Nutlin-3a is an MDM2 antagonist that inhibits the interaction between MDM2 and p53, thereby activating p53 and modulating various cellular stress responses. In our previous study, we showed that nutlin-3a suppresses the hexosamine biosynthetic pathway via GFPT2 downregulation, leading to lysosomal dysfunction and impaired autophagosome–lysosome fusion in KRAS mutant/p53 wild-type lung cancer cells. The SNARE complex—comprising STX17 on autophagosomes, VAMP8 on lysosomes, and SNAP29 bridging the two—mediates the fusion of autophagosomes and lysosomes, the final step of autophagy, known as autophagic flux. Additionally, N-/O-glycosylation of lysosomal proteins has been proposed to potentially influence the stability and interactions of the SNARE complex. Therefore, we investigated the underlying mechanism of nutlin-3a–induced disruption of autophagosome–lysosome fusion.

Localization of autophagosome and lysosome was visualized by confocal microscopy after staining with their respective markers such as LC3 and LAMP1. Expression levels of lysosomal and autophagosome-related molecules along with SNARE complex components were measured by RT-qPCR and western blotting. Further analysis of SNARE complex interactions as well as N-/O-glycosylation of relevant molecules was examined by immunoprecipitation.

Nutlin-3a treatment markedly reduced SNAP29 expression, as confirmed by RT-qPCR and western blotting. Immunoprecipitation analysis further demonstrated an increase in N-/O-glycosylation levels of LAMP1 and LAMP2, along with a significant reduction in the interactions among SNAP29, STX17, and VAMP8. These findings suggest that SNAP29 reduction and glycosylation-modified lysosomes contribute to the disruption of SNARE complex formation, thereby impairing autophagosome–lysosome fusion. Confocal microscopy corroborated these findings by showing reduced SNAP29–VAMP8 colocalization. Additionally, nutlin-3a treatment induced an increase in Galectin-3, a marker of lysosomal membrane permeabilization, while western blot analysis showed TFEB activation and increased expression of CTSB, LAMP1, and LAMP2. Collectively, these results suggest a compensatory mechanism attempting to counteract nutlin-3a–induced lysosomal dysfunction.

Our results demonstrate that nutlin-3a disrupts autophagosome-lysosome fusion by downregulating SNAP29 and impairing SNARE complex assembly in KRAS mutant/p53 wild-type lung cancer cells.