FIGURE 6 from Pim Kinase Inhibitors Increase Gilteritinib Cytotoxicity in FLT3-ITD Acute Myeloid Leukemia Through GSK-3β Activation and c-Myc and Mcl-1 Proteasomal Degradation

S159A-mutated Mcl-1 confers resistance to Mcl-1 downregulation and to apoptosis induction by gilteritinib and AZD1208 combination treatment. A, Ba/F3-ITD cells infected with pBabe-Flag hMcl-1-S159A, containing Mcl-1 with a mutation changing serine to alanine at residue 159, preventing phosphorylation, pBabe-Flag hMcl-1 plasmid, containing wild-type Mcl-1, or pBABE-puro empty vector were treated with either gilteritinib and AZD1208 or DMSO control, and serial samples were immunoblotted for Mcl-1 and vinculin loading control. Densitometric analysis is also shown. B, To measure Mcl-1 protein turnover, cells were pretreated with CHX for 1 hour and then treated with gilteritinib and AZD1208 (+) or DMSO control (−). Serial samples were immunoblotted for c-Myc and vinculin loading control. Densitometric analysis was performed. Mcl-1 was normalized to vinculin and 50% protein turnover timepoints were determined to be 1.75 versus more than 2 hours for gilteritinib and AZD1208 versus DMSO control for empty vector, 1.27 versus 1.6 for wild-type Mcl-1 and more than 2 hours for both for S159A Mcl-1. C, Cells infected with pBabe-Flag hMcl-1-S159A, pBabe-Flag hMcl-1 or pBABE-puro empty vector control were treated with gilteritinib and/or AZD1208, or DMSO control, for 48 hours, and apoptosis was measured. Apoptosis induction by gilteritinib and AZD1208 combination was significantly reduced in cells infected with pBABE-puroS159A compared with empty vector control (**, P