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FIGURE 6 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

S159A-mutated Mcl-1 confers resistance to Mcl-1 downregulation and to apoptosis induction by gilteritinib and AZD1208 combination treatment. A, Ba/F3-ITD cells infected with pBabe-Flag hMcl-1-S159A, containing Mcl-1 with a mutation changing serine to alanine at residue 159, preventing phosphorylation, pBabe-Flag hMcl-1 plasmid, containing wild-type Mcl-1, or pBABE-puro empty vector were treated with either gilteritinib and AZD1208 or DMSO control, and serial samples were immunoblotted for Mcl-1 and vinculin loading control. Densitometric analysis is also shown. B, To measure Mcl-1 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. Densitometric analysis was performed. Mcl-1 was normalized to vinculin and 50% protein turnover timepoints were determined to be 1.75 versus more than 2 hours for gilteritinib and AZD1208 versus DMSO control for empty vector, 1.27 versus 1.6 for wild-type Mcl-1 and more than 2 hours for both for S159A Mcl-1. C, Cells infected with pBabe-Flag hMcl-1-S159A, pBabe-Flag hMcl-1 or pBABE-puro empty vector control 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 pBABE-puroS159A compared with empty vector control (**, P < 0.001).

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.