Figure 1.

T790M-expressing NSCLC cell lines show enhanced signaling downstream of mTOR, incompletely suppressed by the irreversible EGFR inhibitor CL-387,785. A, lysates were prepared from exponentially growing cells and subjected to Western blotting with the indicated antibodies. Arrows demarcate the increased levels of phospho-Akt, phospho-p70S6K, phospho-S6, and cyclin D1 in HCC827_ Del/T790M cells. B, NCI-H1975 cells were infected with lentiviruses encoding shRNAs targeting mTOR (mTOR_A or mTOR_B) or nontargeting shRNA (NT) for 7 d before the preparation of lysates for Western blotting (top), or for 10 d before assessment of viabilityby CCK-8 assay in the absence (bottom left) or presence of 1 µmol/L CL-387,785 for an additional 72 h (bottom right). Statistically significantly decreased viability(P = 0.005, for mTOR_B shRNA expression compared with control) was associated with reduced expression of mTOR, phospho-p70S6K, and phospho-S6. Columns, mean; bars, SD. C, top, exponentially growing H3255 (L858R), NCI-H1975 (L858R/T790M), and NCI-H820 (E746_T751del/T790M) cells were treated with the indicated concentrations of CL-387,785 for 24 h, and lysates were subjected to Western blotting with the indicated antibodies. Higher concentrations of CL-387,785 are required to dephosphorylate mutant EGFR in the T790M-expressing cell lines, associated with persistent expression of phospho-Akt and phospho-S6. Bottom, isogenic HCC827_Del and HCC827_Del/T790M cells were treated with CL-387,785 at the indicated concentrations for 24 h, and lysates were subjected to Western blotting with the indicated antibodies. HA, hemagglutinin. Dephosphorylation of mutant EGFR harboring T790M requires higher drug concentration, with persistence of expression of both phospho-Akt and phospho-S6. D, cell lines were treated with a range of concentrations of CL-387,785, rapamycin, or CL-387,785 + rapamycin for 72 h in triplicate and subjected to CCK-8 assay. The viability of each sample was normalized to that of DMSO-treated cells. Points, average of normalized values for two independent experiments; bars, SD. Combination indices at the IC₅₀ indicate synergy of EGFR and mTOR inhibition in T790M-expressing cells with values of 0.09565, 0.05488, and 0.05628 in NCI-H1975, NCI-H820, and HCC827_Del/T790M cells, respectively.