The integration of specific tyrosine kinase inhibitors (TKI) such as imatinib (Gleevec) into polychemotherapy treatment protocols has significantly improved the response rate in BCR-ABL1+ leukemia patients (initial remission went from 35% to 88%). However, stable remission cannot be sustained in many cases as the leukemic cells pursue several escape mechanisms against TKI treatment; one of them is the occurrence of mutations in the ABL1 kinase domain. Especially, in the BCR-ABL1 T315I mutant, only ponatinib (TKI) is effective , albeit with severe cardiovascular side effects. As Hsp90 facilitates the correct folding of several oncogenic newly synthesized or denatured proteins, among them BCR-ABL1, therefore targeting Hsp90 with small molecule inhibitors would destabilize BCR-ABL1 and can serve as a therapeutic target. We, therefore, next tested the efficiency of 5b in a murine BA/F3 cell line stably expressing clinically relevant mutants (BCR-ABL1 T315I , BCR-ABL1 E255K , and BCR-ABL1 M351T ) with prominent TKI resistance profiles. As expected, similar to K562 cells, exposure of 5b significantly reduced proliferation (Figure ) and induced apoptosis (Figure ) at 6 and 12 µM, comparable to NB (at 0.3 mM) in BA/F3 cells expressing BCR-ABL1 T315I, E255K, or M351T mutants. Additionally, after the application of 5b, BCR-ABL1 oncoprotein was destabilized, and downstream signaling pathways (Akt and Stat5) were blocked with increasing concentrations of 5b, comparable to the human leukemic cell lines (Figure ). Furthermore, 24 h exposure of 5b on BCR-ABL1 T315I, E255K or M351T mutants-expressing BA/F3 cells significantly inhibited the colony formation ability (Figure ). In addition, in our generated human BCR-ABL1+ IM-resistant cell line models (K562-IMr and KCL22-IMr), 28 5b did not differentiate in inducing apoptosis between IM-resistant vs. IM-sensitive clones, proposing a superior effect of 5b in human IM-resistant BCR- ) Exposure of 5b to these cells destabilizes BCR-ABL1 and subsequently its associated downstream signaling pathways. d) BA/F3 BCR-ABL1 mutant-expressing cells were seeded in methylcellulose-based semisolid medium, after 24 h treatment with 5b. Colonies were counted after 14 days. e) K562-IMr cells were treated with the indicated concentration of 5b for 48 h, later dually stained with annexin V/PI, and subsequently measured by FACS. f) Primary CML CD34+ patient cells were treated with the indicated concentration of 5b or IM alone or in combination, and later the viable cells were counted after every 24 h interval for 6 days. g) Apoptosis induction in primary CML CD34+ patient cells after exposure of 5b or IB alone or in combination. g) Primary CML CD34+ patient cells were seeded in methylcellulose medium after 24 h treatment with 5b, IM alone or both in combination. Colonies were counted after 14 days. Columns depict the mean of three independent experiments (n = 3).