Pim kinase inhibitors increase gilteritinib cytotoxicity in FLT3-ITD acute myeloid leukemia through GSK-3β activation and c-Myc and Mcl-1 proteasomal degradation

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) has poor outcomes. FLT3-ITD drives constitutive and aberrant FLT3 signaling, activating STAT5 and upregulating the downstream oncogenic serine/threonine kinase Pim-1. FLT3 inhibitors are in clinical use, but with limited and transient efficacy. We previously showed that concurrent treatment with Pim and FLT3 inhibitors increases Apoptosis induction in FLT3-ITD-expressing cells through post-translational downregulation of Mcl-1. Here we further elucidate the mechanism of action of this dual targeting strategy. Cytotoxicity, Apoptosis and protein expression and turnover were measured in FLT3-ITD-expressing cell lines and AML patient blasts treated with the FLT3 Inhibitor gilteritinib and/or the Pim inhibitors AZD1208 or TP-3654. Pim Inhibitor and gilteritinib co-treatment increased Apoptosis induction, produced synergistic cytotoxicity, downregulated c-Myc protein expression, earlier than Mcl-1, increased turnover of both proteins, which was rescued by Proteasome inhibition, and increased efficacy and prolonged survival in an in vivo model. Gilteritinib and Pim Inhibitor co-treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids, preventing phosphorylation at these sites, did not downregulate these proteins, increase their turnover or increase Apoptosis induction. Moreover, concurrent treatment with gilteritinib and Pim inhibitors dephosphorylated (activated) the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β), and GSK-3β inhibition prevented c-Myc and Mcl-1 downregulation and decreased Apoptosis induction. The data are consistent with c-Myc T58 and Mcl-1 S159 phosphorylation by activated GSK-3β as the mechanism of action of gilteritinib and Pim Inhibitor combination treatment, further supporting GSK-3β activation as a therapeutic strategy in FLT3-ITD AML. .