Erlotinib Induces Cell Death by Blocking NIX-mediated Mitophagy Through Lysosomal Swelling in IDH1-mutant Cholangiocarcinoma

Background/Aim: Metabolic alterations resulting from mutation in tricarboxylic acid cycle enzymes such as isocitrate dehydrogenase 1 (IDH1) have been observed in various cancers. These alterations can be exploited as therapeutic targets to induce metabolic synthetic lethality. This study aimed to characterize the metabolic signature of cholangiocarcinoma (CCA) carrying an IDH mutation and investigate a novel anti-cancer mechanism of erlotinib in inducing cancer cell death through the regulation of altered metabolism. Materials and Methods: Two CCA cell lines were used: SNU-1079, carrying an IDH1 mutation, and SNU-1196, IDH1 wild-type. Metabolic alterations were validated using liquid chromatography–tandem mass spectrometry. The anti-tumor effects of erlotinib on CCA cell lines were evaluated using cell viability, colony formation, and Annexin V/PI staining assays. Mitochondrial physiology was assessed via microscopy and cytofluorometry following MitoTracker loading. Lysosome swelling was confirmed by detecting cytosolic cathepsin B via western blot and immunocytochemistry using LysoTracker. Results: SNU-1079 cells exhibited a mitochondria-independent metabolic feature, suggesting functional mitochondrial alteration. The mitochondrial membrane potential was disrupted, and mitochondrial length was reduced in SNU-1079 cells. This cell line utilized NIX-mediated mitophagy through hyperactivated epidermal growth factor receptor (EGFR) signaling. Erlotinib inhibited EGFR signaling and induced SNU-1079 cell death by interrupting the mitophagic flux. The blockage of mitophagy by erlotinib was associated with lysosomal swelling, indicated by the presence of cytosolic cathepsin B. Conclusion: The CCA cell line carrying an IDH mutation utilized mitophagy as a novel metabolic compensatory mechanism activated through EGFR-specific signaling. Mitophagy acted as a metabolic synthetic lethality partner to the EGFR inhibitor erlotinib. These findings strongly suggest the potential of erlotinib as a therapeutic strategy for patients with IDH1-mutant CCA.