UVRAG-ITCH-ESCRT axis mediates a lysosomal off-switch dampening NOTCH1 signaling in cancer

NOTCH1 signaling is a conserved pathway essential for cell fate decisions, and its dysregulation drives multiple cancers such as T-cell acute lymphoblastic leukemia (T-ALL). Precise control of NOTCH1 signal strength and duration is critical, yet mechanisms fine-tune NOTCH1 signaling remains poorly understood. Here we identify a previously unrecognized endo-lysosomal “off-switch” for NOTCH1 mediated by the UVRAG-ITCH axis. We demonstrate that UVRAG (UV radiation resistance-associated gene), traditionally known for its role in autophagy, acts independently of autophagy to negatively regulate NOTCH1 signaling. UVRAG physically engages the membrane-tethered NOTCH1 extracellular truncation (NOTCH1ΔE), the activated intermediate generated after ligand-induced or mutational S2 cleavage, and recruits the E3 ubiquitin ligase ITCH. The UVRAG-ITCH complex catalyzes non-canonical Lys27-linked ubiquitination of NOTCH1ΔE at a conserved lysine (K1795) in its RAM domain, thereby tagging this intermediate for recognition by endosomal sorting complexes (ESCRT) and routing to lysosomes for degradation. Consequently, UVRAG-ITCH-mediated degradation of NOTCH1ΔE limits its availability for gamma-secretase (S3) cleavage, reducing the generation of the NOTCH1 intracellular domain (NICD) and attenuating downstream transcriptional activity. Disruption of this UVRAG-ITCH-ESCRT checkpoint leads to accumulation of NOTCH1ΔE and excessive NICD production. In T-ALL cells harboring hyperactive NOTCH1 mutations, we found that UVRAG expression is often suppressed. Restoration of UVRAG in T-ALL models reinstated the lysosomal off-switch: it promoted NOTCH1ΔE ubiquitination and degradation, lowered NICD levels, and dampened oncogenic NOTCH1 signaling. UVRAG re-expression in these cells significantly impaired leukemia cell proliferation and self-renewal, reduced the population of leukemia-initiating cells, and even enabled partial differentiation of malignant T-cell blasts. Notably, reinstating UVRAG also sensitized T-ALL cells to gamma-secretase inhibitors (GSIs). The combination of UVRAG reactivation with GSI treatment synergistically induced apoptosis and tumor regression. In xenograft and patient-derived T-ALL models, enforced UVRAG expression combined with GSI therapy dramatically reduced leukemic burden and prolonged survival, underscoring the therapeutic potential of co-targeting this pathway. In conclusion, our study reveals that the UVRAG-ITCH axis functions as a lysosomal off-switch to terminate aberrant NOTCH1 signaling at the membrane intermediate stage. This degradation-based checkpoint offers a novel therapeutic target to restrain NOTCH1-driven malignancies and potentially enhance the efficacy of NOTCH1 pathway inhibitors.