CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion
Shengnan Wang, … , Zhaolin Sun, Deyu Fang
Shengnan Wang, … , Zhaolin Sun, Deyu Fang
Published December 1, 2023
Citation Information: J Clin Invest. 2023;133(23):e167728. https://doi.org/10.1172/JCI167728.
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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 4

ATXN3 inhibition improves antitumor immunity partially through downregulating tumoral PD-L1 expression.

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ATXN3 inhibition improves antitumor immunity partially through downregul...
(A–C) WT or ATXN3-KO LLC1 cells were injected subcutaneously into C57BL/6 mice (n = 10). Tumor growth curve (A), photograph (B), and weight (C) are shown. (D) MFI of surface PD-L1 on LLC1 tumors (n = 5). (E–G) Quantification of CD4+ (E) and CD8+ T cell (F) and Treg (G) percentages (n = 5–10). (H and I) Quantification of cell-surface PD-1 (H) and PD-L1 (I) MFI on CD8+ T cells (n = 5). (J and K) Quantification of cell-surface CTLA-4 MFI (J) and Tim3 percentage (K) in CD8+ T cells (n = 5). (L) MFI of cell-surface LAG3 and percentage in CD8+ T cells (n = 5). (M–O) Intracellular staining of Blimp1+ CD8+ T cell (M), EOMES+ CD8+ T cell (N), and T-bet+ CD8+ T cell (O) percentage in LLC1 tumors (n = 5-7). (P) Quantification of cell-surface CD44+ CD8+ T cell percentage from LLC1 tumors (n = 5–10). (Q) Apoptotic CD8+ T cells in the tumors were analyzed (n = 5–7). (R and S) Representative flow staining and quantification of intracellular cytokine staining of granzyme B+CD8+ and IFN-γ+CD8+ in CD45+ T cell populations from LLC1 tumors (n = 5). (T) Tumor growth curve and tumor photograph of C57BL/6 mice injected subcutaneously with WT and ATXN3-KO LLC1 cells with or without treatment of anti-CD8 depleting antibodies (n = 5). (U) Left: Tumor cell-surface PD-L1 expression. Right: Tumor growth of WT or ATXN3-KO LLC1 cells stably expressing PD-L1 (as shown in the left plot) in C57BL/6 mice (n = 5). A and C–S: 2-tailed unpaired t test; T and U: ordinary 1-way ANOVA. *P < 0.05, **P < 0.01,***P < 0.001.