Checkpoint control of NOTCH via K27-linked ubiquitination by the UVRAG-ITCH-ESCRT pathway

NOTCH signaling is a highly conserved intercellular communication pathway essential for cell fate determination and tissue homeostasis, and its dysregulation contributes to diverse human cancers, including T-cell acute lymphoblastic leukemia (T-ALL)1,2. Although the proteolytic activation of the NOTCH1 receptor is well characterized1, little is known about the mechanisms that regulate the signaling amplitude and response dynamics. Here, we defined the molecular basis by which the activated membrane-tethered NOTCH1 intermediate (NOTCH1ΔE) undergoes stringent checkpoint control mediated by UV radiation resistance-associated gene (UVRAG). We demonstrate that UVRAG acts in concert with the E3 ubiquitin ligase ITCH to specifically bind NOTCH1ΔE and facilitate K27-linked ubiquitination of NOTCH1ΔE at a conserved lysine residue. This ubiquitination directs NOTCH1ΔE toward degradation via the ESCRT-lysosome pathway, limiting the availability of NOTCH1ΔE for subsequent γ-secretase cleavage and nuclear signaling activation. Disruption of the UVRAG-ITCH-ESCRT axis stabilizes NOTCH1ΔE and exacerbated oncogenic signaling in T-ALL with activating NOTCH1 mutations. Re-expression of UVRAG, suppressed in T-ALL, leads to attenuated NOTCH signaling, inhibited leukemia progression, diminished leukemia-initiating cell populations, and enhanced sensitivity to γ-secretase inhibitor therapies across xenograft, patient-derived, and NOTCH1-drivien murine models of T-ALL. Collectively, our findings uncover a previously unappreciated endo-lysosomal control mechanism governing NOTCH1 signaling activation, and highlight the UVRAG–ITCH–ESCRT axis as a promising therapeutic target in NOTCH-dependent malignancies