a, Effects of a dose-response of erlotinib in HCC827 xenograft tumors in immunocompromised mice (n=10 tumors/treatment group). b–f, mRNA expression of indicated genes in HCC827 erlotinib resistant tumor xenografts (treated at the indicated erlotinib doses) relative to vehicle-treated control tumors. The number of tumors analyzed from each treatment cohort is indicated in parentheses. Data are expressed as the mean ± SEM of the fold change relative to the mean expression of the genes in 2 vehicle treated control xenograft tumors.

a, AXL and pAXL protein levels are increased in the absence of pEGFR and increased pMET in HCC827 tumors from each erlotinib treatment cohort compared with vehicle treated tumors. Tumors were harvested for analysis at the completion of the study for each treatment group (Vehicle: Day 45; 6.25 Erl: Day 60; 12.5 Erl: Day 110; 25 Erl: Day 110; 50 Erl: Day 100) b, Acute and transient treatment of HCC827 tumors with erlotinib (12.5 mg/kg/day) decreases pAKT, pERK, pRELa and increases the levels of cleaved Parp. c, Response of parental HCC827 xenograft tumors or HCC827 ER xenograft tumors (n= 10 tumors/group) transduced with a non-target shRNA or an shRNA targeting AXL to treatment with either vehicle or erlotinib (12.5 mg/kg/day). Tumor volumes are expressed as mean ± SEM. d, Validation of AXL knockdown and the effects of erlotinib treatment on pEGFR in representative tumor xenografts (c) by western blot analysis.

a, HCC827 ER1-ER5 sublines are resistant to erlotinib treatment as measured by CellTiterGLO cell viability assay. Data are from 3 independent experiments and are expressed as percent of vehicle treated cells and mean ± SEM. b, Expression of AXL and GAS6 in the ER sublines compared with parental HCC827 cells (data are from Western blot analysis). c, Erlotinib IC₅₀ in HCC827 cell lines (as indicated) measured 48h after treatment with a non-targeting or AXL or GAS6 siRNA. Erlotinib IC₅₀ is shown in parentheses. Data are representative of 3 independent experiments. d, Erlotinib IC₅₀ in HCC827 cell lines (as indicated) measured 48h after treatment with vehicle (control) or with MP-470 (1 µM) or XL-880 (1 µM) and erlotinib. Erlotinib IC₅₀ is shown in parentheses. Data are representative of 3 independent experiments. e, Effects of treatment for 48h with a non-targeting (−) or AXL siRNA in parental or ER1 and ER2 cell lines in the absence and presence of erlotinib on the indicated biomarkers. Data represent 3 independent experiments. f–g, Effects of treatment for 48h with a vehicle or the indicated doses of (f) MP-470 or (g) XL-880 in parental or ER1 and ER2 cell lines in the absence and presence of erlotinib on the indicated biomarkers. Data represent 3 independent experiments. h,Erlotinib IC₅₀ in HCC827 cells measured 5 days after transfection the cDNA constructs encoding the indicated proteins and treated with either vehicle (left) or with XL-880 (1 µM) and erlotinib. Erlotinib IC₅₀ is shown in parentheses. Data are representative of 3 independent experiments. i, Western blot for the indicated proteins in lysates from cells transfected with the indicated cDNA constructs and treated with XL-880 (1 µM) for 3 hours prior to cell lysis (h).

a, Effects of treatment with a non-targeting or the indicated vimentin siRNAs on AXL expression in HCC827 ER3 cells. b, Erlotinb IC₅₀ in HCC827 parental or ER3 cells upon treatment with a non-targeting or the indicated vimentin siRNAs. Erlotinib IC₅₀ is shown in parentheses. Data represent 3 independent experiments. c, Increased migration through a transwell chamber of ER3 compared to parental HCC827 cells in cells treated with a non-targeting or the indicated vimentin (VIM) or AXL siRNAs or XL-880 (1 µM); n=3, data expressed as mean ± SEM. d, Increased adherence to plastic of HCC827 ER3 cells compared to HCC827 parental cells in cells treated with a non-targeting or the indicated vimentin (VIM) or AXL siRNAs or XL-880 (1 µM). 5 100× microscopic fields per cell line were counted. n=3, data expressed as mean ± SEM. e, Western blot for the indicated proteins in lysates from cells transfected with the indicated siRNAs in (c–d).

a–b, Representative expression of the indicated proteins by IHC in (a) case 7 and (b) case 9 shown in Table 2. IHC staining for AXL and GAS6 was scored as shown in Supplemental Figure 14. Vimentin IHC staining was scored using an established, clinically validated protocol26and EGFR T790M and MET amplification were assessed by sequencing and FISH, respectively, using established assays. Scale bars indicate in (a) 100 µM and (b) 10 µM.