CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion
Shengnan Wang, Radhika Iyer, Xiaohua Han, Juncheng Wei, Na Li, Yang Cheng, Yuanzhang Zhou, Qiong Gao, Lingqiang Zhang, Ming Yan, Zhaolin Sun, Deyu Fang
Shengnan Wang, Radhika Iyer, Xiaohua Han, Juncheng Wei, Na Li, Yang Cheng, Yuanzhang Zhou, Qiong Gao, Lingqiang Zhang, Ming Yan, Zhaolin Sun, Deyu Fang
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Research Article Otology

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 advancing our understanding of tumor immune evasion and the improvement of existing antitumor immunotherapies. Herein, we describe a CRISPR-based screening platform and identified ATXN3 as a positive regulator for PD-L1 transcription. TCGA database analysis revealed a positive correlation between ATXN3 and CD274 in more than 80% of human cancers. ATXN3-induced Pd-l1 transcription was promoted by tumor microenvironmental factors, including the inflammatory cytokine IFN-γ and hypoxia, through protection of their downstream transcription factors IRF1, STAT3, and HIF-2α. Moreover, ATXN3 functioned as a deubiquitinase of the AP-1 transcription factor JunB, indicating that ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells largely abolished IFN-γ– and hypoxia-induced PD-L1 expression and consequently enhanced antitumor immunity in mice, and these effects were partially reversed by PD-L1 reconstitution. Furthermore, tumoral ATXN3 suppression improved the preclinical efficacy of checkpoint blockade antitumor immunotherapy. Importantly, ATXN3 expression was increased in human lung adenocarcinoma and melanoma, and its levels were positively correlated with PD-L1 as well as its transcription factors IRF1 and HIF-2α. Collectively, our study identifies what we believe to be a previously unknown deubiquitinase, ATXN3, as a positive regulator for PD-L1 transcription and provides a rationale for targeting ATXN3 to sensitize checkpoint blockade antitumor immunotherapy.

Authors

Shengnan Wang, Radhika Iyer, Xiaohua Han, Juncheng Wei, Na Li, Yang Cheng, Yuanzhang Zhou, Qiong Gao, Lingqiang Zhang, Ming Yan, Zhaolin Sun, Deyu Fang

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Figure 1

Identification of ATXN3 as a PD-L1–positive regulator in cancer cells by unbiased CRISPR screening.

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Identification of ATXN3 as a PD-L1–positive regulator in cancer cells by...
(A) Schematic of the deubiquitinase CRISPR knockout screening workflow. (B) Guides enriched in PD-L1–low and PD-L1–high populations with their fold enrichment. The guide code of each gene for further validation is indicated. (C) Guide hits described were validated by flow using individual guide knockouts. (D) Western blotting validation of ATXN3 knockout and PD-L1 expression with specific sgRNAs in LLC1 cells. WT, cells transfected with empty vector; KO, ATXN3-knockout stable cell strains. (E and F) Representative flow cytometry plots and quantification by MFI of cell-surface PD-L1 in LLC1 cells. (G) Western blotting analysis of ATXN3 and PD-L1 expression in A549 cells with knockout of ATXN3. (H and I) Representative flow cytometry plots and quantification of cell-surface PD-L1 in A549 cells with knockout of ATXN3. (J and K) Cd274 and Atxn3 mRNA levels were analyzed by reverse transcription quantitative PCR (RT-qPCR) in LLC1 cells. (L and M) Cd274 and Atxn3 mRNA levels were analyzed by RT-qPCR in B16 cells. (N) Correlation of CD274 mRNA levels with ATXN3 mRNA levels in lung cancer patients (n = 22). (O) Correlation of CD274 with ATXN3 expression in multiple tumors based on TCGA data (n = 40). C, F, and I–M: 2-tailed unpaired t test; N: Pearson’s correlation analysis. *P < 0.05, **P < 0.01, ***P < 0.001.