CYP3A4 in BMSCs blocked the inhibitory activity of FLT3 TKIs in FLT3/ITD AML in the absence of AML–stromal cellular contact. (A) Expression of phosphorylated FLT3 (P-FLT3) and total FLT3 (T-FLT3) in Molm14 cells by western blotting after 1 hour of incubation with 3 different FLT3 TKIs (20 nM sorafenib, 20 nM gilteritinib, and 3.5 nM quizartinib) that had been incubated with or without bone marrow stroma for 72 hours. The left 3 control lanes are conditioned media without TKIs (just DMSO). (B) Quantification of phosphorylated FLT3 in Molm14 cells. Data are the mean ± SEM of 4 (sorafenib) and 5 (gilteritinib) independent experiments. Quantification of phosphorylated FLT3 with quizartinib treatment is not presented because the experiment was performed once. ***P < .001. CT Stro, control F/STRO; KD Stro, shRNA CYP3A4 knockdown F/STRO; Liq, no F/STRO.