Ligand-induced ubiquitination unleashes LAG3 immune checkpoint function by hindering membrane sequestration of signaling motifs

Yong Jiang; Anran Dai; Yuwei Huang; Hua Li; Jian Cui; Haochen Yang; Lu Si; Tao Jiao; Zhengxu Ren; Ziwei Zhang; Si Mou; Hengrui Zhu; Wenhui Guo; Qiang Huang; Yilin Li; Manman Xue; Jingwei Jiang; Fei Wang; Li Li; Qinying Zhong; Kun Wang; Baichuan Liu; Jinjiao Wang; Gaofeng Fan; Jun Guo; Liang Chen; Creg J Workman; Zhirong Shen; Yan Kong; Dario A A Vignali; Chenqi Xu; Haopeng Wang

doi:10.1016/j.cell.2025.02.014

Ligand-induced ubiquitination unleashes LAG3 immune checkpoint function by hindering membrane sequestration of signaling motifs

Cell. 2025 Mar 6:S0092-8674(25)00199-0. doi: 10.1016/j.cell.2025.02.014. Online ahead of print.

Authors

Yong Jiang¹, Anran Dai², Yuwei Huang³, Hua Li⁴, Jian Cui⁵, Haochen Yang⁴, Lu Si⁶, Tao Jiao⁶, Zhengxu Ren⁴, Ziwei Zhang⁷, Si Mou⁷, Hengrui Zhu⁷, Wenhui Guo⁶, Qiang Huang⁸, Yilin Li⁹, Manman Xue⁴, Jingwei Jiang¹, Fei Wang², Li Li², Qinying Zhong², Kun Wang², Baichuan Liu³, Jinjiao Wang², Gaofeng Fan², Jun Guo⁶, Liang Chen⁸, Creg J Workman⁵, Zhirong Shen¹⁰, Yan Kong¹¹, Dario A A Vignali¹², Chenqi Xu¹³, Haopeng Wang¹⁴

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
² School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
³ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Lingang Laboratory, Shanghai 200031, China.
⁴ Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
⁵ Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA, USA.
⁶ Key Laboratory of Carcinogenesis and Translational Research (Ministry Education), Department of Melanoma and Sarcoma, Peking University Cancer Hospital and Research Institute, Beijing 100142, China.
⁷ BeiGene, Ltd, Beijing 102206, China.
⁸ School of Medicine, Shanghai University, Shanghai 200444, China; State Key Laboratory of New Targets Discovery and Drug Development for Major Diseases, Xi'an 710032, China.
⁹ National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China.
¹⁰ BeiGene, Ltd, Beijing 102206, China. Electronic address: zhirong.shen@beigene.com.
¹¹ Key Laboratory of Carcinogenesis and Translational Research (Ministry Education), Department of Melanoma and Sarcoma, Peking University Cancer Hospital and Research Institute, Beijing 100142, China. Electronic address: ky_puch@163.com.
¹² Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Cancer Immunology and Immunotherapy Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. Electronic address: dvignali@pitt.edu.
¹³ Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China; Shanghai Academy of Natural Sciences (SANS), Shanghai 200031, China. Electronic address: cqxu@sibcb.ac.cn.
¹⁴ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Shanghai Academy of Natural Sciences (SANS), Shanghai 200031, China; State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai 201210, China. Electronic address: wanghp@shanghaitech.edu.cn.

PMID: 40101708
DOI: 10.1016/j.cell.2025.02.014

Abstract

Lymphocyte activation gene 3 (LAG3) has emerged as a promising cancer immunotherapy target, but the mechanism underlying LAG3 activation upon ligand engagement remains elusive. Here, LAG3 was found to undergo robust non-K48-linked polyubiquitination upon ligand engagement, which promotes LAG3's inhibitory function instead of causing degradation. This ubiquitination could be triggered by the engagement of major histocompatibility complex class II (MHC class II) and membrane-bound (but not soluble) fibrinogen-like protein 1 (FGL1). LAG3 ubiquitination, mediated redundantly by the E3 ligases c-Cbl and Cbl-b, disrupted the membrane binding of the juxtamembrane basic residue-rich sequence, thereby stabilizing the LAG3 cytoplasmic tail in a membrane-dissociated conformation enabling signaling. Furthermore, LAG3 ubiquitination is crucial for the LAG3-mediated suppression of antitumor immunity in vivo. Consistently, LAG3 therapeutic antibodies repress LAG3 ubiquitination, correlating with their checkpoint blockade effects. Moreover, patient cohort analyses suggest that LAG3/CBL coexpression could serve as a biomarker for response to LAG3 blockade. Collectively, our study reveals an immune-checkpoint-triggering mechanism with translational potential in cancer immunotherapy.

Keywords: Cbl-b; LAG3; TCR signaling; c-Cbl; cancer immunotherapy; checkpoint; ubiquitination.