Fig. 2

The Hippo transducers TAZ and TEAD mediate FLT3-induced drug resistance in BP-CML. A PCA analysis of the gene expression profiles of K562-mock, FLT3, FLT3-IR, and -NR cells. B Heatmap showing the relative expression of pro-tumorigenic factors within the major oncogenic pathways (color-coded as indicated in the legend) in RNA-seq data from K562-mock, K562-FLT3, K562-FLT3-IR, and NR cells. C Immunoblotting analysis comparing the expression patterns of various proteins in solid tumors and leukemia cell lines. D and E qPCR analyses of WWTR1 (D) and YAP1 (E) mRNA expression levels in K562-mock, FLT3, FLT3-IR, and FLT3-NR cells. **p < 0.01, ***p < 0.001; ns, not significant (p > 0.05). F Immunoblotting analysis of TAZ and YAP in K562-mock, FLT3, and TKI-resistant FLT3-IR, -NR, and -DR cells. G and H Expression of WWTR1 (G) and YAP1 (H) transcript levels in normal CD34 + cells (CD34) and each CML phase were analyzed from the NCBI Gene Expression Omnibus database (accession no. GSE4170). CD34, n = 7; CP, n = 57; AP, n = 9; BP, n = 33. p-values were calculated using Student’s t-test and error bars are means of ± SD. ***p < 0.001; ns, not significant (p > 0.05). I ChIP-seq analysis showing histone H3K27 acetylation (H3K27ac) profiles for the WWTR1 and YAP1 loci in K562-mock and -FLT3-IR cells. The promoter regions for each gene are highlighted. J Immunoblotting analysis of TAZ induction and FLT3 activation in K562-mock and K562-FLT3 cells treated with FLT3 ligand (0, 10, 30, and 100 ng/ml) for 36 h. K Immunofluorescence images of the nuclear localization TAZ (green) and TEAD (red) in K562-FLT3-IR cells. L Immunoprecipitation analysis identifying the protein–protein interactions between endogenous TAZ with TEAD in K562-FLT3-IR cells. Ectopic expression of flag-TAZ or flag-TAZ-4SA (a constitutively active TAZ mutant) were used as positive controls. TAZ knockout (KO) cells were used as a negative control. M Acquisition of TKI resistance in K562-FLT3 cells transduced with shRNAs targeting TAZ or TEAD. Growth curve of cells treated with 1 μM imatinib (left) and a bar graph showing the indicated cell numbers measured on day 12 (right). ***p < 0.001, ****p < 0.0001. N Restoration of TKI sensitivity in K562-FLT3-IR cells transduced with shRNAs targeting TAZ or TEAD. Growth curve analysis of cells treated with 1 μM imatinib (left) and a bar graph showing the indicated cell numbers measured on day 14 (right). ****p < 0.0001. O Colony formation assay in K562-FLT3 cells treated with 3 μM imatinib and 20 ng/ml FL (left column) and K562-FLT3-IR cells treated with 3 μM imatinib for 3 weeks (right column) after knockdown of TAZ or TEAD. P Growth curves (left) and viability (right) of K562-FLT3 cells treated with 1 μM imatinib with or without TEAD inhibitor YBY-15 (30 μM). ****p < 0.0001. D-E, M–N, P All p-values were calculated using one-way ANOVA with Bonferroni corrections for multiple comparisons (D-E, M–N) and Student’s t-test (P). Error bars are means of triplicates ± SD. A p-value of less than 0.05 indicates a statistical difference