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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Mol Cancer Ther. 2021 Feb 10;20(4):676–690. doi: 10.1158/1535-7163.MCT-20-0663

Figure 7. PP2A-activating drug and FLT3 inhibitor combination downregulates c-Myc and Pim-1 expression through GSK-3β activation and AKT inactivation is necessary and sufficient for GSK-3β activation by PP2A-activating drug and FLT3 inhibitor combination.

Figure 7.

A. Combination treatment activates GSK-3α/β. c-Myc, Pim-1, p-GSK-3α/β (S21/S9) and GSK-3α/β protein expression was measured by immunoblotting in MV4-11 cells treated with 15 nM gilteritinib and/or 10 μM DT-061, or DMSO control, showing rapid decrease in p-GSK-3α/β (S21/S9) (GSK-3α/β activation) with combination treatment. This blot is also shown in Figure 2B. B. GSK-3β inhibitor prevents c-Myc and Pim-1 downregulation by combination treatment. c-Myc, Pim-1, p-GSKα/β (S21/S9) and GSKα/β protein expression was measured by immunoblotting in Ba/F3-ITD cells treated with the GSK-3β inhibitor TC-G 24 (17 nM) and/or 15 nM gilteritinib and 10 μM DT-061. TC-G 24 increased p-GSKα/β, demonstrating GSKα/β inactivation. C. GSK-3β inhibitor prevents c-Myc and Pim-1 downregulation by combination treatment in FLT3-ITD AML patient blasts. c-Myc and Pim-1 expression was measured by immunoblotting in blasts treated with the GSK-3β inhibitor TC-G 24 (17 nM) and/or 15 nM gilteritinib and 10 μM DT-061. D. GSK-3β inhibitor prevents apoptosis induction by PP2A-activating drug and FLT3 inhibitor combination. Apoptosis was measured by annexin V/PI staining in Ba/F3-ITD cells cultured for 48 hours with DMSO control, FTY720 or DT-061 and and/or gilteritinib (left) or quizartinib (right), GSK-3β inhibitor TC-G 24 or combinations. GSK-3β inhibitor prevented apoptosis induction (P<0.0005). E. Constitutive AKT activation prevents GSK-3β activation. p-GSK-3α/β and GSK-3α/β protein expression was measured in parental Ba/F3-ITD and Ba/F3-ITD cells infected with empty vector or myristoylated AKT1 treated with 1 nM quizartinib and 2 μM FTY720. F. AKT inhibitor activates p-GSK-3α/β. p-GSK-3α/β and GSK-3α/β expression was measured in Ba/F3-ITD cells treated with the AKT inhibitor MK-2206 (5 μM) and/or 15 nM gilteritinib and 2 μM FTY720.