Fig. 6. Pie chart representation of the tissue type distribution of the 500 cell lines screened for sensitivity to the tyrosine kinase inhibitors. The corresponding percentage of total lines are indicated after the tissue type.

Fig. 7. Immunoblots are shown by using antibodies directed against the indicated proteins and their phosphorylated counterparts. The effect of erlotinib treatment on downstream survival signaling pathways in a small-cell lung cancer cell line (KHM-3S), a gastric cancer cell line (Nugc-4) and three NSCLC cell lines (PC9, JPC-3, and HCC-827) is shown. All five of these cell lines harbor activating mutations in the EGFR kinase domain. Note that at the time point shown (6 h), treatment of the Nugc-4 cell line did not result in a decrease in Akt phosphorylation; however, treatment of the cell line with 50 nM erlotinib for 1, 3, 6, and 24 h demonstrated complete suppression of phosphorylation at 1 and 3 h, followed by an increase almost to baseline levels by 24 h (data not shown).

Fig. 8. Sensitivity to kinase inhibitors is associated with inhibition of downstream prosurvival signaling pathways. (A) Immunoblot demonstrating that levels of MET and phospho-MET are well correlated among PHA665752-sensitive (denoted by *) and PHA665752-insensitive tumor cell lines in a panel of 21 gastric cancer cell lines examined. (B) Immunoblots indicating that treatment of sensitive gastric cancer cell lines with 200 nM of either erlotinib (E) or PHA665752 (PHA) for 24 h is associated with down-regulation of prosurvival signaling pathways only in response to the MET inhibitor PHA665752 (not in response to erlotinib). Treatment of the MET-amplified lung cancer cell line EBC-1 with the MET inhibitor results in suppression of key prosurvival signaling pathways. (C) Immunoblot demonstrating expression of total MET and phospho-MET (Y1234/35) in a panel of PHA665752-sensitive (Hs746T) and -resistant cell lines. MET gene copy number is indicated below each cell line. (D) Immunoblots demonstrating the effect of treatment of three PHA665752-resistant cell lines with a range of the MET inhibitor (PHA) for 24 h on phospho-MET as well as downstream signaling pathways. Note that drug treatment does not suppress downstream signaling in these cell lines, despite the fact that in one line (NCI-H1573), MET phosphorylation is efficiently inhibited by drug treatment.

Fig. 9. Effect of BRAF and NRAS mutations on sensitivity to BRAF inhibition. (A) Sensitivity of human cancer cell lines harboring activating NRAS mutations to 200 nM of the BRAF inhibitor AZ628 was established, The eight cell lines with NRAS mutations among the 500 analyzed are shown here ranked according to their sensitivity to the inhibitor. (B) Immunoblot analysis of ERK1/2 phosphorylation in selected cell lines in response to a range of concentrations of AZ628. Immunoblots to the left of the figure represent activated ERK1/2 (phospho-ERK1/2), and the levels of total ERK1/2 proteins are shown in the immunoblots on the right. The identity of cell lines and their sensitivity to AZ628 is indicated to the left of the figure. (C) Immunoblot analysis of the effect of a range of concentrations of AZ628, U0126 (MEK inhibitor) and Sorafenib on ERK1/2 phosphorylation in the M14 melanoma cell line.

Fig. 10. Immunoblots demonstrating the effect of treatment of three EGFR mutant cell lines with either erlotinib (20 and 200 nM) or the Src inhibitor AZD0530 (0.2 and 1 mM) for 6 h on EGFR activation and downstream signaling.

Fig. 11. HER2 gene amplification is correlated with sensitivity to a HER2 inhibitor and suppression of signaling pathways. (A) Pie chart representation of the sensitivity of 500 human cancer cell lines to treatment with 200 nM of the irreversible EGFR/HER2 kinase inhibitor, HKI-272. The drug effect was calculated as the fraction of untreated cells present after 72 h of treatment. The effect of treatment with 200 nM erlotinib is indicated in the adjacent column. Details regarding the most sensitive cell lines are shown in the chart. Cell lines demonstrating HER2 amplification are denoted with an asterisk if there is substantial gene amplification (copy number > 4). (B) Immunoblots indicating that sensitivity to HKI-272 (denoted by asterisk) is well correlated with high-level expression of total and phospho-HER2 in a panel of cancer cell lines tested. These lines appear to be "HER2-addicted". (C) Immunoblots indicating the effect of treating HER2-amplified cell lines with erlotinib or the irreversible EGFR/HER2 inhibitor HKI-272 for 24 h on downstream survival signaling pathways. Note that downstream survival signaling in several HER2-addicted cell lines is unaffected by erlotinib treatment.