Investigating mechanisms of acquired resistance to the AKT inhibitor capivasertib (AZD5363)

The PI3K/AKT/mTOR (PAM) pathway is a major driver of cell growth, proliferation and survival and is hyperactivated in 70% of ovarian cancers. PAM inhibitors, such as the AKT-kinase inhibitor capivasertib (AZD5363; currently in Phase III clinical trials), have therefore been `developed as novel cancer therapeutics. Acquired resistance to kinase inhibitors is a major threat to their clinical success, thus understanding resistance mechanisms can support their utility in the clinic. The aim of this thesis was to elucidate resistance mechanisms to capivasertib, using the A2780 human ovarian carcinoma parental and capivasertib-resistant cell lines as models, which harbour hyperactivating PAM pathway mutations.

The isogenic capivasertib-resistant subclone, A2780 254R-B, exhibited cross-resistance to both allosteric and multiple ATP-competitive AKT inhibitors. Additionally, they were also cross-resistant to allosteric and kinase inhibitors of mTORC, although no resistance was observed for the cap-dependent protein synthesis inhibitor, 4EGI-1. Furthermore, 4EBP1 expression was reduced and S6RP S235/6 phosphorylation was capivasertib-resistant, which correlated with overexpression of p90RSK. The induced interaction of 4EBP1 and eIF4E with increased 4EBP1 phosphorylationinhibition was not as great in 254R-B (4-fold) compared to PAR (9-fold), which was associated with a 9-fold increase in cap-dependent protein synthesis, determined by dual-luciferase report assay. Overexpression of 4EBP1 in 254R-B cells reduced resistanceto capivasertib by 6-11 fold. Exogenous 4EBP1 additionally reverted the 4EBP1-eIF4E interaction in 254R-B cells sensitive to 4EBP1 phosphorylation inhibition, suggesting that the resistance mechanism may also be dependent on an mTORC1-independent kinase.

Another isogenic capivasertib-resistant subclone, A2780 254R-D, exhibited cross-resistance to ATP-competitive but not allosteric inhibitors of AKT which suggests that resistance may involve a non-AKT target of capivasertib.

Overall, this thesis provides evidence that acquired resistance to capivasertib is associated with increased cap-dependent protein synthesis, mediated through reduced 4EBP1 activity. Reduced 4EBP1 expression in tumours may therefore be used as a predictive biomarker for screening patients for acquired resistance to capivasertib.