AbstractCaMKK2 is an enzyme which has been implicated in PCa tumorigenesis and progression by the regulation of glycolysis through activation of its substrate AMPK. However phenotypic effects observed in CaMKK2 KO mice and cell lines with CaMKK2 inhibition or silencing were not rescued by AMPK, suggesting that activation of AMPK is not the only physiologically relevant function of CaMKK2 in PCa. It has potential as a possible therapeutic target in androgen responsive and castrate resistant disease but the only commercially available inhibitor, STO-609, exhibits promiscuity and direct inhibition of AMPK. Thus, to progress the understanding and druggability of CaMKK2 in PCa we investigated novel interactors and functions of the kinase and developed novel inhibitors to improve on STO-609.
We have identified and validated two novel interactors of CaMKK2; Gemin4 and ARCN1. Gemin4 is a member of the SMN complex whose function is in miRNA biogenesis and formation of the spliceosome. ARCN1 is the delta subunit of the COPI coatomer complex which traffics proteins from the Golgi to the ER. The Gemin4 interaction is mediated by the Novel CaMKK2 Binding Motif; a nine aa consensus sequence present within Gemin4 which mediates interactions of import to the CaMKK2 and Gemin4 interactome. ARCN1 is dependent on the presence of CaMKK2 for its activity.
We have identified a role of CaMKK2 in PCa in modulating the balance between protein trafficking and organelle maintenance. CaMKK2 depletion in an androgen responsive setting results in increased Golgi disorganisation, ER expansion, abortive autophagy, impaired lysosomal acidification, elongation of mitochondria and an increase in ROS. This is likely mediated via interaction with ARCN1 and subsequent impact of COPI coatomer complex stability.
CaMKK2 does have a role in metabolic regulation in PCa cells. However, we have found that this is mediated via its role in maintenance of organelle function rather than via AMPK. CaMKK2 depletion has no effect on p-AMPK levels and also results in increased glucose uptake and dysregulated metabolism which shifts toward a more glycolytic phenotype.
We have developed two novel inhibitors of CaMKK2 which show increased potency and selectivity when compared to STO-609 in both LNCaP and PC3 with regards to viability, proliferation and cell death (Inhibitor #2 and A050). We observed synergy when Inhibitor #2 was combined with the anti-diabetic drug metformin in both LNCaP and PC3 and observed synergy when A050 was combined with metformin in PC3.
The novel functions of CaMKK2 identified in this project show that CaMKK2 is not simply a metabolic regulator in PCa but a central hub through which protein trafficking and organelle maintenance are balanced. Further investigation is required but will have interesting implications in the prostate cancer field and beyond as CaMKK2 inhibition studies have largely focused on reducing CaMKK2 in the brain to treat obesity. The two novel inhibitors identified in this study have promising in vitro effects and will require further investigation to validate effects on an in vivo model system.
|Date of Award||Jul 2020|
|Supervisor||David Waugh (Supervisor), Ian Mills (Supervisor) & Emma Evergren Mills (Supervisor)|