Redox signalling in myeloid leukaemia
Moloney, Jennifer Noreen
University College Cork
FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase (RTK) expressed in approximately 90% of acute myeloid leukaemia (AML) patients. Internal tandem duplication of sequences in the juxtamembrane domain of the FLT3 receptor (FLT3-ITD) is the most prevalent FLT3 mutation accounting for 15-35% of AML cases. FLT3-ITD expressing cells produce elevated levels of reactive oxygen species (ROS), particularly NADPH oxidase 4 (NOX4) - and p22phox -generated ROS which act as pro-survival signals. Increased ROS production in AML is linked to enhanced cell survival and proliferation as well as a differentiation block. Little was known of the mechanism in which the FLT3-ITD oncoprotein activates NOX4-generated hydrogen peroxide (H2O2) and thus this PhD project was designed to elucidate the mechanism. The FLT3-ITD mutation results in ligand-independent constitutive activation of the FLT3 receptor at the plasma membrane and impaired trafficking of the FLT3 receptor in compartments of the endomembrane system, such as the endoplasmic reticulum (ER). Firstly, we investigated FLT3-ITD-induced activation of aberrant prosurvival signalling cascades resulting in the activation and generation of NOX4- and p22phox -generated H2O2 at the plasma membrane and ER. To this end, receptor trafficking inhibitors, tunicamycin and brefeldin A were employed and resulted in ER retention of FLT3-ITD in the FLT3-ITD expressing AML MV4-11 cell line. Inhibition of FLT3-ITD cell surface expression resulted in decreased NOX4 and p22phox protein levels, suggesting an important role for FLT3-ITD subcellular localisation in the generation of pro-survival ROS. We found that PI3K/AKT signalling only occurs downstream of FLT3-ITD at the plasma membrane and is required for the generation of NOX4- and p22phox -generated pro-survival H2O2 in AML. Taken together, these findings identify that FLT3-ITD at the plasma membrane is responsible for the production of NOX4- and p22phox -generated H2O2. Next, we investigated and identified the pro-survival signalling pathways downstream of FLT3-ITD at the plasma membrane and the ER. The PI3K/AKT and ERK1/2 signalling pathways are activated and GSK3β signalling is inhibited downstream of FLT3-ITD at the plasma membrane. STAT5 signalling is activated downstream of FLT3-ITD at the ER. Activation of the ERK1/2 pathway results in the inhibition of GSK3β signalling through phosphorylation of serine at position 9. NOX4 is a major source of ROS in AML. Given its constitutive activity we investigated its subcellular localisation. We show for the first time that FLT3-ITD expressing patient samples and cells express the NOX4 splice variant D (NOX4D 28 kDa). FLT3-ITD expressing AML cells express NOX4D in the nuclear membrane where it is contributing to endogenous H2O2 and may be involved in genetic instability. We have also identified that prototype NOX4 and p22phox colocalise to the nuclear membrane of MV4-11 and 32D/FLT3-ITD cells. Glycosylation of NOX4 and NOX4D is critical for their oncogenic effects. We have shown that the PI3K/AKT and STAT5 pathways are responsible for the production of NOX4D-generated prosurvival H2O2 in FLT3-ITD expressing AML. In summary, this thesis elucidates the mechanism in which activation and localisation of FLT3-ITD stimulates the PI3K/AKT and STAT5 pro-survival signalling pathways. This in turn leads to elevated production of NOX4-, NOX4Dand p22phox -generated H2O2 in AML which may contribute to DNA damage and genetic instability. My work therefore presents FLT3-ITD at the plasma membrane and NOXs as attractive therapeutic targets in the treatment of AML.
FLT3-ITD , NADPH oxidase 4 (NOX4) , NOX4 splice variant D 28 kDa (NOX4D) , p22phox , Acute myeloid leukaemia , Oncogene , Pro-survival reactive oxygen species , Nuclear membrane , Hydrogen peroxide
Moloney, J. N. 2018. Redox signalling in myeloid leukaemia. PhD Thesis, University College Cork.