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In CLL cells, NOTCH1 activation may occur through mutation\u2010independent mechanisms involving ligand interaction or cell\u2010intrinsic, ligand\u2010independent pathways that are not yet fully elucidated. To explore ligand\u2010independent activation, we examined the involvement of NOTCH1 endocytic trafficking in generating the active intracellular domain (N1\u2010ICD) in CLL cells. Using proximity ligation assay, we demonstrated that the NOTCH1 extracellular domain (N1\u2010ECD), transmembrane subunit (N1\u2010TM), and N1\u2010ICD colocalize with Rab5 and Rab7, indicating NOTCH1 internalization and cleavage within endosomal compartments. Experiments with the endocytosis inhibitor Pitstop\u20102 demonstrated that NOTCH1 internalization is essential for N1\u2010ICD generation. We provided evidence that N1\u2010ICD generation occurs in Rab5 and Rab7 endosomal membranes. Treatment with chloroquine reduced N1\u2010ICD, due to impaired endosomal acidification affecting enzymatic activity. Presenilin\u20101, the catalytic subunit of the \u03b3\u2010secretase complex responsible for N1\u2010ICD generation, was found in early endosome compartments and colocalized with Rab5, Rab7, N1\u2010TM, and N1\u2010ICD. CLL cells expressing N1\u2010ICD showed higher Rab5, Rab7, and presenilin\u20101 levels, increased Rab5 membrane association, and presenilin\u20101 activity versus N1\u2010ICD\u2010negative cells which showed increased lysosomal targeting. Silencing Rab5 or Rab7 by siRNA, or inhibiting Rab prenylation with psoromic acid, led to reduced N1\u2010ICD levels and increased apoptosis in CLL cells. Interestingly, NOTCH1\u2010mutated cases showed similar NOTCH1 trafficking. These findings identify for the first time a Rab\u2010dependent endocytic trafficking as a key regulator of NOTCH1 activation and a potential therapeutic target in CLL.<\/jats:p>","DOI":"10.1002\/hem3.70440","type":"journal-article","created":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T13:40:35Z","timestamp":1785246035000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rab GTPases drive ligand\u2010independent NOTCH1 activation via altered endocytic trafficking in chronic lymphocytic leukemia"],"prefix":"10.1002","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4008-2752","authenticated-orcid":false,"given":"Filomena","family":"De Falco","sequence":"first","affiliation":[{"name":"Department of Medicine and Surgery Institute of Hematology and Center for Hemato\u2010Oncology Research (CREO), University of Perugia and Santa Maria della Misericordia Hospital Perugia 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