On March 19, 2025, the research team led by Prof. Wensheng Wei at Peking University and Changping Laboratory published a research paper titled “Massively parallel interrogation of human functional variants modulating cancer immunosurveillance” in Signal Transduction and Targeted Therapy. The study leveraged high-throughput screening technology at single-base resolution and systematically identified numerous key functional sites regulating the expression of PD-L1 and HLA-I for the first time. This work constructed a novel molecular network of tumor immune regulation, providing a critical theoretical foundation for precision immunotherapy.

 

Anti-PD-1/PD-L1 immune checkpoint blockade (ICB) therapy has revolutionized the clinical cancer treatment, while abnormal PD-L1 or HLA-I expression in tumor patients can significantly impact the therapeutic efficacy¹. In recent years, several studies have employed CRISPR screens to uncover regulators of PD-L1 and HLA-I in cancer cells^2-5. However, due to the limited resolution of canonical CRISPR/Cas9 screens, these approaches mainly provide insights into the functional roles of regulators at the gene level. Somatic mutations occurring within cancer cells that modulate these critical immune regulators are closely associated with tumor progression and ICB therapy outcomes. According to the International Cancer Genome Consortium (ICGC) database, single-nucleotide variants account for over 90% among various somatic mutations, yet their functional relevance remains poorly understood. A comprehensive interpretation of cancer immune-related mutations is still lacking, with a vast number of clinical variants remaining functionally uncharacterized.

 

Wei’s laboratory previously constructed an ABEmax-based sgRNA library comprising approximately 820,000 sgRNAs, comprehensively targeting all feasible potential phosphorylation sites (serine, threonine, and tyrosine) in the human genome⁶. In this study, researchers utilized this sgRNA library to conduct high-throughput functional screening in human malignant melanoma A375 cells (Figure 1a), identifying thousands of novel functional sites that regulate the expression of PD-L1 and HLA-I (Figure 1b–d). Through large-scale individual validation, the study confirmed that these sites include novel regulatory sites in both known genes and previously uncharacterized regulatory genes, many of which are closely associated with phosphorylation modifications, particularly genes involved in the IFNγ signaling pathway.

 

In-depth analysis revealed that these mutations exert multifaceted effects, extending beyond their involvement in phosphorylation modification processes. They can alter mRNA or protein stability, DNA binding capacity, protein-protein interactions, and enzymatic catalytic activity, potentially leading to gene inactivation or augmentation. These multi-layered regulatory effects ultimately modulate the expression levels of immune molecules, providing new perspectives for deciphering the dynamic regulation of the tumor immune microenvironment.

Figure 1. High-throughput screening based on the ABEmax system identifies functional sites regulating PD-L1 and HLA-I expression.

 

This study also identified multiple key mutations that simultaneously regulate the expression of PD-L1 and HLA-I, such as the clinically relevant mutation SETD2_Y1666. SETD2 is the primary histone methyltransferase responsible for catalyzing H3K36 trimethylation (H3K36me3). The research found that mutation at the Y1666 site, while not affecting the mRNA and protein expression levels of SETD2, severely impair its catalytic activity of H3K36me3, leading to the synchronous upregulation of PD-L1 and HLA-I. These results suggest that the SETD2_Y1666 mutation may influence tumor immune escape by modulating chromatin states.

 

Further animal experiments demonstrated that in melanoma and colon cancer models, this mutation significantly enhanced the infiltration and cytotoxicity of CD8+ T cells and effectively improved the efficacy of anti-PD-1 therapy (Figure 2a–b). Additionally, in various human tumor cell lines (melanoma A375 and A875, fibrosarcoma HT1080, breast cancer MCF-7), this mutation consistently exhibited immune regulatory effects, highlighting its broad-spectrum value in clinical applications (Figure 2c). This finding aligns with recent clinical studies, which indicate that patients with different cancer types carrying SETD2 inactivation mutations exhibit more pronounced responses to ICB therapy.

 

Figure 2. Representative clinically relevant sites identified through screening and their immune regulatory functions in various tumor models and cell lines.

 

By integrating multi-cohort data from ICGC and COSMIC databases, this study demonstrated that identified key sites show strong clinical relevance, with approximately 40% matching known clinical mutations (Figure 2d). This study establishes the first comprehensive resource of functional regulators modulating cancer immunosurveillance at the amino acid and base levels, providing clinical biomarkers for predicting tumor immune responses and ICB treatment outcomes. These findings offer novel perspectives for clinical diagnosis, ICB therapy, and the development of innovative drugs for cancer treatment.

 

Dr. Ying Liu (Associate Researcher at Changping Laboratory), Dr. Yongshuo Liu (former postdoctoral fellow at Peking University, now Associate Researcher in the Department of Laboratory Medicine at Shandong Cancer Hospital), Xuran Niu (Ph.D. student at the School of Life Sciences, Peking University), Ang Chen (Ph.D. student at the Academy for Advanced Interdisciplinary Studies) and Dr. Yizhou Li (postdoctoral fellow at Changping Laboratory) are co-first authors of this paper. The research was supported by Changping Laboratory, the National Natural Science Foundation of China, the Peking-Tsinghua Center for Life Sciences, the Taishan Scholars Program, and the China Postdoctoral Science Foundation.

 

Paper link: https://www.nature.com/articles/s41392-025-02171-5

 

References

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2     Mezzadra, R. et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature 549, 106-110, doi:10.1038/nature23669 (2017).

3     Suresh, S. et al. eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer. Nat Cancer 1, 533-545, doi:10.1038/s43018-020-0056-0 (2020).

4     Dersh, D. et al. Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas. Immunity 54, 116-131 e110, doi:10.1016/j.immuni.2020.11.002 (2021).

5     Gu, S. S. et al. Therapeutically Increasing MHC-I Expression Potentiates Immune Checkpoint Blockade. Cancer Discov 11, 1524-1541, doi:10.1158/2159-8290.CD-20-0812 (2021).

6     Li, Y. et al. Functional profiling of serine, threonine and tyrosine sites. Nat Chem Biol, doi:10.1038/s41589-024-01731-0 (2024).