Fig. 1: Characterisation of the cell death-inducing effects of various AKT inhibitors and of the W80A mutation on allosteric AKT inhibitor response.

a Allosteric AKT inhibitors have higher cell death-inducing activity compared to ATP-competitive inhibitors. A panel of cancer cell lines with the various PI3K/AKT-activating lesions (shown in the table on the left) was treated with three allosteric AKT inhibitors (MK-2206, miransertib and ARQ 751) and two ATP-competitive AKT inhibitors (GSK690693 and ipatasertib) at the indicated doses. The fraction of dead cells following 4 days of drug treatment is shown (cell death %). b Allosteric and ATP-competitive inhibitors have comparable effects on AKT kinase inhibition. The effects of AKT inhibitors on AKT and AKT substrate phosphorylation was assessed in MDA-MB-361 cells by immunoblot using the indicated antibodies following 18 h of treatment. c The cell death-inducing effects of the allosteric AKT inhibitor ARQ 751 and the ATP-competitive inhibitor ipatasertib were compared in washout experiments. Cells were treated with the indicated doses of drug for either 30 min or 1 h. The drug was then removed and cells were extensively washed before allowing them to grow for an additional 72 h. As a control, cells were also continuously exposed to drug for the duration of the experiment. The fold change in cell numbers (left) and the fraction of dead cells following 3 days of drug treatment (right) is shown. d The W80A mutation confers resistance to some but not all allosteric inhibitors in an isoform-selective manner. AKT1 W80A or AKT2 W80A was ectopically expressed in MDA-MB-361 cells. Cells were treated with MK-2206, miransertib or ARQ 751 at the indicated doses. The fold change in cell numbers (left) and the fraction of dead cells following 4 days of drug treatment (right) is shown. e Cells were also treated for 18 h and lysed. Lysates were analysed by immunoblot with the indicated antibodies. f HCT116 AKT1/2 DKO cells were stably transduced with wild-type or W80A variants of either AKT1 or AKT2. Cells were then treated with the indicated drugs and lysed. Lysates were analysed by immunoblot with the indicated antibodies. n.s., not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.