Figure 6. ARAF overexpression causes acquired resistance of EGFR mutant lung cancers to EGFR inhibitors. See also Figure S6 and Table S1–S3.

(A) Treatment timeline for three NSCLC patients with tumor ARAF amplification. (B and C) Fluorescence in situ hybridization for ARAF and BRAF in the patient specimens. Red, ARAF; green, BRAF; orange, centromeric of chromosome X. Scale bar, 5 μM.

(D) Control or ARAF knockdown PDX-138 cells were subcutaneously injected into NSG mice. Erlotinib was given at 25 mg/kg once a day. Bars, mean±SEM, n=5, two-tailed Student’s t test.

(E) PC9 or HCC827 cells expressing vector or doxycycline-inducible ARAF were seeded in 96 well plates at a density of 2,000 cells/well and supplemented with 100 ng/ml doxycycline. After 24 hours induction, cells were treated with erlotinib for 72 hours. Cell survival was normalized to untreated controls. Drug concentrations inducing 50% inhibition in survival (IC₅₀ nmol/L) are indicated. Bars, mean±SD triplicate of experiments.

(F) PC9 cells expressing vector or doxycycline-inducible ARAF were treated with 100 ng/ml doxycycline for 24 hours. Cells were then treated with 100 nM erlotinib, 3 μM SHP099 or in combination.

(G) PC9 cells expressing vector or doxycycline-inducible ARAF were seeded in 96 well plates at a density of 2,000 cells/well and supplemented with 100 ng/ml doxycycline. Cells were then treated with 100 nM erlotinib, 3 μM SHP099, or in combination. Bars, mean±SD triplicate of experiments.

(H) PC9 cells expressing doxycycline-inducible ARAF were subcutaneously injected into nude mice. Mice were fed either normal or doxycycline containing food. Erlotinib was given at 12.5 mg/kg once a day, SHP099 at 75 mg/kg once a day. Bars, mean±SEM, n=5.

(I) PDX-138 cells were subcutaneously injected into NSG mice. Erlotinib was given at 25 mg/kg once a day, SHP099 at 150 mg/kg once a day. Bars, mean±SEM, n=5.