Drug screening and phosphotyrosine proteomic profiling to characterize response to RET inhibition in TPC1 cells. (A) Results from a drug screen testing 209 compounds in TPC1 cells for effects on viability. Select kinase inhibitors are highlighted in red, including cabozantinib. AUC, area under the curve. (B) Short-term cellular viability after exposure to cabozantinib in TPC1 cells (red) is compared to the average dose response of a panel of cancer cell lines (PanCan, gray, n = 52). (C) Results from a drug screen comparing TPC1 cells (red) to a panel of 52 cancer cell lines (gray) after treatment with cabozantinib and select kinase inhibitors. (A–C) The drug screens performed were replicated in two independent experiments, and a representative experiment is shown. (D) Western blot analysis of ERK activation and RET phosphorylation in TPC1 cells treated with 100 nM cabozantinib (XL184) or 100 nM BLU6864 (RETi). Cells were harvested at the indicated times; the 24 + 1 timepoint indicates a treatment of an additional 1 h with the indicated compound after an initial 24 h of treatment. Representative blots are shown from four independent experiments. (E) TPC1 cells treated with BLU6864 were compared to vehicle-treated cells, and phosphotyrosine peptides were analyzed by mass spectrometry. Peptides corresponding to JAK1 (Y1034) and STAT3 (Y705) are highlighted. (F) Western blot analysis of JAK and ERK activation in TPC1 cells treated with XL184, ruxolitinib, or AZD1480 (JAK2i). Treatment time is indicated, the 6 + 1 timepoint indicates a treatment of 1 h with either ruxolitinib (JAK2i; lane 6) or AZD1480 (lane 7) after a 6-h treatment with XL184. Representative blots are shown from three independent experiments.