Subcellular localization of the FLT3-ITD oncogene plays a significant role in the production of NOX- and p22phox-derived reactive oxygen species in acute myeloid leukemia

Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) receptor is the most prevalent FLT3 mutation accounting for 20% of acute myeloid leukemia (AML) patients. FLT3-ITD mutation results in ligand-independent constitutive activation of the receptor at the plasma membrane and 'impaired trafficking' of the receptor in compartments of the endomembrane system, such as the endoplasmic reticulum (ER). FLT3-ITD expressing cells have been shown to generate increased levels of Reactive Oxygen Species (ROS), in particular NADPH Oxidase (NOX)-generated ROS which act as pro-survival signals. The purpose of this study is to investigate FLT3-ITD production of ROS at the plasma membrane and ER in the FLT3-ITD expressing AML cell line MV4-11. Receptor trafficking inhibitors; Tunicamycin and Brefeldin A induce ER retention of FLT3-ITD, resulting in a decrease in protein expression of NOX4 and its partner protein p22^phox, thus demonstrating the critical importance of FLT3-ITD localization for the generation of pro-survival ROS. NOX-generated ROS contribute to total endogenous hydrogen peroxide (H₂O₂) in AML as quantified by flow cytometry using the cell-permeable H₂O₂-probe Peroxy Orange 1 (PO1). We found that PI3K/Akt signaling only occurs when FLT3-ITD is expressed at the plasma membrane and is required for the production of NOX-generated ROS. ER retention of FLT3-ITD resulted in NOX4 deglycosylation and p22^phox protein degradation.

Acute myeloid leukemia; FLT3-ITD; NADPH oxidase; Oncogene; Pro-survival reactive oxygen species; p22(phox).