290 - Subcellular Localization of the FLT3-ITD Oncogene Is Critical for the Production of NOX4- Generated Hydrogen Peroxide in Leukemia

Abstract

Reactive oxygen species (ROS)-dependent signaling is implicated in leukemia and plays a role in the initiation and disease progression. Internal tandem duplication of the juxtamembrane of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most prevalent mutation, accounting for 20% of 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 intracellular compartments, such as the endoplasmic reticulum (ER). FLT3-ITD expressing cells have been shown to generate increased levels of ROS, in particular NOX-generated ROS which act as pro-survival ROS. The purpose of this study is to investigate FLT3-ITD production of hydrogen peroxide (H2O2) 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 p22phox. Thus demonstrating the critical importance of FLT3-ITD localization for the generation of pro-survival ROS. NOX-generated ROS contribute to total endogenous H2O2 in AML as quantified by flow cytometry using the cell-permeable H2O2-probe PO1. Mitochondrial- and cyclooxygenase-generated ROS do not contribute to endogenous H2O2 in AML. 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 p22phox protein degradation by the ubiquitin proteasome pathway. To conclude, for FLT3-ITD to generate its oncogenic effects it has to be located at the plasma membrane. We would like to acknowledge The Children’s Leukaemia Research Project for generous funding to support this work.