Seung Un Seo ^a,1, Seon Min Woo ^a,1, Seul Gi Lee ^a, Min Yeong Kim ^b, Hyun Shik Lee ^c, Yung Hyun Choi ^b, Sang Hyun Kim ^d, Young-Chae Chang ^e, Kyoung-jin Min ^f, Taeg Kyu Kwon ^a,g,*

^a Department of Immunology, School of Medicine, Keimyung University, Daegu, 42601, South Korea ^b Department of Biochemistry, College of Korean Medicine, Dong-Eui University, Busan, 47227, South Korea ^c School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daegu, 41566, South Korea ^d Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, 41944, South Korea ^e Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu, 42472, South Korea ^f New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu, 41061, South Korea ^g Center for Forensic Pharmaceutical Science, Keimyung University, Daegu, 42601, South Korea

^* Corresponding author.
¹ These authors contributed equally to the present study.

Cathepsin K inhibitor (odanacatib; ODN) and cathepsin K knockdown (siRNA) enhance oxaliplatin-induced apoptosis through p53-dependent Bax upregulation. However, its underlying mechanisms remain unclear. In this study, we elucidated the mechanism behind enhancement of oxaliplatin-induced apoptosis by ODN. We also investigated the molecular mechanisms of ODN-induced Bax upregulation. Here, we demonstrated that ODN-induced Bax upregulation required p53, but it was independent of p53 transcriptional activity. Various mutants of the DNA-binding domain of p53 induced Bax upregulation in ODN-treated cells. p53 functional domain analysis showed that the C-terminal domain of p53 participates in the physical interaction and stabilization of Sp1, a major transcription factor of Bax. We screened a specific siRNA encoding 50 deubiquitinases and identified that BAP1 stabilizes Sp1. The knockdown or catalytic mutant form of BAP1 abolished the ODN-induced upregulation of Sp1 and Bax expression. Mechanistically, ODN induced BAP1 phosphorylation and enhanced Sp1-BAP1 interaction, resulting in Sp1 ubiquitination and degradation. Interestingly, ODN-induced BAP1 phosphorylation and DNA damage were modulated by the production of mitochondrial reactive oxygen species (ROS). Mitochondrial ROS scavengers prevented DNA damage, BAP1-mediated Sp1 stabilization, and Bax upregulation by ODN. BAP1 downregulation by siRNA inhibited apoptosis induced by the combined treatment of ODN and oxaliplatin/etoposide. Therefore, Sp1 is a crucial transcription factor for ODN-induced Bax upregulation, and Sp1 stabilization is regulated by BAP1.