(A) Parental and resistant HCC827 PFR5 cells treated with increasing concentrations of PF00299804. Cell viability relative to untreated controls measured after 72 hours. Each data point represents the mean ±SD of 6 wells. (B) HCC827 and HCC827 PFR5 and PFR6 cells were treated for 6 hours with 1 μM PF00299804 or gefitinib, PHA-665,752, or their combination. Cell lysates were immunoblotted to detect indicated proteins. (C) Upper, HCC827 PFR6 cells treated with increasing concentrations of PF00299804, PF2341066, or their combination. Lower, HCC827 PFR6 cells treated with increasing concentrations of gefitinib alone or in combination with PF2341066. Cell viability relative to untreated controls measured after 72 hours. Each data point represents the mean ±SD of 6 wells. (D) HCC827 PFR xenogafts in nu/nu mice were treated with PF2341066, PF00299804, or their combination. Tumors measured twice weekly. Only combination treatment led to tumor shrinkage and was the most effective treatment in vivo (p < 0.0001). Treatment was stopped after 56 days (arrow) and no tumor re-growth was observed in 35 weeks. Each data point represents the mean ±SD for 5 mice.

(A) MET copy number determined by quantitative PCR. Parental (Par) HCC827 and MET amplified HCC827 GR (GR5) cells were used as negative and positive controls, respectively. Each column represents the mean ±SD for 3 independent experiments. (B) Parental HCC827 cells and PFR clones were immunoblotted to detect indicated proteins. (C) Genome wide view of copy number changes generated using Human Mapping 250K Sty single nucleotide polymorphism (SNP) array and analyzed using the dChip program (see Experimental Procedures). HCC827 GR clones were compared with HCC827 PFR and HCC827 parental clones. Blue curve indicates degree of amplification of each SNP from 0 (left) to 8 (right). (D) Chromosome 7 view of copy number changes in HCC827 parental, GR and PFR cells. Arrow indicates MET oncogene.

(A, B) HCC827 cells treated with (A) increasing concentrations of gefitinib alone or in combination with PHA-665,752, in the absence or presence of HGF (50ng/mL), or (B) increasing concentrations of gefitinib alone or in combination with the indicated concentrations of HGF. Cell viability relative to untreated controls measured after 72 hours. Each data point represents the mean ±SD of 6 wells. (C) HCC827 cells were treated for 6 hours with 1μM gefitinib alone or in combination with the indicated concentrations of HGF. Cell lysates were immunoblotted to detect indicated proteins. (D) Cells were treated for 6 hours with gefitinib (HCC827, PC-9), PF00299804 (H1975), or lapatinib (BT-474, SKBR3), alone or in combination with HGF (50ng/mL). All drugs were used at 1μM. Cell lysates were immunoblotted to detect indicated proteins. *indicates cross-reaction by the p-EGFR antibody against p-HER2. Cell lines in which HGF rescued viability are labeled in blue, and cell lines in which HGF did not rescue viability are labeled in red. (E) HCC827-HGF xenografts in nu/nu mice treated with PF2341066, gefitinib, or their combination and tumors measured twice weekly. Some growth inhibiton was observed with gefitinib alone, however only combination treatment led to complete tumor shrinkage (p = 0.002). Each data point represents the mean ±SD for 5 mice. See also .

(A) IC₅₀ values for viability curves () in the presence or absence of HGF and IGF. Cells were treated with increasing concentrations of the appropriate TKI alone (red) or in combination with 50ng/mL HGF (blue) or 75ng/mL IGF (green). (B) Cells were treated for 6 hours with gefitinib (HCC827, PC-9, A431, HN11), PF00299804 (H1975) or lapatinib (BT-474, SKBR3) alone or in combination with HGF (50ng/mL) or IGF (75ng/mL). All drugs were used at 1μM. Cell lysates immunoblotted to detect indicated proteins. BT-474 and SKBR3 cell lysates were run on the same gel, and no MET or IGF-1Rβ was detected in SKBR3 cells relative to BT-474 cells. See also for quantification.

(A) Cells treated for 6 hours with gefitinib (HCC827, PC-9), PF00299804 (H1975), or lapatinib (BT-474, SKBR3), alone or in combination with HGF (50ng/mL). All drugs were used at 1μM. Cell extracts were immunoprecipitated with an anti-p85 antibody followed by Western blot with anti-p-Tyr, anti-ERBB3 and anti-p85 antibodies. (B) HCC827 cells were transfected with a negative control or GAB1 siRNA for 48 hours. Transfected cells were treated for 6 hours with gefitinib (1μM) alone or in combination with HGF (50ng/mL). Cell lysates were immunoblotted to detect indicated proteins. See also . (C) HCC827 cells were transfected with GAB1 siRNA or a negative control siRNA for 48 hours, then treated with increasing concentrations of gefitinib, alone or in combination with 50ng/mL HGF. Left, cell viability relative to untreated controls measured after 72 hours. Each data point represents the mean ±SD of 6 wells. Right, plot of IC₅₀ values corresponding to cell viability curves.

(A) HCC827 cells treated with HGF (50ng/mL) and 1μM gefitinib are resistant to gefitinib (HCC827-50 cells). After the removal of HGF, stably resistant HGF-independent HCC827-50GR cells survive in 1μM gefitinib alone. In contrast, parental HCC827 cells do not survive when treated with 1μM gefitinib. (B) Parental HCC827 cells and HCC827-50 cells (pre-treated with gefitinib in combination with HGF (50ng/mL) for 14 days) were grown in media alone (No Rx) or media treated with 1μM gefitinib (+Gef) for 7 days. Viable cells were visualized and quantified using Syto60 staining. (C) Fluorescence in situ hybridization (FISH) of MET/EGFR/CEP7 probe set with HCC827 and HCC827-50GR cells. MET (orange) EGFR (green) CEP7 (aqua). Metaphase spread (bottom) shows multiple copies of EGFR and MET (arrow) on individual chromosomes. Scale bars represent 10μm. (D) HCC827-50GR cells (upper) and HCC827-50GR cells grown in media alone (without gefitinib) for 8 weeks, 50GR 8wks R5 (lower), were treated with increasing concentrations of gefitinib or PHA-665,752 or their combination for 72 hours. Cell viability was measured relative to untreated controls. Each data point represents the mean ±SD of 6 wells. (E) HCC827 cells and stably resistant HCC827-50GR cells were treated for 6 hours with gefitinib, PHA-665,752, or their combination. All drugs were used at 1μM. Cell lysates were immunoblotted to detect indicated proteins. See also and and .

(A) Fluorescence in situ hybridization (FISH) of MET/EGFR/CEP7 probe set. MET (red) EGFR (green) CEP7 (aqua). Left, tumor sections from control HCC827 xenograft models that do not express HGF showed normal MET copy number. Right, tumor sections from one of three HCC827-HGF xenografts treated with gefitinib () showed significant MET amplification (arrow). (B) High-throughput FISH analysis of HCC827 cells identifies a subpopulation harboring MET amplification (arrow). MET (RP-11-95I120; red); 7qter (RP-11-6903; green). All scale bars represent 10μm. (C) Parental HCC827 cells and three independent clones harbor a small percentage of MET amplified cells. No pre-existing MET amplification was detected in H3255 or PC-9 cell populations. (D) Left, HCC827 cells were spiked with approximately 0.1% of GFP labeled HCC827 cells or GFP labeled MET amplified HCC827 GR6 cells. Each population was grown in either media alone or media treated with gefitinib (1μM) with HGF (50ng/mL). Cells were collected after 19 days and GFP levels were quantified using FACS. Each data point for cells treated with gefitinib+HGF represents the mean ±SD for 3 independent wells. Fold change is the ratio of Day 19 to Day 0 (%GFP). Right, diagrammatic depiction of results. See also .

(A) Summary of tumors from geftinib/erlotinib treated patients, including 16 paired, and 11 drug resistant samples only. Samples were evaluated for EGFR mutational status, MET amplification and HGF expression *Specimen contained less < 30% tumor cells. **MET amplification defined by qPCR as previously described (). Data on EGFR T790M and MET amplification in resistant specimens only from patients 1-4 and 17-19 has been previously published (). N/A; not available. (B) FISH analysis of pre-treatment sample from patient 10 shows evidence of a subset of MET amplified cells (arrow) before exposure to an EGFR TKI. MET (RP-11-95I120; orange); CEP 7 (aqua). Scale bars represent 10μm. See also .