{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T18:14:39Z","timestamp":1788545679347,"version":"build-2803163510"},"reference-count":0,"publisher":"American Society of Hematology","content-domain":{"domain":["ashpublications.org"],"crossmark-restriction":true},"short-container-title":[],"abstract":"<jats:p>CAR T-cells targeting CD19 have revolutionized the treatment of relapsed\/refractory B-cell malignancies. However, approximately 50% of patients relapse. Patient-derived data indicates that CD19+ relapse is linked to poor CAR T-cell proliferation and\/or persistence. We aimed to improve CD19-CAR T-cell survival by manipulating Notch1, a receptor that is activated following T-cell receptor ligation and is essential for upregulating pro-inflammatory and survival genes in T-cells. However, its role in the context of CAR T-cells remains poorly understood. We found that both lentiviral overexpression of Notch1's intracellular domain (N1ICD) and Notch1 genetic knockout resulted in defective CAR-T cell activation and proliferation. This suggests that CAR T-cells require a precise, balanced level of Notch1 signaling for strong effector function. The PEST domain, located at the most distal C-terminus of the Notch1 intracellular domain, regulates the receptor's half-life through ubiquitylation. To augment the levels of active Notch1 and prolong the receptor's signaling, we genetically deleted this domain in the endogenous N1ICD of CAR T-cells. Notch1 PEST-deleted CAR T-cells showed enhanced proliferation post-activation, leading to increased cytotoxicity against CD19+ tumors in vitro. Transcriptionally, Notch1 PEST deletion resulted in upregulation of interferon response pathways and proliferative genes during CAR activation, consistent with Notch1 activation. In B-ALL xenograft in vivo models, treatment with Notch1 PEST-deleted CAR T-cells resulted in greater tumor reduction and increased CAR-T expansion compared to control CAR T-cells. Our findings demonstrate a critical role of Notch1 in CAR-T effector function and provide a novel strategy to augment endogenous Notch1 activity by leveraging native gene regulation.<\/jats:p>","DOI":"10.1182\/blood.2026033694","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T17:18:48Z","timestamp":1788369528000},"update-policy":"https:\/\/doi.org\/10.1182\/blood.2019cm0000","source":"Crossref","is-referenced-by-count":0,"title":["Rational Engineering of Notch1 Signaling to Boost the Proliferation of CD19-BB\u03b6 CAR-T"],"prefix":"10.1182","author":[{"given":"Merle Kincaid","family":"Phillips","sequence":"first","affiliation":[{"name":"Mass General Brigham, Boston, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5214-233X","authenticated-orcid":false,"given":"Filippo","family":"Birocchi","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felix","family":"Korell","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Charlestown Navy Yard, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diego","family":"Salas-Benito","sequence":"additional","affiliation":[{"name":"Mass General Brigham, Boston, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1379-0876","authenticated-orcid":false,"given":"Viktoria","family":"Blumenberg","sequence":"additional","affiliation":[{"name":"Cellular Immunotherapy Program, Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, Charlestown, Massachusetts, United 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States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3726-1840","authenticated-orcid":false,"given":"Valentina M","family":"Supper","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Boston, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4818-2082","authenticated-orcid":false,"given":"Kathleen ME","family":"Gallagher","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1175-521X","authenticated-orcid":false,"given":"Stefanie R","family":"Bailey","sequence":"additional","affiliation":[{"name":"United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trisha R","family":"Berger","sequence":"additional","affiliation":[{"name":"MGH \/ Harvard Medical School, Charlestown, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3111-0221","authenticated-orcid":false,"given":"Mark B","family":"Leick","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Boston, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1532-4414","authenticated-orcid":false,"given":"Adele","family":"Mucci","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Charelstown, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7578-0393","authenticated-orcid":false,"given":"Marcela V.","family":"Maus","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"234","published-online":{"date-parts":[[2026,9,2]]},"container-title":["Blood 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