Figure 4.

Transcriptional changes and levels of apoptosis in ponatinib-resistant cells following treatment with catalytic mTOR inhibitors. A) Schematic diagram demonstrating the activity of allosteric (blue-green) and catalytic (red) mTOR inhibitors. B) KCL22^Pon-Res cells were cultured with 150 nM dasatinib, 100 nM ponatinib, 10 nM rapamycin, 500 nM PI-103, or 100 nM NVP-BEZ235 or untreated (Untr). Phosphorylation of CRKL, RPS6, and 4E-BP1 was measured four hours following drug treatment. C) KCL22^Pon-Res cells were cultured ± 2 µM imatinib, 2 µM nilotinib, 150 nM dasatinib, 100 nM ponatinib, 10 nM rapamycin, or 100 nM NVP-BEZ235, and apoptosis was measured following 72 hours of drug treatment. Error bars = SD. Three independent experiments were performed. D–G) KCL22^Pon-Res cells were cultured ± 100 nM NVP-BEZ235, alone and in combination with 100 nM ponatinib for 24 hours, and RNA was harvested for RNA-seq. D) The transcriptional response of KCL22^Pon-Res cells to NVP-BEZ235 alone (left) and in combination with ponatinib (right) is represented by Volcano plots (up- and downregulation are indicated by magenta and green, respectively; light and dark colors correspond to q-value thresholds of 0.05 and 0.01, respectively; statistically nonsignificant changes are colored gray). E) A proportional Venn diagram represents the overlap in statistically significant response (q ≤ 0.05) to ponatinib alone (dark blue), NVP-BEZ235 alone (green), and the combination (light blue) in the KCL22^Pon-Res cells. F) A proportional Venn diagram represents the overlap in statistically significant response (q ≤ 0.05) to ponatinib in KCL22 cells (red) and NVP-BEZ235 in KCL22^Pon-Res cells (green). G) A direct comparison of the transcriptional responses of all 1718 genes to treatment common to both experiments; identical expression is shown by the red line, and the true linear relationship is indicated by the blue line. One independent experiment was performed in quadruplicate. Untr = untreated.