Internal tandem duplication (ITD) in Fms-like tyrosine kinase 3 (FLT3) is 
frequently observed in acute myeloid leukemia (AML). Quizartinib, gilteritinib, 
and midostaurin are inhibitors against FLT3-ITD that have good efficacy for 
FLT3-ITD-positive AML patients. Long-term administration leads to drug 
resistance through acquired tyrosine kinase domain (TKD) mutations in FLT3-ITD, 
such as N676K, F691L, D835V, and Y842C. Here, our screen to detect inhibitors 
capable of overcoming resistance to FLT3 inhibitors identified heat shock 
protein (HSP) 90 inhibitors as potential candidates. Although Ba/F3 cells 
expressing FLT3-ITD with TKD mutations (Ba/F3-ITD+N676K, Ba/F3-ITD+F691L, 
Ba/F3-ITD+D835V, and Ba/F3-ITD+Y842C) showed various resistance patterns to FLT3 
inhibitors compared with Ba/F3-ITD cells that express FLT3-ITD lacking TKD 
mutations, they were more sensitive to HSP90 inhibitors than Ba/F3 cells. 
Notably, the Ba/F3-ITD+D835V cells were the most sensitive to HSP90 inhibitors. 
Treatment with HSP90 inhibitors downregulated FLT3 and its downstream signaling 
and induced G1 arrest followed by apoptosis in Ba/F3-ITD+N676K, Ba/F3-ITD+F691L, 
Ba/F3-ITD+Y842C, and especially Ba/F3-ITD+D835V cells at lower concentrations 
compared with Ba/F3-ITD cells. The downregulation of FLT3-ITD+D835V was caused 
by rapid proteolysis in autophagy. Similar results were also observed in the 
quizartinib-resistant MV4-11 cells, QR1 and QR2, which were established by 
culturing cells in the presence of quizartinib and harbored FLT3-ITD+D835H and 
FLT3-ITD+D835V, respectively, in a single allele. Interestingly, the efficacies 
of HSP90 inhibitors in QR cells are reversely correlated with that of quizartib, 
but not to gilteritinib and midostaurin. Collectively, HSP90 inhibitors are good 
candidates to overcome drug resistance in AML with various FLT3-ITD TKD 
mutations.