Phospho-reprogramming mitochondrial respiration underlies sunitinib resistance in renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. While surgery is effective for localized ccRCC, advanced disease necessitates the use of additional therapies such as immune checkpoint inhibitors or tyrosine kinase inhibitors (TKIs). Emergent resistance limits TKI efficacy, and prognosis in patients with refractory disease is poor. Recent work indicates that metabolic reprogramming underlies TKI resistance, however the details are poorly understood. Phosphoproteomics study has shown that sunitinib-resistant ccRCC tumors exhibit differential phosphorylation of the electron transport chain Complex II subunit SDHA and the mitochondrial chaperone TRAP1. TRAP1 controls metabolic flux by regulating the activity of the mitochondrial respiratory machinery, indicating a role for TRAP1 phosphorylation in tuning this activity. The objective of our work was to determine the impact of SDHA & TRAP1 phosphorylation on metabolism and TKI resistance.Transient transfection of SDHA phospho-mutants in wild-type 293 or TRAP1 phospho-mutants in TRAP1 knockout 293 cells was used to model phosphorylation changes in SDHA and TRAP1. Immunoblotting and immunoprecipitation were used to evaluate protein expression and interaction. Seahorse Mito stress test analysis was used to measure the respiratory capacity of 293 cells expressing TRAP1 and SDHA phospho-mutants, while MTT assay was used to measure the viability of 293 cells expressing TRAP1 phospho-mutants in response to TKI treatment. MTT assay was additionally used to measure proliferation of naïve and TKI-resistant normal kidney and ccRCC cell lines upon TRAP1 inhibition with Gamitrinib-TPP. We observed that expression of TRAP1 and SDHA phospho-mutants modulated respiration and impacted sensitivity to the TKIs sunitinib and cabozantinib, suggesting a common resistance mechanism. Furthermore, sunitinib-resistant ccRCC cell lines exhibited differing sensitivity to a small molecule TRAP1 inhibitor, suggesting a potential therapeutic intervention. Post-translational modifications regulate the activity of many metabolic proteins, including TRAP1 and SDHA, thereby tuning cellular metabolic flux. Differential phosphorylation of these proteins is correlated with ccRCC sensitivity to sunitinib, and metabolic dysregulation underlies TKI resistance. We found that blocking phosphorylation of SDHA and TRAP1 suppressed respiration, and TRAP1 phospho-null mutation decreased cell sensitivity to TKIs. Furthermore, we observed that sunitinib-resistant ccRCC cell lines demonstrated increased sensitivity to TRAP1 inhibition. Collectively, our work demonstrates the potential for novel combination therapies for targeting TKI-resistant ccRCC.