NADPH Oxidases Are Important Regulators of Growth and Migration in Myeloid Neoplasms.

Abstract 2190

Poster Board II-167

Myeloid neoplasms are frequently associated with transforming tyrosine kinase oncogenes, including BCR-ABL in chronic myelogenous leukemia (CML), JAK2V617F in myeloproliferative disorders and FLT3.ITD in acute myeloid leukemia (AML). Cells transformed by tyrosine kinase oncogenes have been found to display increased levels of intracellular reactive oxygen species (ROS). Elevated ROS levels have been implicated in genomic instability through oxidative DNA damage, but also regulate cellular functions through redox-sensitive proteins. The majority of intracellular ROS in non-phagocytic cells are produced within the mitochondria. Nevertheless, recent studies indicate a role for NADPH oxidases (NOX) in transformation. There are five NOX genes (NOX1-5) and two genes encoding the related dual oxidases (DUOX1-2) in humans. We asked whether NOXs alter cell growth, migration and ROS production in hematologic malignancies transformed by tyrosine kinase oncogenes. Real-time PCR analysis in patient-derived cell lines KU812 (BCR-ABL), HEL (JAK2V617F) and Molm13 (FLT3.ITD), showed expression of NOX2, NOX4, and NOX5. Murine cells are readily transformed by BCR-ABL, JAK2V617F and FLT3.ITD, even though the gene for NOX5 is absent in mice. Therefore, we targeted NOX2 and NOX4 in KU812, HEL and Molm13 cells using lentiviral-based shRNAs. In addition, we also performed knockdown of p22^phox, a common subunit that is required for stability and functioning of NOX1-4, thus also controlling for functional redundancy between the NOX genes. Interestingly, we found that p22^phox knockdown reduced cell growth by 16.0-32.0% in KU812, 27.3-50.9% in HEL, and 31.3-54.2% in Molm13 cells (p<0.05, n=3). Similar to cell growth, a drastic reduction in spontaneous migration compared to control cells was observed in KU812 (74.3-87.6%, p<0.05) HEL (47.1-67.6%, p<0.05) and Molm13 (51.0-75.6%, p<0.05) cells with p22^phox knockdown. Surprisingly, knockdown of NOX2, NOX4, or p22^phox did not significantly reduce intracellular ROS levels. Further, mitochondrial electron transport chain inhibitors led to a decrease in intracellular ROS levels and oxygen consumption in KU812, HEL and Molm13 cells. Using patient samples, a similar decrease in ROS was observed in response to complex III inhibitors myxothiazol (CML: 57.2±9.4%; polycythemia vera: 36.2±0.4%; AML: 51.8±5.2%; p<0.05, n=4) and stigmatellin (CML: 62.5±7.8%; PV: 48.1±4.2%; AML: 53.9±5.5%; p<0.05, n=4). Consistent with the above data, KU812, HEL and Molm13 cells treated with their respective tyrosine kinase inhibitors, showed a reduction in MitoSOX Red fluorescence, an indication for reduced mitochondrial superoxide radicals (KU812: -38.9%; HEL: -31.3%; Molm13: -41.9%), further implicating mitochondria in the excess generation of intracellular ROS. Overall, these data suggest that expression of NOX proteins is required for optimal growth and migration of myeloid leukemia cells and therefore hints at novel targets for drug development that may aid traditional therapy. (supported in part by NIH/NCI grant 5R01CA134660-02)