Nuclear membrane-localised NOX4D generates pro-survival ROS in FLT3-ITD-expressing AML

// Jennifer N. Moloney 1 , Ashok Kumar Jayavelu 2, 3 , Joanna Stanicka 1 , Sarah L. Roche 1 , Rebecca L. O’Brien 1 , Sebastian Scholl 4 , Frank-D. Bohmer 2 and Thomas G. Cotter 1 1 Tumour Biology Laboratory, School of Biochemistry and Cell Biology, Bioscience Research Institute, University College Cork, Cork, Ireland 2 Institute of Molecular Cell Biology, CMB, Jena University Hospital, Jena, Germany 3 Current address: Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany 4 Department of Haematology/Oncology, Clinic for Internal Medicine II, Jena University Hospital, Jena, Germany Correspondence to: Thomas G. Cotter, email: t.cotter@ucc.ie Keywords: acute myeloid leukaemia; FLT3-ITD; pro-survival reactive oxygen species; NOX4 splice variant D/NOX4D 28 kDa; nuclear membrane Received: July 27, 2017     Accepted: October 02, 2017     Published: November 01, 2017 ABSTRACT Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most prevalent genetic aberration present in 20-30% of acute myeloid leukaemia (AML) cases and is associated with a poor prognosis. FLT3-ITD expressing cells express elevated levels of NADPH oxidase 4 (NOX4)-generated pro-survival hydrogen peroxide (H 2 O 2 ) contributing to increased levels of DNA oxidation and double strand breaks. NOX4 is constitutively active and has been found to have various isoforms expressed at multiple locations within a cell. The purpose of this study was to investigate the expression, localisation and regulation of NOX4 28 kDa splice variant, NOX4D. NOX4D has previously been shown to localise to the nucleus and nucleolus in various cell types and is implicated in the generation of reactive oxygen species (ROS) and DNA damage. Here, we demonstrate that FLT3-ITD expressing-AML patient samples as well as -cell lines express the NOX4D isoform resulting in elevated H 2 O 2 levels compared to FLT3-WT expressing cells, as quantified by flow cytometry. Cell fractionation indicated that NOX4D is nuclear membrane-localised in FLT3-ITD expressing cells. Treatment of MV4-11 cells with receptor trafficking inhibitors, tunicamycin and brefeldin A, resulted in deglycosylation of NOX4 and NOX4D. Inhibition of the FLT3 receptor revealed that the FLT3-ITD oncogene is responsible for the production of NOX4D-generated H 2 O 2 in AML. We found that inhibition of the PI3K/AKT and STAT5 pathways resulted in down-regulation of NOX4D-generated pro-survival ROS. Taken together these findings indicate that nuclear membrane-localised NOX4D-generated pro-survival H 2 O 2 may be contributing to genetic instability in FLT3-ITD expressing AML.