUSC). The close association between hypoxic environments and tumor immune escape, with PD-L1 serving as a key marker, has garnered considerable attention. This study aims to delve into the molecular mechanisms underlying the regulation of PD-L1 deubiquitination by the hypoxia-induced HIF-1α/USP3 pathway, shedding light on the immune escape mechanisms in LUSC. Methods: NSCLC cells were cultured under hypoxia condition and the expression of USP3, HIF-1α and PD-L1 were measured by Western blot. Chromatin Immunoprecipitation (ChIP) and Co-immunoprecipitation (CO-IP) were employed to elucidate the direct regulatory mechanisms of the HIF-1α/USP3 pathway in PD-L1 deubiquitination. In vitro T cell-mediated killing assays was used to investigate the functional roles of USP3 in antitumor immunity in NSCLC. Results: We demonstrated that hypoxia score was increased in LUSC and negatively correlated with CD8+ T cell infiltration. Hypoxia as a major factor triggering LUSC cell immunosuppression and against T cell surveillance via stabilization of PD-L1. Mechanistically, USP3, induced by HIF-1α, is required for hypoxia-mediated PD-L1 stabilization in LUSC cells. USP3 inhibits the ubiquitination and degradation of PD-L1. The combination of USP3 inhibitors with PD-1/PD-L1 monoclonal antibodies significantly enhances the anti-LUSC effect, possibly through promoting the infiltration of CD8+ T and NK cells. Conclusion: This research unveils a novel mechanism by which hypoxia, through the HIF-1α/USP3 pathway, regulates PD-L1 deubiquitination, thereby promoting immune escape in LUSC. Furthermore, our study provides a new strategy involving USP3 inhibitors in combination with PD-1/PD-L1 monoclonal antibodies, offering novel insights for molecular targeted therapy and reversal of drug resistance in LUSC. Citation Format: Xiuwen Zhang, Hua Huang, Chen Ding, Chen Chen, Yongwen Li, Hongyu Liu, Chen Jun. Hypoxia-induced HIF-1α/USP3 pathway promoting immune escape in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4040.