Figure 3.
Compound testing on KIT D816V iPSC/ESC-derived hematopoietic cells and evaluation of effect of nintedanib on SM patient samples. (A) Drug response curves (0-10 μM) for nintedanib, midostaurin, and imatinib on KIT D816V (n = 1-4) and control (n = 2) iPSC-derived KIT+ hematopoietic cells from patient 1. IC50 values were calculated based on averaged titration curves obtained for cells derived from different iPSC lines. IC50 values for nintedanib were 27 to 105 nM for KIT D816V cells and 262 to 542 nM for control cells. (B) Averaged drug response ± standard deviation of KIT D816V and control iPSC-derived KIT+ cells treated with 100 nM or 1 μM nintedanib, midostaurin, imatinib, or rigosertib for 66 hours. Vehicle (DMSO)-treated cells were used as control (0 nM). Patient 1: n = 6-12. Patient 2: n = 3-9. Patient 3: n = 6-9. *P ≤ .05, **P < .001, ***P ≤ .0001, drug responses of KIT D816V vs control KIT+ cells at the same drug concentration, Welch’s t test. (C) Representative western blot analysis of KIT receptor signaling upon nintedanib or midostaurin treatment of KIT D816V iPSC–derived hematopoietic cells. Cells were treated with 1 μM compound for 4 hours prior to analysis. Vehicle (DMSO)-treated cells were used as control. Positions of molecular weight markers are indicated. (D) Nintedanib response curves (0-10 µM) for SM primary sample (patient 10) and a healthy donor (HD; n = 2). MNCs were subjected to MACS, and KIT+ and KIT− cells were treated with nintedanib for 66 hours, followed by viability measurement using a CellTiter Glo assay. Vehicle (DMSO)-treated cells were used as control. Nintedanib shows cytotoxicity to healthy donor cells only at concentrations closer to 10 µM, whereas cell viability is severely compromised at concentrations > 100 nM. (E) Averaged response of 7 to 11 SM primary samples and 6 to 8 HD primary samples to 1 µM nintedanib or midostaurin treatment. MNCs were treated as described in (D). Nintedanib treatment led to a significant decrease in the viability of KIT+ SM MNCs, whereas midostaurin targeted KIT+ and KIT− cells equally. Additionally, nintedanib did not have a significant impact on the viability of HD cells, in contrast to midostaurin, which led to a significant reduction in the viability of KIT+ and KIT− HD cells. *P ≤ .0003, Welch’s t test.