FLT3-activating mutations are one of the most frequent genetic aberrations in acute myeloid leukemia (AML).1 Internal tandem duplication (FLT3-ITD) mutations are associated with the worst prognosis, whereas tyrosine kinase domain (FLT3/TKD) mutations have an uncertain prognostic impact, but represent a resistance mechanism to FLT3 tyrosine kinase inhibitors (FLT3 TKIs).2 Although there is evidence that FLT3 TKIs have improved outcomes for AML patients, their development has been impeded by numerous obstacles in the past 15 years. The first generation of FLT3 TKIs, such as lestaurtinib, midostaurin, and sorafenib, were multikinase inhibitors that lacked potency. [3][4][5][6] Conversely, quizartinib, a highly potent TKI against type III receptor tyrosine kinases, also inhibits c-Kit, exacerbating myelosuppression. 7 Others failed because of short in vivo half-lives. 8 Last, sustained FLT3 inhibition can result in the emergence of resistance-conferring FLT3/TKD point mutations, most often at residue D835. Gilteritinib (previously referred to as ASP2215) is a pyrazinecarboxamide derivative being studied in AML clinical trials because of its potential selectivity, potency, and activity against all classes of FLT3-activating mutations (see supplemental Figure 1, available on the Blood Web site). In this study, we investigated the activity of gilteritinib and compared it with 4 other FLT3 TKIs in clinical development: midostaurin, sorafenib, quizartinib, and crenolanib. [10][11][12][13] We tested the inhibitory activity of gilteritinib against different forms of FLT3 in leukemia cells by immunoblotting (summarized in Figure 1A; representative blots shown in supplemental Figure 2A). When tested in media, the 50% inhibitory concentration (IC 50 ) for inhibition of the wild-type receptor was 5 nM; the ITD-mutated form ranged from 0.7 to 1.8 nM depending on the cell context. In plasma, the IC 50 for inhibition of the FLT3-ITD receptor ranged from 17 to 33 nM (supplemental Figure 2B). Using a panel of FLT3 point mutations known to confer resistance to type II inhibitors, such as sorafenib and quizartinib, we also found that gilteritinib had similar degrees of inhibitory activity against commonly identified TKD mutations ( Figure 1A). It also had activity against the gatekeeper mutation at F691, although at a relatively higher IC 50 .In addition to FLT3, data from the kinase selectivity assay (supplemental Figure 1) indicated that gilteritinib has activity against receptor tyrosine kinase Axl, which may modulate the activity of FLT3 in AML.14 We confirmed that the drug inhibits Axl (supplemental Figure 2C), although the IC 50 against this receptor is 41 nM, approximately 20-fold higher than what we observed for the ITD-mutated FLT3 receptor.One obstacle in the development of FLT3 inhibitors has been the inability to achieve sustained FLT3 inhibition in vivo. 8,15,16 We used the plasma inhibitory activity assay to confirm sustained in vivo inhibition, 4 with plasma collected at trough time points from patients receiving gilte...
