Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors

Abstract

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

Shwachman–Bodian–Diamond syndrome (SBDS) protein is a direct inhibitor of protein phosphatase 2A (PP2A) activity and overexpressed in acute myeloid leukaemia

“…We have previously shown that PP2A activity is reduced in myeloid cells and patients blasts harbouring constitutively active c-KIT/D816V and FLT3/ITD compared with GM-CSF-dependent FD-EV cells and activity is increased using FTY720 and AAL(S) [3][4][5]. Here, we confirmed this and showed that shRNA-mediated knockdown of SBDS (shSBDS) increased PP2A activity ( Fig.…”

“…The family of serine/threonine phosphatases (PP2A) frequently shows reduced activity in myeloid leukaemias [1,2]. This is particularly the case in acute myeloid leukaemias (AML) driven by overexpression or constitutively active c-KIT and FLT3, where PP2A inhibition is required for cell transformation which enhances the activation of oncogenic signalling pathways and promotes anti-apoptotic processes [2][3][4]. In myeloid malignancies, pharmacological activation of PP2A using Forskolin, the immunosuppressant FTY720 (reviewed in [2]) and the non-immunosuppressive chiral-deoxy analogue of FTY720, AAL(S) has a potential therapeutic benefit [4,5].…”

FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions

“…FLT3 activation inhibits activity of the tumor suppressor serine/threonine phosphatase protein phosphatase 2A (PP2A), and PP2A activating drugs , including the immunomodulating agent fingolimod (FTY720), FDA-approved for relapsing multiple sclerosis, are cytotoxic toward cells with FLT3-ITD and produce synergistic cytotoxicity with FLT3 inhibitors in cells with FLT3-ITD in vitro [72, 73], including in the presence of bone marrow stroma [73]. PP2A activating drugs do not decrease phosphorylation of FLT3-ITD and actually increase phosphorylation of STAT5, but significantly decrease phosphorylation of AKT and ERK [72].…”

Targeting PP2A in cancer: Combination therapies

“…Combination therapy in both cases resulted in synergistic cell death measured by cell viability. Similarly, Smith et al reported PP2A inhibition downstream of FLT3-ITD expressing AML via decreased expression of PP2A-A and investigated the ability of PP2A reactivation in sensitizing response to FLT3 inhibitors [38]. This study utilized FTY720 and AAL(S), a related analog that lacks immunomodulatory effects, in combination with multiple FLT3 inhibitors.…”