H2O2 Production Downstream of FLT3 Is Mediated by p22phox in the Endoplasmic Reticulum and Is Required for STAT5 Signalling

John F Woolley,Ruth Naughton,Lavinia Bhatt,Joanna Stanicka,Christopher J Chang,Thomas G Cotter,Bryan C Dickinson,David R Gough,Vladimir V Kalinichenko

doi:10.1371/journal.pone.0034050

Abstract

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H2O2-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H2O2 after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H2O2 in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H2O2 production in AML cells is mediated by p22phox and is critical for STAT5 signalling.

Highlights

Aberrant signalling through receptor tyrosine kinases (RTKs) is known to be a significant pathway in tumour development and perpetuation [1]
MV4-11 and MOLM-13 are established IL-3 independent myelomonocytic leukemia cell lines homozygous and heterozygous for FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), respectively. Both of these cell lines have been used as models of FLT3-ITD acute myeloid leukemia (AML) in numerous studies to date and display a higher endogenous reactive oxygen species (ROS) level compared to cells with non-mutated receptors [10]
We show that treatment of MV4-11 and MOLM-13 cells with PKC412 resulted in decreased endogenous ROS, and this decrease in ROS was not observed when either HL-60 cells were treated with PKC412 (Figure 1a)

Summary

Introduction

Aberrant signalling through receptor tyrosine kinases (RTKs) is known to be a significant pathway in tumour development and perpetuation [1]. The most prevalent mutation of FLT3 is the internal tandem duplication (ITD) of the juxtamembrane domain conferring constitutive activation of the tyrosine kinase domain, leading to autophosphorylation of the receptor and subsequent phosphorylation of substrate proteins [2]. Of significant clinical relevance is the association of FLT3-ITD with increased chemoresistance in AML patients [5]. Constitutive activation of FLT3 switches on downstream signalling pathways such as PI3K/Akt, Ras/Raf/ MAPK and Jak/STAT [6]. Activation of these pathways in myeloid cells is known to promote survival, proliferation, and transformation [7,8,9]. FLT3-ITD expressing cell lines are known to have increased levels of endogenous reactive oxygen species (ROS) [10], it is still unclear precisely what advantage this confers on the cells

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