Figure 4.
Nintedanib targets KIT D816V MCs. (A) Flow cytometry analysis of KIT D816V and control iPSC-derived CD45+/KIThigh MCs at >30 days of differentiation (patient 1). (B) KIT D816V MCs (red) show higher expression of FCER1A compared with control cells (blue) (upper panel; dashed line is shown as a reference). Mean fluorescence intensity (MFI) of FCER1A (n = 6-9), KIT (n = 18-19), and CD45 (n = 15-17) on KIT D816V MCs and controls (lower panels) * P = .017, Welch’s t test. (C) Representative composite images of acidic toluidine blue–stained cytospins (left panels) or smears (right panels) of FACS-sorted KIT D816V and control MCs (image assembly indicated by dotted lines). Homogenous population of multilobulated promastocytes with (arrowheads) or without (arrows) metachromatic granules were observed. Scale bars, 25 μm. (D) Same as in (C), but stained for tryptase. Cells with high (arrowheads) and low (arrows) number of tryptase-positive granules were observed. Scale bars, 25 μm. For full representative images of (C) and (D) see supplemental Figure 18. (E) Quantitative RT-PCR analysis for KIT D816V and control FACS-sorted MCs (n = 9-15 and n = 3-7, respectively). mRNA expression of MC-specific chymase 1 (CMA1), tryptase α/β 1 (TPSAB1), tryptase β2 (TPSB2), carboxypeptidase A3 (CPA3), FCER1A, and KIT is depicted. KIT D816V MCs show higher FCER1A mRNA expression compared with unmutated KIT cells. *P = .014, Welch’s t test. (F) Drug response curves of FACS-sorted KIT D816V and control MCs (n = 5 and n = 4, respectively, derived from 2 KIT D816V iPSC lines and 1 control iPSC line) treated with nintedanib, midostaurin, or imatinib. Nintedanib IC50 values were 34 and 633 nM for KIT D816V and control cells, respectively. Midostaurin IC50 values were 48 and 570 nM for KIT D816V and control cells, respectively. (G) Averaged drug response ± standard deviation of KIT D816V and control iPSC–derived FACS sorted MCs (n = 9-15 and n = 12, respectively) treated with 100 nM or 1 μM nintedanib, midostaurin, or imatinib. *P = .02, **P ≤ .0006, drug responses of KIT D816V vs control MCs at the same drug concentration, Welch’s t test. ns, not significant.

Nintedanib targets KIT D816V MCs. (A) Flow cytometry analysis of KIT D816V and control iPSC-derived CD45+/KIThigh MCs at >30 days of differentiation (patient 1). (B) KIT D816V MCs (red) show higher expression of FCER1A compared with control cells (blue) (upper panel; dashed line is shown as a reference). Mean fluorescence intensity (MFI) of FCER1A (n = 6-9), KIT (n = 18-19), and CD45 (n = 15-17) on KIT D816V MCs and controls (lower panels) * P = .017, Welch’s t test. (C) Representative composite images of acidic toluidine blue–stained cytospins (left panels) or smears (right panels) of FACS-sorted KIT D816V and control MCs (image assembly indicated by dotted lines). Homogenous population of multilobulated promastocytes with (arrowheads) or without (arrows) metachromatic granules were observed. Scale bars, 25 μm. (D) Same as in (C), but stained for tryptase. Cells with high (arrowheads) and low (arrows) number of tryptase-positive granules were observed. Scale bars, 25 μm. For full representative images of (C) and (D) see supplemental Figure 18. (E) Quantitative RT-PCR analysis for KIT D816V and control FACS-sorted MCs (n = 9-15 and n = 3-7, respectively). mRNA expression of MC-specific chymase 1 (CMA1), tryptase α/β 1 (TPSAB1), tryptase β2 (TPSB2), carboxypeptidase A3 (CPA3), FCER1A, and KIT is depicted. KIT D816V MCs show higher FCER1A mRNA expression compared with unmutated KIT cells. *P = .014, Welch’s t test. (F) Drug response curves of FACS-sorted KIT D816V and control MCs (n = 5 and n = 4, respectively, derived from 2 KIT D816V iPSC lines and 1 control iPSC line) treated with nintedanib, midostaurin, or imatinib. Nintedanib IC50 values were 34 and 633 nM for KIT D816V and control cells, respectively. Midostaurin IC50 values were 48 and 570 nM for KIT D816V and control cells, respectively. (G) Averaged drug response ± standard deviation of KIT D816V and control iPSC–derived FACS sorted MCs (n = 9-15 and n = 12, respectively) treated with 100 nM or 1 μM nintedanib, midostaurin, or imatinib. *P = .02, **P ≤ .0006, drug responses of KIT D816V vs control MCs at the same drug concentration, Welch’s t test. ns, not significant.

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