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FIGURE 1 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

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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

Pim inhibitor and gilteritinib combination treatment is synergistic in cells with FLT3-ITD. A,C, 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. Apoptosis was analyzed by Annexin V and PI staining, measured by flow cytometry. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01. B,D, 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.

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.