Fluvastatin reverses resistance to FLT3 tyrosine kinase inhibitors mediated by multiple mechanisms. (A) Resistance to lestaurtinib induced by stimulation with FLT3 ligand (FL) is overcome by prior treatment with fluvastatin. BaF3 FLT3/ITD cells were treated with or without 1μM fluvastatin for 24 hours followed by incubation in lestaurtinib for 1 hour and then stimulated with 20 ng/mL FL for 5 minutes. Densitometry of the pFLT3 bands plotted in response to treatment is shown. (B) BaF3 cells expressing a FLT3/ITD, D835Y, or FLT3/ITD N676K resistance mutation were treated with sorafenib for 48 hours and viability was assessed by MTT assay. (C) The same cells were treated for 24 hours with increasing concentrations of fluvastatin to study FLT3 glycosylation or (D) for 48-96 hours and analyzed by MTT assay. Cells were simultaneously treated with either (E) lestaurtinib or (G) fluvastatin and 10 ng/mL interleukin-3 for 48 hours after which cell growth was evaluated by MTT. (F) Activation of IL-3 receptor signaling pathways was assessed by treating cells with or without 1μM fluvastatin for 24 hours followed by incubation with or without lestaurtinib for 1 hour and a 10-minute stimulation with or without 10 ng/mL IL-3 followed by SDS-PAGE Western blotting analysis of STAT5, AKT, and MAPK activation states. Results are representative of at least 3 experiments.