Figure 5. Low neurofibromin expressing cells respond to erlotinib when combined with MEK inhibition.

A. NF1 silenced cells are insensitive to EGFR or MEK inhibitor alone but sensitive to combined drug treatment. Cells expressing control shRNA (shSC) or shRNA targeting NF1 (shNF1#2) were treated with indicated concentrations of AZD-6244 alone (solid line) or in combination with 30 nM erlotinib (dotted line) for 72hrs before cell viability was measured using Cell Titer Blue. Cell survival is shown normalized to untreated controls. Error bars denote SEM.

B. Levels of pERK and pAKT are analyzed by western blotting after treating the shSC- or shNF1#2-infected cells with 30nM erlotinib or 1µM MEK inhibitor AZD-6244 (MEKi) alone or in combination for 1hr. A short and long exposure is shown of pERK. Total ERK and AKT are shown as loading control.

C. T790M-positive erlotinib resistant PC9 (PC9^T790M) cells remain insensitive to erlotinib when treatment is combined with the MEK inhibitor. PC9^T790M and parental PC9 cells are treated with indicated concentrations of MEK inhibitor AZD-6244 alone (straight line) or in combination with 30nM erlotinib (dotted line) for 72hrs before cell viability was measured and survival normalized to untreated controls. Error bars denote SEM.

D. Levels of pERK and pAKT are analyzed by western blotting after PC9 and PC9^T790M cells were treated with 30nM erlotinib or 1µM AZD-6244 (MEKi) alone or in combination for 1hr. A short and long exposure is shown of pERK. Total ERK and AKT are shown as loading control.

E. NF1 silenced xenograft tumors are sensitive to erlotinib in combination with MEK inhibition, but insensitive to either of both drugs alone. Mice were subcutaneously injected with shSC- or shNF1#2-infected PC9 cells. Once tumors were detectable, mice were randomized to 4 different treatment regimens: vehicle, erlotinib, AZD-6244 (MEKi) or combined erlotinib and AZD-6244 (erl + MEKi) as described in Experimental Procedures. Data in the figure represent the mean ± SEM tumor volumes for each treatment group over time. P-values are indicated for the mice receiving erlotinib alone or combined with AZD-6244 and analyzed against the vehicle control group.

F. PC9^T790M xenograft tumors are insensitive to erlotinib in combination with MEK inhibition. Mice were injected with PC9^T790M cells. Once tumors were detectable, mice were treated by 4 different treatment regimens as in Figure 5E.