AbstractThe PI3K-Akt signalling pathway is central to cell proliferation and growth, and hyperactivating mutations in this pathway are found in a large number of cancers. Downstream of PI3K, Akt is heavily studied, however the highly related SGK family of protein kinases have recently been identified as driving cancer cell growth in certain contexts. In particular, my research interest and the focus of this thesis is the role these kinases, in particular SGK3, has in resistance to Class I PI3K and Akt inhibitors in certain ER+ breast cancers.
The first major aim of my PhD was to develop a method by which the signalling of SGK3 can be specifically modulated in cancer cells. Due to the high homology between the SGK family of protein kinases, chemical inhibition of SGK3 specifically has not been possible. Further, model cancer cell lines have been intractable to genome modification. I therefore, in collaboration with the Ciulli lab, designed and characterised SGK3-PROTAC1, a Proteolysis Targeting Chimera (PROTAC) which targeted SGK3 for specific ubiquitylation by VHL E3 ligase and subsequent proteasomal degradation. I demonstrated by quantitative Mass Spectrometry that SGK3-PROTAC1 mediated degradation was highly specific. Rapid, reversible degradation of SGK3 provided a tool for specifically modulating SGK3 in cancer cells. Further analysis using this compound in ZR-75-1 cells suggested some possible, previously unknown functions of SGK3 in cAMP signalling and mitochondrial metabolism. On degradation of SGK3, I observed a downregulation of the phosphodiesterase PDE4D and upregulation of a host of proteins mediating Oxidative Phosphorylation in mitochondria. These effects were observed only on degradation of the protein and not inhibition of its kinase activity, suggesting putative kinase-independent roles of SGK3. These findings would be extremely exciting to follow up in future work, and demonstrate the power of the PROTAC approach.
Given the potential of the PROTAC approach to probe biological systems, I also collaborated with the Ciulli lab to generate HaloPROTAC-E. This compound utilizes the bacterial dehalogenase activity of HaloTag7 to specifically induce degradation of any endogenously Halo-tagged protein. I demonstrated that HaloPROTAC-E was able to induce degradation of the entire VPS34 complex to impact on downstream biology. This compound will be a useful tool within the unit and wider field to study proteins with no known ligands or validate target proteins for potential PROTAC development.
The second aim of my PhD was to study how SGK3 is activated in the context of PI3K inhibition. In this thesis, I studied the mTORC2 component mSin1, and provide preliminary data to show that certain regions of this protein may explain the differential regulation of Akt and SGK3. I also performed a preliminary assay to study interactors regulating the activity of Class III PI3K VPS34. Together, these experiments represent important preliminary results in understanding the activation of SGK3 and how it mediates Class I PI3K and Akt inhibitor resistance.
|Date of Award||2020|
|Sponsors||Biotechnology and Biological Sciences Research Council & AstraZeneca|
|Supervisor||Dario Alessi (Supervisor) & Claire Crafter (Supervisor)|