Figure 5: ATM loss-of-function dampens AKT/mTOR signalling.
From: MEK inhibitors block growth of lung tumours with mutations in ataxia–telangiectasia mutated
(a,b) Drug synergy experiment in AALE cells with ATM inhibitor (KU60019) and trametinib (40 nM). Displayed is relative cell viability for KU60019 treatment alone and for co-treatment with trametinib. Error bars indicate s.d. (n=3). (b) Deviation from Bliss additivity calculated from experiment in a. Error bars indicate s.d. (n=3). (c) Fold induction of apoptotic cells after 3-day compound or vehicle (DMSO) treatment, as measured by annexin V positivity. Error bars indicate s.d.’s (n=4). **P<0.01, ***P<0.001, two-way ANOVA with Holm Sidak multiple comparisons correction. (d) Western blot analysis of ATM knockout and control NCI-H322 cells treated with TAK-733 (1.0 μM, 6 h) with indicated antibodies (pERK (T202/204); pAKT (S473); p-mTOR (S2448); p4EBP1 (T37/46); pS6K (T389)). Shown is a control and two independent knockout clones. For d, a representative blot for tubulin is used as loading control. (e) Drug synergy experiment on NCI-H322 control (+/+) and ATM knockout (−/−) cells treated with trametinib simultaneously with DMSO or two different concentrations of AKT inhibitor (MK2206). Relative cell viability is shown as dose–response curves. Effect of single MK2206 treatment on cell viability in the absence of trametinib is shown below as the bar graph. Deviation from Bliss additivity calculated for both control (grey bars) and knockout (red bars) cells for indicated concentrations is shown bottom right. Error bars indicate s.d. (n=3). (f) Phosphorylation of indicated targets (pERK (T202/204); pAKT (S473); p-mTOR (S2448); p4EBP1 (T37/46); pS6K (T389)) in response to TAK-733 treatment (1.0 μM, 6 h) is shown as a fold change compared with DMSO-treated baseline (dashed line). Data were determined by quantification of digital western blot images in ImageJ for six wild-type (black symbols) and seven ATM mutant (red symbols) lung cancer cell lines. Median for each group is displayed as the horizontal line. Two-sided t-test was used to calculate the P values.
