{
  "abstract": "Background CD19-targeted CAR T-cell therapies have shown success in treating B-cell malignancies, with response rates up to 90%. However, up to half of patient’s relapse, often with CD19+ disease, suggesting T-cell intrinsic deficits. Prior studies indicate that CAR T-cell persistence and proliferation are key predictors of efficacy. Genetic modifications that mimic hyperproliferative states, such as TET2 loss or overexpression of BATF, have been explored to enhance CAR T-cell function. Notch1, a key regulator of T-cell development and proliferation, is upregulated post-TCR stimulation and has been implicated in T-cell leukemia via mutations to its PEST domain, which regulates protein degradation. While canonical Notch1 signaling has been shown to improve CAR T-cell metabolism and memory phenotype, its precise modulation in engineered CD8+ T-cells remains understudied.Methods CD19-BBz CAR T-cells were engineered with CRISPR-Cas9 to knockout Notch1 or truncate the endogenous PEST domain. Lentiviral vectors were used for CAR and Notch1 intracellular domain (N1ICD) overexpression for transgene delivery. T-cell proliferation, cytotoxicity, and activation markers were assessed via flow cytometry, luciferase reporters, cytokine secretion assays, and live cell imaging. CAR T-cell function was evaluated in vitro and in NALM6 xenograft murine models.Results Notch1 knockout in CD19-BBz CAR T-cells impaired proliferation, cytokine production, and cytotoxicity, consistent with previous findings in CD8+ T cells. Surprisingly, constitutive overexpression of N1ICD also impaired CAR expression, viability, and function. In contrast, truncation of the Notch1 PEST domain—mimicking T-ALL mutations—prolonged N1ICD stability post-activation, resulting in enhanced proliferation, survival, cytokine secretion, and tumor cell killing. These CAR T-cells persisted better in vivo and improved leukemia clearance and mouse survival.Conclusions Notch1 signaling must be tightly regulated for optimal CAR T-cell function. Both knockout and overexpression impair effector activity, underscoring the need for precise modulation. Truncating the PEST domain stabilizes N1ICD in an activation-dependent manner, enhancing CAR T-cell proliferation and anti-tumor efficacy. These findings identify Notch1 PEST editing as a promising strategy to boost CAR T-cell function and highlight the broader utility of Cas9-mediated domain editing in cell therapy.",
  "authors": [
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Charlestown, MA, USA",
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA"
      ],
      "name": "Merle K Phillips"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital, Boston, MA, USA"
      ],
      "name": "Filippo Birocchi"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Felix Korell"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Diego Salas-Benito"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Charlestown, MA, USA",
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA"
      ],
      "name": "Giulia Escobar"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Amanda A Bouffard"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Magdi Elsallab"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Charlestown, MA, USA",
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital, Boston, MA, USA"
      ],
      "name": "Charlotte Graham MBChB"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Yueyang Fan"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Hana Takei"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Alexander J Armstrong"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Aiyana Parker"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Valentina M Supper"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Kathleen M Gallagher"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Stefanie R Bailey"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Trisha R Berger"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital Cancer Center, Boston, MA, USA"
      ],
      "name": "Adele Muci"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital, Boston, MA, USA"
      ],
      "name": "Mark B Leick"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Broad Institute of MIT and Harvard, Cambridge, MA, USA",
        "Massachusetts General Hospital, Charlestown, MA, USA"
      ],
      "name": "Marcela V Maus"
    }
  ],
  "title": "265 Augmenting notch1 to enhance CD19-BBz CAR-T therapy",
  "uid": "2db84908-fe6b-58b5-95ae-24ed4f8fac1b"
}