Concurrent FLT3 Inhibitor and PP2A Activating Drug Treatment Induces Synergistic Cytotoxicity in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication through Proteasomal Degradation of Pim-1 and c-Myc

Introduction: In 30% of acute myeloid leukemia (AML) patients, internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) causes constitutive and aberrant FLT3 signaling, and these patients have short relapse-free and overall survival. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. FLT3 activation also inhibits the tumor suppressor protein phosphatase 2A (PP2A). FLT3 inhibitors and PP2A-activating drugs have been shown to induce synergistic cytotoxicity in cells with FLT3-ITD. To address mechanisms underlying this effect, we studied effects of combination therapy on the oncogenic serine/threonine kinase Pim-1 and the transcription factor c-Myc, both of which are upregulated in cells with FLT3-ITD and are also PP2A substrates.

Methods: Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and/or the PP2A-activating drug fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Drug combination effects were measured by combination index determined by the Chou-Talalay method using CompuSyn software. Apoptosis was measured by Annexin V/propidium iodide staining detected by flow cytometry. c-Myc and GAPDH control mRNA was measured by real-time polymerase chain reaction. Pim-1 kinase, c-Myc, phospho-c-Myc^Ser62, phospho-c-Myc^Thr58, phospho-STAT5^Tyr694, STAT5, phospho-PP2A^Tyr307, PP2A, phospho-BAD^Ser112 and BAD levels were measured by immunoblotting. Cycloheximide treatment was used to assess protein stability. Protein expression and stability were measured with and without the proteasome inhibitor MG-132. Pim-1 kinase was inhibited with the pan-Pim inhibitor AZD1208. Ba/F3-ITD cells were infected with pMX-Flag-K67M kinase-dead (KD) Pim-1 and empty pMX retroviral vectors and with pBABE-ER-cMYC and with empty pBABE-ER retroviral vectors.

Results: Concurrent treatment with 15 nM gilteritinib or 1 nM quizartinib and FTY720 2 µM in cell lines and 4 µM in patient samples decreased growth and increased apoptosis of cells with FLT3-ITD, relative to single drug treatments, and produced synergistic cytotoxicity. FLT3 inhibition was confirmed by decrease in phospho-STAT5 and PP2A activation by decreased phospho-PP2A. Concurrent treatment decreased expression of both Pim-1 and c-Myc protein, but not c-Myc mRNA, in Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD, relative to single drug treatments. Additionally, selective decrease in phospho-Myc^Ser62, a stable c-Myc phosphoprotein that is dephosphorylated by PP2A, was seen, with persistence of phospho-c-Myc^Thr58. FLT3 inhibitor and PP2A activator combination treatment was found to decrease stability of c-Myc and Pim-1 protein, in relation to single drugs. Moreover, pretreatment with the proteasome inhibitor MG-132 abrogated downregulation of Pim-1 and c-Myc protein expression and decrease in Pim-1 and c-Myc protein stability in Ba/F3-ITD cells treated with FLT3 inhibitor and PP2A activator. Pretreatment with the pan-Pim kinase inhibitor AZD1208, with Pim-1 inhibition confirmed by decreased phospho-BAD^S112 had no effect on c-Myc downregulation, and c-Myc was similarly downregulated in Pim-1 kinase-dead cells as in parental and empty-vector cells, demonstrating that combination treatment effects on c-Myc are not Pim-1 kinase-dependent. Additionally, FLT3 inhibitor and PP2A-activating drug combination induced apoptosis in 30% of cells with c-Myc overexpression, compared to 60% of parental and empty vector-infected cells. Finally, c-Myc overexpression did not abrogate Pim-1 downregulation by combination treatment.

Conclusions: Concurrent FLT3 inhibitor and PP2A activating drug treatment induces synergistic cytotoxicity in AML cells with FLT3 internal tandem duplication through proteasomal degradation of Pim-1 and c-Myc, and effects on Pim-1 and c-Myc are independent. The data support in vivo testing of FLT3 inhibitor and PP2A-activating drug combinations and development of a clinical trial.