CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1 positive regulator for tumor immune evasion

Abstract Regulation of tumoral PD-L1 expression is critical to advance our knowledge in understating the tumor evasion of immunosurveillance and for improvement of antitumor immunotherapy. While several E3 ubiquitin ligases and ubiquitin-specific peptidases regulate PD-L1 protein stability, the deubiquitinases involved in PD-L1 transcription for cancer cell immune evasion have not been identified. Herein, we developed a CRISPR-based screening platform and identified ATXN3 as a top positive regulator for PD-L1 transcription. Analysis of TCGA database revealed a statistically significant positive correlation between ATXN3 and CD274 expression in more than 80% human cancers, indicating that ATXN3 is a positive regulator for PD-L1 transcription in broad spectra of human cancers. ATXN3 promotes PD-L1 transcription induced by multiple tumor microenvironmental (TME) factors including inflammatory cytokine IFN-γ and hypoxia through protecting their downstream transcription factors IRF1, STAT3 and HIF-2α. In addition, ATXN3 functions as a deubiquitinase of PD-L1 transcription factors YAP-1 and JunB, indicating ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells resulted in reduced PD-L1 expression and consequently enhanced antitumor immunity in mice, which are partially revised by reconstitution of PD-L1 expression. Further, tumoral ATXN3 suppression improves the preclinical efficacy of checkpoint blockade antitumor immunotherapy and partially dependent on CD8+ T cells. Importantly, ATXN3 expression is increased in human lung adenocarcinoma and melanoma and its levels are positively corelated with PD-L1 as well as its transcription factor IRF1 and HIF-2α. Collectively, our study identifies a previously unknown deubiquitinase ATXN3 as a positive regulator for PD-L1 transcription and provides a rationale for ATXN3 targeting in sensitizing checkpoint blockade antitumor immunotherapy. Citation Format: Shengnan Wang, Radhika Lyer, Deyu Fang. CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1 positive regulator for tumor immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 618.