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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 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 <0.0001).
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.
