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.
View: Text | PDF
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

×

Figure 3

ATXN3 selectively functions as a HIF-2α deubiquitinase to promote tumoral PD-L1 transcription.

Options: View larger image (or click on image) Download as PowerPoint
ATXN3 selectively functions as a HIF-2α deubiquitinase to promote tumora...
(A and B) A549 cells were cultured under normoxia and hypoxia (hyp) (1% pO2) for 48 hours, and surface PD-L1 levels were analyzed by flow cytometry and quantification. (C) ATXN3 specifically interacts with HIF-2α. HA–HIF-2α expression plasmid was cotransfected with or without FLAG-ATXN3 into HEK293T cells. Their interactions were examined by co-IP with anti-FLAG antibodies and by Western blotting with anti-HA antibodies. (D) The interaction between ATXN3 and HIF-1α was tested in transfected HEK293T cells. (E) Endogenous interaction between ATXN3 and HIF-2α in A549 cells. (F) HA-ubiquitin, FLAG–HIF-2α, and Myc-ATXN3 plasmids were cotransfected into HEK293T cells. HIF-2α ubiquitination was determined by immunoprecipitation of HIF-2α with anti-FLAG antibodies and immunoblotting with anti-HA antibody. (G and H) HIF-2α was cotransfected with or without ATXN3 plasmids into HEK293T cells. The transfected cells were treated with CHX for different times. The protein levels of HIF-2α (top panel) and ATXN3 (middle panel) were analyzed by Western blotting. β-Actin was used as a loading control (bottom panel). (I and J) Immunoblot analysis of HIF-2α protein stability in WT and ATXN3-KO A549 cells. (K) ATXN3 enhances hypoxia-induced PD-L1 expression through protecting HIF-2α from ubiquitination-induced protein degradation. B: Ordinary 1-way ANOVA; H and J: 2-tailed unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.