Effects of single and combined menin-MLL and FLT3 inhibition on FLT3 and phosphorylated FLT3 protein levels. (A) FLT3 and MEIS1 mRNA expression in human MOLM13 (left) and MV411 (middle) cells and murine Npm1^CA/+Flt3^ITD/+ (right) leukemia cells after single or combinatorial treatment with MI-503 (2.5 µM; 4 days for MOLM13 and Npm1^CA/+Flt3^ITD/+ cells and 3 days for MV411 cells) and FLT3 inhibitors (quizartinib, 3 nM; ponatinib, 100 nM, 24 hours) as assessed by qRT-PCR. Bar graphs represent the mean with SD of 3 independent experiments, each performed in technical triplicate. (B) Immunoblot analysis of FLT3 and phosphorylated (p)FLT3 in MOLM13 cells (left) and MV411 cells (right) upon treatment with 2.5 µM MI-503 (for 3 and 4 days in MV411 and MOLM13 cells), quizartinib (3 nM, 24 hours), or the 2 combined. One representative blot of 3 independent experiments is shown. Numbers indicate the DMSO-normalized quantification of western blot signals, relative to the loading control, performed by densitometry with ImageJ software. (C) Immunoblot analysis of STAT5 and phosphorylated (p)STAT5 in MOLM13 (left) and MV411 (right) cells after treatment as described in panel B. One representative blot of 3 independent experiments is shown. Numbers indicate the DMSO-normalized quantification of western blot signals, relative to the loading control, performed by densitometry with ImageJ software. pFLT3 (D) and pSTAT5 (E) protein expression in human MOLM13 and MV411 and murine Npm1^CA/+Flt3^ITD/+ cells after treatment with MI-503 (2.5 µM; 4 days for MOLM13 and Npm1^CA/+Flt3^ITD/+ cells; 3 days for MV411 cells), FLT3 inhibitor (quizartinib, 3 nM, and ponatinib, 100 nM; 24 hours) or their combination, as assessed by flow cytometry. One representative histogram of 3 independent experiments is shown. The colored numbers in the flow histograms indicate the percentage of pFLT3⁺ and pSTAT5⁺ cells, respectively.