Figure 7.

INCB3619 inhibits EGFR autocrine signaling in NCI-H1666 and sensitizes it to gefitinib

A: INCB3619 inhibits EGFR ligand activation from NCI-H1666 measured by the A431 reporter assay. Left panel: control, buffer; TGFα, 10 ng/ml TGFα; TGFα + C225, TGFα added to the A431 cells pretreated with C225 for 10 min. Right panel: control, medium alone; DMSO, conditioned medium from NCI-H1666 treated with DMSO for 48 hr; 2 µM 3619 or 10 µM 3619, conditioned medium from NCI-H1666 treated with INCB3619 at indicated concentrations for 48 hr. Phospho- and total EGFR signals were quantitated, and the ratios are presented relative to control.

B: INCB3619 inhibits EGFR ligand-dependent signaling in NCI-H1666 cells. A549 cells were serum starved for 4 hr before being treated with indicated concentrations of various compounds for 72 hr. For INCB3619, 50 ng/ml of TGFα was added to reverse the signaling effect.

C: INCB3619 sensitizes NCI-H1666 cells to gefitinib. NCI-H1666 cells in serum-free medium were treated with various concentrations of gefitinib and INCB3619 for 72 hr, and their viability was measured and plotted as a percentage of viable cells relative to DMSO mock treatment. Data are presented as mean values with error bars (SD; n = 3).

D: Combination effect of INCB3619 and gefitinib on the EGFR autocrine signaling. NCI-H1666 cells in serum-free medium were treated with DMSO alone, 2 µM INCB3619, 2 µM INCB3619 in the presence of 50 ng/ml TGFα, 0.5 µM gefitinib, or 0.5 µM gefitinib plus 2 µM INCB3619. Phospho- and total ERK1/2 signals were quantitated, and the ratios are presented relative to control.