Dynamic O-GlcNAcylation coordinates etoposide-triggered tumor cell pyroptosis by regulating p53 stability.

O-GlcNAcylation, a modification of nucleocytoplasmic proteins in mammals, plays a critical role in various cellular processes. However, the interplay and their underlying mechanisms in chemotherapy-induced tumor regression between O-GlcNAcylation and pyroptosis, a form of programmed cell death associated with innate immunity, remains unclear. Here, we observed that during the etoposide-induced pyroptosis of SH-SY5Y and A549 cells, overall O-GlcNAcylation levels are substantially reduced. Pharmacological inhibition or genetic manipulation of O-GlcNAcylation, such as OGT inhibition or OGA overexpression, sensitized these cells to etoposide-induced pyroptosis both in vitro and in vivo. Mechanistically, mutations at S96 and S149 residues attenuated p53 O-GlcNAcylation, weakening its interaction with MDM2, reducing p53 ubiquitination, and increasing protein stability. These results suggest that S96 may be a putative O-GlcNAcylation site. Therefore, p53 target genes-Fas, DR-5, Puma, and PIDD-were transcriptionally upregulated, leading to activation of the caspase-3-GSDME axis and promoting etoposide-induced pyroptosis in various tumor cells. This study demonstrates a previously uncharacterized association between O-GlcNAcylation and chemotherapy-induced pyroptosis, offering potential therapeutic interventions for pyroptosis-related diseases.