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posted on 2024-02-16, 14:20 authored by Jonelle K. Lee, Aditi Chatterjee, Mario Scarpa, Christopher M. Bailey, Sandrine Niyongere, Prerna Singh, Moaath K. Mustafa Ali, Shivani Kapoor, Yin Wang, Giovannino Silvestri, Maria R. Baer <p>Pim inhibitor and gilteritinib combination treatment is synergistic in cells with FLT3-ITD. <b>A,</b><b>C,</b> Apoptosis induction. Ba/F3-ITD, MV4-11, and MOLM-14 cells were treated with the FLT3 inhibitor gilteritinib (15 nmol/L for Ba/F3-ITD and 10 nmol/L for MV4-11 and MOLM-14) and/or the Pim inhibitor AZD1208 (1 µmol/L; A) or TP-3654 (1 µmol/L; C) or DMSO control for 48 hours in triplicate experiments<b>.</b> Apoptosis was analyzed by Annexin V and PI staining, measured by flow cytometry. ****, <i>P</i> < 0.0001; ***, <i>P</i> < 0.001; **, <i>P</i> < 0.01. <b>B,</b><b>D,</b> Cytotoxicity. Ba/F3-ITD cells seeded at 5,000 cells/well and MV4-11 and MOLM-14 cells at 10,000 cells/well in 96-well plates were treated for 48 hours with gilteritinib and/or AZD1208 (B) or TP-3654 (D) as single drugs and in combinations at the concentrations shown, in triplicate. Cytotoxicity was measured by the WST-1 assay, and drug combination effects were determined by Chou-Talalay analysis. Synergism was defined by combination index values < 0.8.</p>
Funding
U.S. Department of Veterans Affairs (VA)
CU | National Cancer Institute, Cairo University (NCI)
History
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.
