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. 2018 Sep 4;2:PO.18.00126. doi: 10.1200/PO.18.00126

Fig 4.

Fig 4.

Expression of RET fusions that induce resistance to osimertinib with a concomitant increase in sensitivity to cabozantinib. PC9 cells were infected with lentivirus that harbored an empty plasmid (pCX4.1-empty) or pCX4.1 with either CCDC6-RET or KIF5B-RET complementary DNA (cDNA), and cells that expressed the plasmids were selected to generate stable cell lines. (A) Cell extracts were immunoblotted (indicated by IB) for (left panel) total RET (left panel) or (right panel) phosphorylated RET. (B) Cell lines were treated with either osimertinib 0.05 µM or cabozantinib or 0.25 µM for 1 h; cell extracts were prepared and then immunoblotted for the indicated proteins. (C) Cells were treated with increasing concentrations (as indicated on the figure) of (left panel) osimertinib or (right panel) cabozantinib for 96 h, and then growth was determined. (D) Cells were treated with the indicated concentrations of osimertinib in the presence of cabozantinib 0.5 µM for 96 h, and then growth was determined. (E) 50% inhibitory concentration (IC50) values were determined by nonlinear regression of growth data. (F) Cells were treated with the indicated concentrations of (left panel) osimertinib or (right panel) cabozantinib for 48 hours, and then caspase 3/7 enzymatic activity determined. (G) PC9 cells that expressed either pCX4.1-empty vector or pCX4.1–CCDC6-RET were treated with the indicated concentrations of cabozantinib in the presence of osimertinib 0.1 µM for 48 h, and then caspase 3/7 enzymatic activity was determined. Results are expressed as the fold change in caspase 3/7 activity compared with the corresponding cell line treated with osimertinib 0.1 µM alone. All experiments were conducted at least two times, and data represent the mean ± standard deviation. (*) P < .05 compared with untreated control. (†) P < .05 compared with the corresponding cell line treated with osimertinib 0.1 µM only. All data were analyzed by two-way Anova with the Tukey multiple comparison test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; −cabo, without cabozanitinib; +cabo, with cabozanitinib.