Efficacy of AX in TKI-resistant leukemic cell line models. (A) IM-resistant K562, KCL22, SUP-B15, and BA/F3-expressing BCR-ABL1^{T315I, T315I (PNr)} along with their respective normal cell lines were treated with second- and third-generation TKIs (dasatinib, nilotinib, radotinib, bosutinib, befetinib, and ponatinib) at 7 different concentrations (ranging from 50 nM to 25 µM) for 72 hours. Later, the average IC₅₀ was determined and plotted on a heat map. (B) BA/F3 cells expressing BCR-ABL1^{T315I, T315I (PNr), M351T, and E255K} mutants, K562 IMr, KCL22 IMr, and SUP-B15 IMr cells were treated with the indicated concentration of AX (48 hours) and later enzymatic activity of caspase-3/7 were examined by caspase-3/7 dependent-Glo assay (absorbance at 405 nm). (C) Likewise, in human leukemia cell lines, AX causes downregulation of BCR-ABL1 and subsequently its associated downstream signaling pathways, including Stat5a, Akt, and Bcl-2 in BA/F3 cells expressing BCR-ABL1^{T315I, T315I (PNr), M351T, and E255K} mutants. (D) Normal BA/F3 cells, BA/F3-expressing BCR-ABL1^{T315I and T3151 (PNr)} mutants, and K562 IMr cells were seeded in methylcellulose medium containing respective compounds at indicated concentration after treatment in liquid medium for 24 hours. Colonies were counted after 14 days. Significance analysis of normally distributed data with variance similar between groups used paired, 2-tailed Student t test. *P < .05, **P < .005, ***P < .001.