SUMOylated Reptin maintains low intracellular ROS levels by activating mitophagy to drive gemcitabine resistance in gallbladder cancer.

Gemcitabine resistance remains a major challenge in the treatment of gallbladder cancer (GBC). Here, we elucidate a novel mechanism underlying gemcitabine resistance in GBC, centered on a self-reinforcing mitophagy/ROS/SENP3/Reptin loop. Gemcitabine-resistant GBC cells maintain significantly lower intracellular reactive oxygen species (ROS) levels than wild-type cells through enhanced mitophagy. This finding delineates a novel transcriptional mechanism that drives mitophagy under low ROS conditions. Mechanistically, this low ROS state decreases the protein abundance of the ROS sensor Sentrin/SUMO-specific protease 3 (SENP3), thereby promoting SUMOylation of its substrate, RuvB-like AAA+ ATPase 2 (Reptin), at the K456 site. SUMOylated Reptin translocates to the nucleus, where it acts as a transcriptional activator to specifically upregulate PTEN-induced putative kinase 1 (PINK1), a key mitophagy regulator. Enhanced PINK1 expression further amplifies mitophagy, effectively scavenging ROS and perpetuating the low ROS state that drives resistance, thus sustaining the gemcitabine-resistant phenotype. Clinically, low SENP3 expression and high Reptin expression correlate with poor gemcitabine response and shorter overall survival in GBC patients. Targeting this pathway, the Reptin ATPase inhibitor CB-6644 effectively suppressed PINK1 transcription, inhibited mitophagy, increased ROS accumulation, and reversed gemcitabine resistance both in vitro and in vivo. These findings identify the mitophagy/ROS/SENP3/Reptin loop as a core resistance mechanism in GBC, highlight SENP3 and Reptin as predictive biomarkers, and establish CB-6644 as a promising therapeutic agent to overcome gemcitabine resistance by disrupting this adaptive pathway.