Rab GTPases drive ligand-independent NOTCH1 activation via altered endocytic trafficking in chronic lymphocytic...

Clinically relevant NOTCH1 activation is frequently observed in chronic lymphocytic leukemia (CLL), even in the absence of gene mutations, raising questions about the underlying mechanisms. In CLL cells, NOTCH1 activation may occur through mutation-independent mechanisms involving ligand interaction or cell-intrinsic, ligand-independent pathways that are not yet fully elucidated. To explore ligand-independent activation, we examined the involvement of NOTCH1 endocytic trafficking in generating the active intracellular domain (N1-ICD) in CLL cells. Using proximity ligation assay, we demonstrated that the NOTCH1 extracellular domain (N1-ECD), transmembrane subunit (N1-TM), and N1-ICD colocalize with Rab5 and Rab7, indicating NOTCH1 internalization and cleavage within endosomal compartments. Experiments with the endocytosis inhibitor Pitstop-2 demonstrated that NOTCH1 internalization is essential for N1-ICD generation. We provided evidence that N1-ICD generation occurs in Rab5 and Rab7 endosomal membranes. Treatment with chloroquine reduced N1-ICD, due to impaired endosomal acidification affecting enzymatic activity. Presenilin-1, the catalytic subunit of the γ-secretase complex responsible for N1-ICD generation, was found in early endosome compartments and colocalized with Rab5, Rab7, N1-TM, and N1-ICD. CLL cells expressing N1-ICD showed higher Rab5, Rab7, and presenilin-1 levels, increased Rab5 membrane association, and presenilin-1 activity versus N1-ICD-negative cells which showed increased lysosomal targeting. Silencing Rab5 or Rab7 by siRNA, or inhibiting Rab prenylation with psoromic acid, led to reduced N1-ICD levels and increased apoptosis in CLL cells. Interestingly, NOTCH1-mutated cases showed similar NOTCH1 trafficking. These findings identify for the first time a Rab-dependent endocytic trafficking as a key regulator of NOTCH1 activation and a potential therapeutic target in CLL.