FIGURE 5 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

T58A-mutated c-Myc confers resistance to c-Myc downregulation and to apoptosis induction by gilteritinib and AZD1208 combination treatment. A, Ba/F3-ITD cells infected with pCDH-MSCV-MycT58A-EF1a-copGFP, containing c-Myc with a mutation changing threonine to alanine at residue 58, preventing phosphorylation, pCDH-MSCV-Myc(WT)-EF1a-copGFP, containing wild-type c-Myc, or pCDH-MSCV-MCS-EF1-copGFP empty vector were treated with either gilteritinib and AZD1208 or DMSO control and serial samples were immunoblotted for c-Myc and vinculin loading control. Densitometric analysis is also shown. B, To measure c-Myc 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. Densitometry was performed. c-Myc was normalized to vinculin and 50% protein turnover timepoints were determined to be 1.035 versus 1.2 hours for gilteritinib and AZD1208 versus DMSO control for empty vector, 0.6 versus 0.72 for wild-type c-Myc and more than 2 hours for both for T58A c-Myc. C, Cells infected with pCDH-MSCV-MycT58A-EF1a-copGFP, pCDH-MSCV-Myc(WT)-EF1a-copGFP or pCDH-MSCV-MCS-EF1-copGFP empty vector 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 pMSCVpuro-Flag-cMyc T58A, compared with empty vector control (***, P