Mitochondria serve as the primary source of intracellular reactive oxygen species (ROS), which play a critical role in orchestrating cell death pathways such as pyroptosis in various types of cancers. PINK1-mediated mitophagy effectively removes damaged mitochondria and reduces detrimental ROS levels, thereby promoting cell survival. However, the regulation of pyroptosis by PINK1 and ROS in neuroblastoma remains unclear. In this study, we demonstrate that inhibition or deficiency of PINK1 sensitizes ROS signaling and promotes pyroptosis in neuroblastoma cells via the BAX-caspase-GSDME signaling pathway. Specifically, inhibition of PINK1 by AC220 or knockout ofimpairs mitophagy and enhances ROS production, leading to oxidation and oligomerization of TOMM20, followed by mitochondrial recruitment and activation of BAX. Activated BAX facilitates the release of CYCS (cytochrome c, somatic) from the mitochondria into the cytosol, activating CASP3 (caspase 3). Subsequently, activated CASP3 cleaves and activates GSDME, inducing pyroptosis. Furthermore, inhibition or deficiency of PINK1 potentiates the anti-tumor effects of the clinical ROS-inducing drug ethacrynic acid (EA) to inhibit neuroblastoma progression. Therefore, our study provides a promising intervention strategy for neuroblastoma through the induction of pyroptosis.AC220, quizartinib; ANOVA, analysis of variance; ANXA5, annexin A5; BAX, BCL2 associated X, apoptosis regulator; BAK1, BCL2 antagonist/killer 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; COX4/COX IV, cytochrome c oxidase subunit 4; CS, citrate synthase; CSC, cancer stem cell; CYCS, cytochrome c, somatic; DTT, dithiothreitol; DNA, deoxyribonucleic acid; EA, ethacrynic acid; Fer-1, ferroptosis inhibitor ferrostatin-1; FLT3, fms related tyrosine kinase 3; GSDMD, gasdermin D; GSDME, gasdermin E; kDa, kilodalton; LDH, lactate dehydrogenase; MFN1, mitofusin 1; MFN2, mitofusin 2; mito, mitochondria; mito-ROS, mitochondrial ROS; mtKeima, mitochondria-targeted monomeric keima-red; ml, microliter; MT-CO2, mitochondrially encoded cytochrome c oxidase II; NAC, antioxidant N-acetyl-L-cysteine; Nec-1, necroptosis inhibitor necrostatin-1; OMA1, OMA1 zinc metallopeptidase; OMM, outer mitochondrial membrane; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PI, propidium iodide; PINK1, PTEN induced kinase 1; PRKN/Parkin, parkin RBR E3 ubiquitin protein ligase; Q-VD, Q-VD-OPH; ROS, reactive oxygen species; sg, single guide; sh, short hairpin; STS, staurosporine; TOMM20, translocase of outer mitochondrial membrane 20; TIMM23, translocase of inner mitochondrial membrane 23; μm, micrometer; μM, micromolar. PINK1in vivo Abbreviation:
