Figure 6.
Synergistic inhibition of proliferation and FLT3 activation after combined treatment with next-generation menin-MLL and FLT3 inhibitors. (A) Dose-response curves of MOLM13 and MV411 cells treated with quizartinib for 24 hours, comparing cells transduced with short hairpin RNAs against MEN1 with control-transduced cells (shLUC). (B) mRNA expression levels of MEN1, MEIS1, and FLT3 in MOLM13 and MV411 cells with MEN1 knock down or control-transduced cells, assessed 48 hours after transduction. (C) Dose-response curves from cell-viability assays after 7 days of treatment with VTP-50469 in human (left) and murine (right) leukemia cells. Viable (4′,6-diamidino-2-phenylindole [DAPI]-negative) cells were assessed by flow cytometry. (D-E) Dose-response curves from cell viability assays of MV411 and MOLM13 cells comparing VTP-50469 (VTP; 3 days for MV411 and 4 days for MOLM13 cells), quizartinib (Qz, 24 hours; D), gilteritinib (Gil, 24 hours; E), and combinatorial VTP-50469 (3 or 4 days) and FLT3 inhibition (24 hours) treatment. Dashed lines indicate IC50 values. (F) Dose-response curves from cell viability assays of Npm1CA/+Flt3ITD/+ cells comparing VTP-50469 (VTP; 6 days), ponatinib (Po, 24 hours, [left]), or gilteritinib (Gil, 24 hours; [right]) with their combination (6 days VTP50469, 24 hours FLT3 inhibition). Dashed lines indicate IC50 values. (G-H) FLT3 and MEIS1 mRNA expression in murine Npm1CA/+Flt3ITD/+ (G), human MV411 (H; left), and MOLM13 (H; right) leukemia cells after single or combined treatment with VTP-50469 (100 nM; 4 days for MOLM13 and Npm1CA/+Flt3ITD/+ cells and 3 days for MV411 cells) and FLT3 inhibitors (quizartinib, 3 nM and ponatinib, 100 nM; 24 hours) as assessed by qRT-PCR. Bar graphs represent the mean with standard deviation of 3 independent experiments, each performed in technical triplicate. (I) Immunoblot analysis of FLT3 and phosphorylated (p)FLT3 in MV411 cells (left) and MOLM13 cells (right) after treatment with VTP-50469 (100 nM; 3 and 4 days in MV411 and MOLM-13, respectively) and quizartinib (3 nM, 24 hours), or their combination. Numbers indicate the DMSO-normalized quantification of western blot signals, relative to the loading control, performed by densitometry using the ImageJ software tool.

Synergistic inhibition of proliferation and FLT3 activation after combined treatment with next-generation menin-MLL and FLT3 inhibitors. (A) Dose-response curves of MOLM13 and MV411 cells treated with quizartinib for 24 hours, comparing cells transduced with short hairpin RNAs against MEN1 with control-transduced cells (shLUC). (B) mRNA expression levels of MEN1, MEIS1, and FLT3 in MOLM13 and MV411 cells with MEN1 knock down or control-transduced cells, assessed 48 hours after transduction. (C) Dose-response curves from cell-viability assays after 7 days of treatment with VTP-50469 in human (left) and murine (right) leukemia cells. Viable (4′,6-diamidino-2-phenylindole [DAPI]-negative) cells were assessed by flow cytometry. (D-E) Dose-response curves from cell viability assays of MV411 and MOLM13 cells comparing VTP-50469 (VTP; 3 days for MV411 and 4 days for MOLM13 cells), quizartinib (Qz, 24 hours; D), gilteritinib (Gil, 24 hours; E), and combinatorial VTP-50469 (3 or 4 days) and FLT3 inhibition (24 hours) treatment. Dashed lines indicate IC50 values. (F) Dose-response curves from cell viability assays of Npm1CA/+Flt3ITD/+ cells comparing VTP-50469 (VTP; 6 days), ponatinib (Po, 24 hours, [left]), or gilteritinib (Gil, 24 hours; [right]) with their combination (6 days VTP50469, 24 hours FLT3 inhibition). Dashed lines indicate IC50 values. (G-H) FLT3 and MEIS1 mRNA expression in murine Npm1CA/+Flt3ITD/+ (G), human MV411 (H; left), and MOLM13 (H; right) leukemia cells after single or combined treatment with VTP-50469 (100 nM; 4 days for MOLM13 and Npm1CA/+Flt3ITD/+ cells and 3 days for MV411 cells) and FLT3 inhibitors (quizartinib, 3 nM and ponatinib, 100 nM; 24 hours) as assessed by qRT-PCR. Bar graphs represent the mean with standard deviation of 3 independent experiments, each performed in technical triplicate. (I) Immunoblot analysis of FLT3 and phosphorylated (p)FLT3 in MV411 cells (left) and MOLM13 cells (right) after treatment with VTP-50469 (100 nM; 3 and 4 days in MV411 and MOLM-13, respectively) and quizartinib (3 nM, 24 hours), or their combination. Numbers indicate the DMSO-normalized quantification of western blot signals, relative to the loading control, performed by densitometry using the ImageJ software tool.

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