TP-3654 enhances efficacy of gilteritinib in vivo. NRG mice injected intravenously with MV4-11-luc cells were treated with gilteritinib and/or TP-3654, or vehicle control, with first treatment day defined as day 1. A, Serial images of mice in first experiment, treated with gilteritinib 7.5 mg/kg and/or TP-3654 50 mg/kg, or vehicle control, once every other day for three doses, followed by 2 days rest, each week. B, Changes in photon intensity, measured by bioluminescence imaging, over time, with P = 0.0043, comparing gilteritinib and TP-3654 combination versus gilteritinib alone on day 60 by two-way ANOVA with Sidak multiple comparison test. C, Survival curves, with P = 0.0027 by Kaplan–Meier analysis. Median survival was 51, 44, 51, and 65 days for mice treated with vehicle, TP-3654, gilteritinib, and gilteritinib and TP-3654 combination, respectively. D, Serial images of mice in second experiment, treated with gilteritinib 15 mg/kg and/or TP-3654 50 mg/kg, or vehicle control, daily for 5 days each week. E, Change in photon intensity, measured by bioluminescence imaging, over time, with P < 0.0001, comparing gilteritinib and TP-3654 combination versus gilteritinib alone by day 35 by two-way ANOVA.
Funding
U.S. Department of Veterans Affairs (VA)
CU | National Cancer Institute, Cairo University (NCI)
ARTICLE ABSTRACT
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 posttranslational 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 cotreatment 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 cotreatment 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.
FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3β activation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.
