NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells
Background: NADPH oxidase is a hydrogen peroxide-generating enzyme, involved in redox signaling in cancer. Results: NADPH oxidase-generated hydrogen peroxide increases DNA damage and genomic instability. Conclusion: NADPH oxidase-derived hydrogen peroxide mediates DNA damage in acute myeloid leukemia (AML). Significance: The mechanism of DNA damage generation is important for studying aggressive AML phenotypes. Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22 phox , a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22 phox and p22 phox -interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22 phox , and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H 2 O 2 )-specific dye, peroxy orange 1 (PO1), and nuclear H 2 O 2 , as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H 2 O 2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22 phox localize to the nuclear membrane in MV4–11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22 phox mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.