Inhibitory activity of crenolanib against FLT3 TKD mutations in primary AML samples. (A) Blasts from patient 2 (de novo D835Y), patient 3 (de novo D835Y), and patient 4 (relapsed D835V) were incubated for 1 hour with crenolanib and then lysed, immunoprecipitated for FLT3, and analyzed for phospho- and total FLT3 by immunoblotting. (B) Blasts from the same patients in panel 2 were treated with crenolanib in 96-well plates for 72 hours, and cytotoxicity was analyzed by MTT. (C) Blasts from patient 4 were also treated with quizartinib and sorafenib in this analysis. In a separate immunoblot experiment (inset), blasts from patient 4 were treated with 20 nM of quizartinib, crenolanib, and sorafenib for 1 hour and then lysed, immunoprecipitated for FLT3, and analyzed by immunoblot for phospho- and total FLT3. (D) Patient 5 had a FLT3/ITD mutation and was treated with sorafenib, responded to treatment, and then relapsed. Blasts collected after relapse harbored a D835F mutation, along with the original FLT3/ITD mutation. These blasts were treated with crenolanib, quizartinib, or sorafenib in 96-well plates for 72 hours, and cytotoxicity was analyzed by MTT.