{
  "abstract": "Background Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty (SB) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked.Methods We developed transgenic mice carrying Doxycycline (Dox)-inducible SB mutagenesis system (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8+ T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8+ T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively.Results Under chronic stimulation, SB-mutagenized CD8+ T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2, known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation.Conclusions Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.",
  "authors": [
    {
      "affiliations": [
        "University of Minnesota Twin Cities Department of Genetics Cell Biology and Development, Minneapolis, Minnesota, USA",
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Chang-Jung Lee"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Alex T Larsson"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Tyler A Jubenville"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Wendy A Hudson"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Zach J Seeman"
    },
    {
      "affiliations": [
        "T Cell Engineering Laboratory Program, Mayo Clinic Rochester, Rochester, Minnesota, USA",
        "Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Rochester, Rochester, Minnesota, USA"
      ],
      "name": "Carli Stewart"
    },
    {
      "affiliations": [
        "University of Minnesota Twin Cities Center for Immunology, Minneapolis, Minnesota, USA",
        "Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota Twin Cities, Minneapolis, Minnesota, USA"
      ],
      "name": "Alexander K Tsai"
    },
    {
      "affiliations": [
        "University of Minnesota Twin Cities Center for Immunology, Minneapolis, Minnesota, USA",
        "University of Minnesota Twin Cities Department of Microbiology and Immunology, Minneapolis, Minnesota, USA"
      ],
      "name": "Adam Burrack"
    },
    {
      "affiliations": [
        "T Cell Engineering Laboratory Program, Mayo Clinic Rochester, Rochester, Minnesota, USA",
        "Division of Hematology, Mayo Clinic Rochester, Rochester, Minnesota, USA"
      ],
      "name": "Brooke L Kimball"
    },
    {
      "affiliations": [
        "T Cell Engineering Laboratory Program, Mayo Clinic Rochester, Rochester, Minnesota, USA",
        "Division of Hematology, Mayo Clinic Minnesota, Rochester, Minnesota, USA"
      ],
      "name": "Elizabeth L Siegler"
    },
    {
      "affiliations": [
        "Department of Immunology, Mayo Clinic Rochester, Rochester, Minnesota, USA"
      ],
      "name": "Vianca V Vianzon"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Erin E Nolan"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Yu-Ling Yang"
    },
    {
      "affiliations": [
        "University of Minnesota Twin Cities Department of Genetics Cell Biology and Development, Minneapolis, Minnesota, USA",
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Christopher M Stehn"
    },
    {
      "affiliations": [
        "University of Minnesota Twin Cities Department of Genetics Cell Biology and Development, Minneapolis, Minnesota, USA",
        "University of Minnesota Twin Cities Genomics Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Daryl M Gohl"
    },
    {
      "affiliations": [
        "University of Minnesota Twin Cities Genomics Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Margaret Donovan"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA",
        "University of Minnesota Twin Cities Institute for Health Informatics, Minneapolis, Minnesota, USA"
      ],
      "name": "Nuri A Temiz"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Flavia E Popescu"
    },
    {
      "affiliations": [
        "Genentech Inc, South San Francisco, California, USA"
      ],
      "name": "Søren Warming"
    },
    {
      "affiliations": [
        "Genentech Inc, South San Francisco, California, USA",
        "Department of Research, MedGenome Inc, Foster City, California, USA"
      ],
      "name": "Somasekar Seshagiri"
    },
    {
      "affiliations": [
        "Mouse Genetics Laboratory, University of Minnesota Twin Cities, Minneapolis, Minnesota, USA"
      ],
      "name": "Yun You"
    },
    {
      "affiliations": [
        "Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, Iowa, USA",
        "Department of Anatomy and Cell Biology, University of Iowa Hospitals and Clinics, Iowa city, Iowa, USA"
      ],
      "name": "Jesse D Riordan"
    },
    {
      "affiliations": [
        "Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, Iowa, USA",
        "Department of Anatomy and Cell Biology, University of Iowa Hospitals and Clinics, Iowa city, Iowa, USA"
      ],
      "name": "Adam Dupuy"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "Branden Moriarity"
    },
    {
      "affiliations": [
        "Department of Immunology, Mayo Clinic Rochester, Rochester, Minnesota, USA"
      ],
      "name": "Laura Rogers"
    },
    {
      "affiliations": [
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA",
        "University of Minnesota Twin Cities Center for Immunology, Minneapolis, Minnesota, USA"
      ],
      "name": "Ingunn Stromnes"
    },
    {
      "affiliations": [
        "Department of Immunology, Mayo Clinic Rochester, Rochester, Minnesota, USA",
        "Division of Hematology, Department of Medicine, Mayo Clinic Rochester, Rochester, Minnesota, USA"
      ],
      "name": "Saad Kenderian"
    },
    {
      "affiliations": [
        "University of Minnesota Twin Cities Department of Genetics Cell Biology and Development, Minneapolis, Minnesota, USA",
        "University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota, USA"
      ],
      "name": "David Largaespada"
    }
  ],
  "title": "Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8+ T-cell exhaustion, persistence in vivo, and CAR-T cell function under tumor-associated chronic antigen stimulation",
  "uid": "b58d5f6b-49e0-5f27-8ddd-5ae8d93d04c4"
}