68Targeting the BBOX1-TBK1-mTORC1 axis in clear cell renal cell carcinoma

Abstract Background Clear cell renal cell carcinoma (ccRCC), a metabolic disease originating from renal proximal convoluted tubule (PCT) epithelial cells, remains incompletely understood regarding its initiating signaling events. Methods We use clinical data-driven analysis, combined with pathological validations to identify γ-butyrobetaine hydroxylase 1 (BBOX1), a key enzyme in carnitine synthesis predominantly expressed in PCT cells, as a tumor suppressor in ccRCC. We also conducted substantial in vitro and in vivo functional studies to understand the role of BBOX1 in ccRCC tumorigenesis. In terms of mechanistic studies, we combined a variety of methods, including transcriptomics, metabolomics, and IP-mass spectrometry, to uncover the underlying mechanisms of BBOX1 in regulating metabolism and ccRCC development. Results BBOX1 expression is lost during ccRCC malignant transformation, and its restoration reduces cell viability in physiological medium and inhibits xenograft tumor growth. Transcriptomic analyses reveal that BBOX1 suppresses critical metabolic pathways, including mTORC1 signaling and glycolysis in ccRCC. Further, we identify TANK-binding kinase 1 (TBK1) as an essential mediator of mTORC1 and glycolysis activation and as a target of BBOX1-mediated tumor suppression. Mechanistically, BBOX1 disrupts TBK1 activation by preventing its interaction with the upstream activator doublecortin-like kinase 2 (DCLK2). In a therapeutic approach, we developed a novel TBK1 Proteolysis-targeting chimera (PROTAC) to target ccRCC tumorigenesis. Conclusions This BBOX1-TBK1-mTORC1 axis unveils a previously uncharacterized mechanism in ccRCC metabolic dysregulation and highlights potential therapeutic strategies.