Fig. 6

Combined inhibition of BCR::ABL1 and FLT3 suppresses BP-CML tumor growth. A Cell images of TKI-resistant K562-FLT3-IR, -NR, and -DR cells treated with either BCR::ABL1 TKI (1 μM imatinib, 20 nM nilotinib, 1 μM dasatinib), 30 nM quizartinib (QZ), 0.1 μM midostaurin (MD) or the combination of each BCR::ABL1 TKI with either quizartinib or midostaurin for 7 days. B Measurements of cell growth (left) and cell viability (day 18) (right) of K562-FLT3-IR cells subjected to single treatments with vehicle (con), 1 μM imatinib (IM), 30 nM quizartinib (QZ), or the combination of imatinib and quizartinib (IM + QZ). n = 3 per group. *p < 0.05, ****p < 0.0001; ns, not significant. C, Immunoblotting analysis of FLT3-pSTAT-TAZ signaling components in K562-FLT3-IR cells subjected to single treatments with 1 μM imatinib (IM), 30 nM quizartinib (QZ), or the combination of imatinib and quizartinib (IM + QZ) for 16 h. D Cell images of TKI-resistant K562-FLT3-IR, -NR, and -DR cells treated with the indicated BCR::ABL1 inhibitors as in (A) or with 10 nM ponatinib (PN). E Cell viability measurement for K562-FLT3-IR cells treated with 1 μM imatinib (IM) or 10 nM ponatinib (PN) treatment. ****p < 0.0001; ns, not significant (p > 0.05). F Immunoblotting analysis of FLT3-pSTAT-TAZ signaling components in K562-FLT3-IR cells treated with 1 μM imatinib (IM), 20 nM nilotinib (NL), 1 μM dasatinib (DS), or 10 nM ponatinib (PN) for 16 h. G Experimental procedure to analyze the therapeutic efficacy of combined treatment of imatinib and quizartinib (IM + QZ) or single treatment of ponatinib (PN) in K562-FLT3-IR xenograft model. H Tumor growth analysis of K562-FLT3-IR-derived xenografts in NOD/SCID mice treated with either vehicle (con), 100 mg/kg imatinib (IM), 30 mg/kg quizartinib (QZ), 15 mg/kg ponatinib (PN), or the combination of imatinib and quizartinib (IM + QZ). All drugs were delivered by daily oral gavage. n = 3 per group. I and J Tumor images (I) and tumor weights (J) of excised K562-FLT3-IR-derived tumors from each group (n = 7). ****p < 0.0001; ns, not significant. K Analysis of FLT3 driver mutations by cDNA sequencing of BP-CML patient samples. L Cell viability measurement of FLT3⁺ imatinib-resistant BP-CML patient-derived BMMCs subjected to combined treatment with imatinib (IM) and midostaurin (MD) for 5 days. n = 3 per group. ***p < 0.001. Independent patient information and results are shown in Fig. S5I. M Cell viability measurement of FLT3⁺ imatinib-resistant BP-CML patient-derived BMMCs subjected to treatment with either imatinib (IM) or ponatinib (PN) for 5 days. n = 5 per group. ****p < 0.0001; ns, not significant. Independent patient information and results are shown in Fig. S5J. B, E, J, L-M All p-values were calculated using one-way ANOVA with Bonferroni corrections for multiple comparisons and error bars are means ± SD. A p-value of less than 0.05 indicates a statistical difference