{
  "abstract": "Background Adoptive T cell immunotherapy has shown curative potential in cancers once considered untreatable. However, its effectiveness in solid tumors is often limited by multifactorial T cell dysfunction, particularly terminal exhaustion and mitochondrial impairment. These dysfunctions are interconnected and difficult to reverse by targeting a single pathway. To overcome this challenge, there is a need to uncover master regulators that can simultaneously modulate multiple aspects of T cell biology to help restore durable anti-tumor activity.Methods Using a systems immunology-driven approach, we analyzed hundreds of transcriptomic datasets from exhausted T cells in cancer and chronic infections. This guided design of an in vivo CRISPR-Cas9 screen targeting components of protein homeostasis and degradation pathways to identify regulators of T cell exhaustion. In parallel, we conducted a computation-guided in vivo CRISPR screen focused on proteostasis regulators correlated with mitochondrial dysfunction using single-cell RNA-seq data from TILs of 316 patients across 21 cancer types and bulk RNA-seq from mouse ACT models. Integrative analysis of both the analyses and screens identified a previously uncharacterized E3 ubiquitin ligase with dual roles in exhaustion and mitochondrial regulation. Functional validation was performed by using genetic perturbation, proteomics, metabolic assays, single-cell RNA-seq, and multiple ACT models.Results An E3 ligase emerged as a critical node in regulation of T cell function/differentiation. Chronic TCR stimulation suppressed its expression, impairing its ability to mediate Lys48-linked ubiquitination of TOX, a master transcription factor in exhaustion. The resulting TOX accumulation disrupted progenitor-to-terminal Tex subset balance. Concurrently, loss of this E3 ligase led to excessive accumulation of the mitochondrial phosphatase PGAM5, driving Drp1-dependent mitochondrial fragmentation and collapse of mitochondrial respiration and ATP production. Genetic deletion of the E3 ligase exacerbated exhaustion and impaired anti-tumor function. Conversely, enforced E3 ligase expression restored mitochondrial fitness, reduced TOX protein levels, and significantly improved T cell persistence and therapeutic efficacy in vivo. Single-cell analyses of human tumors supported clinical relevance across diverse cancers.Conclusions We have identified a multifunctional E3 ligase that is a key regulator coordinating both transcriptional and metabolic axes of T cell function/dysfunction. Enforced expression of this E3 ligase offers a novel strategy to mitigate T cell exhaustion and mitochondrial dysfunction and enhance adoptive T cell therapy. These findings uncover a proteostasis checkpoint in T cell biology and point to new opportunities for next-generation cancer immunotherapies.",
  "authors": [
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Yapeng Su"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Hongcheng Cheng"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Xiaoli Pan"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Yue Xu"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Jing Du"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA"
      ],
      "name": "Daniel Chen"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Xiaomeng Dai"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Ermei Xie"
    },
    {
      "affiliations": [
        "University Hospital of Lausanne, Lausanne, Lausanne, Switzerland"
      ],
      "name": "Raphael Gottardo"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Research Center, Seattle, WA, USA",
        "University of Washington School of Medicine, Seattle, WA, USA",
        "Departments of Immunology and Medicine, University of Washington, Seattle, WA, USA"
      ],
      "name": "Philip D Greenberg"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Suzhou Institute of Systems Medicine, Suzhou, Jiang Su, China"
      ],
      "name": "Guideng Li"
    }
  ],
  "title": "364 Systems immunology-guided in vivo CRISPR screens reveal a multifunctional E3 ligase that enhances adoptive T cell therapy for solid tumors",
  "uid": "0ed17876-6e69-55b6-9a25-4083ebc1f0e2"
}