Activity of crenolanib against WT and ITD-mutated FLT3 in vitro. (A) FLT3/ITD cell lines (Molm14 and MV411) and FLT3 WT cell lines (SEMK2) were treated with crenolanib for 1 hour and then cells were lysed, immunoprecipitated for FLT3, and analyzed by immunoblotting for phospho- and total FLT3. (B) An aliquot of the lysate of Molm14s treated with crenolanib from (A) was reserved for analysis of downstream signaling molecules including pAKT, pMAPK, pSTAT5, and the corresponding total protein content. (C) Cytotoxicity of these doses of crenolanib in FLT3/ITD cell lines was analyzed by MTT assay. HL-60 cells, which express extremely limited WT FLT3, were used as a control. (D) Apoptosis induced by crenolanib and sorafenib 48 hours after treatment of Molm14 cells with drug was analyzed by Annexin V flow cytometry. (E) Patient blasts containing a FLT3/ITD mutation were treated with crenolanib, lysed, and immunoblotted for phospho- and total FLT3.