Bx795

A549 cells were cultured in 12‐well plates at a density of 0.4 × 10⁶ cells per well with F12K containing 10% FBS and 0.1% PS for 24 h. Cells were washed once with DPBS and inoculated in serum‐free and PS‐free F12K medium containing TPCK‐trypsin (0.5 μg/ml) with PR/8 at an MOI of 1 for 1 h. The inoculum was replaced with serum‐free and PS‐free F12K medium containing TPCK‐trypsin (0.5 μg/ml) with indicated amounts of chemical inhibitors. After 24 h, RNAs were isolated for real‐time PCR. The following chemical inhibitors were purchased from Tocris (Pittsburgh, PA, USA) except BX795, which was obtained from Selleckchem (Houston, TX, USA) and used at indicated concentrations: STAT1 inhibitor, fludarabine (30 µM, Catalog No. 3495); JAK1/2 inhibitor, ruxolitinib (4 µM, Catalog No. 7064); NF‐κb inhibitors, pyrrolidinedithiocarbamate ammonium (PDTC) (30 µM, Catalog No. 0727) and Bay 11–7821 (10 µM, Catalog No. 1744), c‐Myc inhibitor, 10058‐F4 (10 µM, Catalog No. 4406), and TBK1 inhibitor, BX795 (4 µM, Catalog No.S1274).

NCI-H1975-OsiR isogenic cells were cultured and expanded in osimertinib (1 µM) containing media. For sensitive xenograft, NCI-H1975 at 5 × 10⁶ cell density were injected subcutaneously into 6–8-week-old NSG mice. When tumor size reached approximately 100 mm³, tumor-bearing mice were randomized and treated with osimertinib, 5 mg/kg or 10 mg/kg, 5 days a week for 3 weeks. To develop resistant xenograft, NCI-H1975-OsiR cells (5 × 10⁶) were injected subcutaneously into 6–8-week-old NSG mice followed by osimertinib treatment starting from the day following tumor cell injection so tumors developed under osimertinib pressure. Mice were treated with designated concentration of osimertinib from the first day of tumor cell injection throughout the entire experiment, and tumor sizes were measured twice a week by caliper. To evaluate the effect of the PDK1-selective inhibitor, BX795, 5 tumor-bearing mice/group were either left untreated, treated with osimertinib alone (5 mg/kg), treated with PDK1 inhibitor BX 795 (25 mg/kg) (Selleckchem, Houston, TX), or with the combination. Tumor volume was measured using the formula V = ab²/2 where a is the largest diameter and b is the smallest. At end of the experiment, residual tumor tissues were harvested for further analysis.

Inhibitors of the IFN response, BX795,^{14 (link)} TPCA-1,^{15 (link)} and ruxolitinib (Rux)^{16 (link)} (Selleck Chemicals, Munich, Germany), were prepared as 10-mM stocks in DMSO. Actinomycin D (AMD) and cyclohexamide (CHX) (Sigma, Dorset, UK) were prepared as 10-mM stocks in DMSO and ethanol, respectively. The Small Diversity Set Compound Library (Dundee Drug Discovery Unit [DDU], University of Dundee, UK) was used for HTS and consists of 15,667 compounds at 10 mM in DMSO. Hit compounds StA-IFN-1 (4-(1-acetyl-1H-indol-3-yl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one) (Chembridge, San Diego, CA), StA-IFN-2 (4-{[4-(thieno[3,2-d]pyrimidin-4-yl)-1,4-diazepan-1-yl]methyl}benzonitrile) (Enamine, Monmouth Jt., NJ), StA-IFN-4 (2-[(4,5-dichloro-6-oxo-1(6H)-pyridazinyl)methyl]-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one) (Enamine, Monmouth, NJ), and StA-IFN-5 (6-methyl-4-phenyl-N-(pyridin-4-yl)quinazolin-2-amine) (Mcule, Budapest, Hungary) were stored as 10-mM stocks in DMSO. All compounds were used at the indicated concentrations.