PP2A Activators Enhance Efficacy of FLT3 Inhibitors in FLT3-ITD Acute Myeloid Leukemia Cells through AKT Inactivation-Dependent Pim-1 and c-Myc Proteasomal Degradation

Introduction

fms-like tyrosine like kinase 3 internal tandem duplication (FLT3-ITD), present in acute myeloid leukemia (AML) cells of 30% of patients, results in constitutive and aberrant FLT3 signaling and, clinically, short disease-free survival. Efficacy of FLT3 inhibitors is limited and transient, but may be enhanced by dual targeting of FLT3-ITD signaling pathways. The tumor suppressor protein phosphatase 2A (PP2A) is inhibited in cells with FLT3-ITD. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated and also stabilized by PP2A inhibition in cells with FLT3-ITD. Pim-1 contributes directly to FLT3-ITD proliferative and anti-apoptotic effects, and also phosphorylates and stabilizes FLT3-ITD in a positive feedback loop. Moreover FLT3-ITD, PP2A and Pim-1 all regulate the transcription factor c-Myc. PP2A-activating drugs enhance efficacy of FLT3 inhibitors. We sought to identify mechanisms underlying the efficacy of this combination.

Methods

Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and blasts from patients with AML with FLT3-ITD were cultured with the FLT3 inhibitors gilteritinib (15 nM) or quizartinib (1 nM) and/or the PP2A-activating drugs FTY720 (2-4 µM) or DT-061 (10 µM), or with DMSO control. Pim-1, c-Myc, p-AKT (S473 and T308) and AKT protein expression was measured by immunoblotting, along with p-STAT5 (Y694), STAT5, p-PP2A (Y307) and PP2A expression. To study post-translational regulation, cells were cultured with cycloheximide (100 µg/mL) with and without the proteasome inhibitor MG-132 (20 µM). Ubiquitinated c-Myc was measured by co-immunoprecipitation and immunoblotting with c-Myc and ubiquitin antibodies. Ba/F3-ITD cells were stably transfected with estrogen receptor (ER)-c-Myc, kinase-dead Pim-1 or myristoylated AKT plasmids or corresponding empty vectors. Apoptosis was detected by Annexin V and propidium iodine staining, measured by flow cytometry. Cells were also cultured with the pan-Pim kinase inhibitor AZD1208 (1 µM), the Myc inhibitor 10058-F4 (100 µM) or the pan-AKT inhibitor MK-2206 (5 µM).

Results

Concurrent treatment of Ba/F3-ITD and MV4-11 cells and primary AML cells with FLT3-ITD with a FLT3 inhibitor (gilteritinib or quizartinib) and a PP2A-activating drug (FTY720 or DT-061) decreased growth and increased apoptosis induction, relative to treatment with single drugs. Concurrent FLT3 inhibitor and PP2A-activating drug treatment decreased expression of both Pim-1 and c-Myc protein. Concurrent treatment decreased Pim-1 half-life from 15 to 5 minutes, and c-Myc half-life from 30 to 5 minutes, while half-lives were restored by concurrent treatment with the proteasome inhibitor MG-132. Concurrent treatment was also shown to increase c-Myc ubiquitination. Effects of concurrent treatment on Pim-1 and c-Myc were independent, as transfection with kinase-dead Pim-1 or treatment with Pim inhibitor AZD1208 did not alter c-Myc downregulation, and c-Myc overexpression or treatment with Myc inhibitor 10058-F4 did not alter Pim-1 downregulation. Concurrent treatment with FLT3 inhibitor and PP2A-activating drug did not alter expression of c-Myc deubiquitinases, but rapidly decreased AKT S473 and T308 phosphorylation. FLT3 inhibitor and PP2A activator co-treatment did not induce downregulation or increased turnover of Pim-1 and c-Myc protein or apoptosis in cells with constitutive AKT activation caused by transfection of myristoylated AKT. Moreover, AKT inhibition downregulated Pim-1 and c-Myc protein expression, decreased Pim-1 and c-Myc protein half-lives from 15 to 5 minutes and 30 to 10 minutes, respectively, and induced apoptosis of cells with FLT3-ITD, replicating the effects of FLT3 inhibitor and PP2A activator co-treatment.

Conclusion

PP2A activators enhance the efficacy of FLT3 inhibitors in AML cells with FLT3-ITD through AKT inactivation-dependent increased Pim-1 and c-Myc proteasomal degradation, which is a novel mechanism. The data support further preclinical and clinical testing of this dual targeting approach to treatment of AML with FLT3-ITD.