Oxidation of protein-tyrosine phosphatases in oncogenic cell transformation

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Cancer cells of different entities can produce elevated levels of reactive oxygen species (ROS). We have previously shown that this occurs in cells of Acute Myeloid Leukemia (AML), which harbor FLT3ITD, an oncoprotein found in ~30% of AML patients and an established driver of leukemic cell transformation. One of the consequences of ROS formation is the reversible oxidation - and thereby inactivation - of an important negative regulator of FLT3, the protein-tyrosine phosphatase (PTP) PTPRJ/DEP-1. This process causally contributes to leukemic cell transformation. We have now uncovered a pathway downstream of FLT3ITD, which leads to ROS formation and PTP oxidation. STAT5, which is strongly activated downstream of FLT3ITD and a critical mediator of transformation, binds directly to the promoter of NADPH-oxidase 4 (NOX4) and drives its elevated expression, ROS formation and PTP oxidation. Consequently, interference with NOX4 expression or activity inhibits leukemic cell transformation in vitro and in vivo (Jayavelu et al., Leukemia 2016). Moreover, two novel targets for FLT3ITD-driven ROS formation were identified: The PTP SHP-1/PTPN6 and the FLT3ITD kinase molecule itself. Further experiments in AML cell lines revealed that SHP-1 plays a role as a negative regulator of cell adhesion. SHP-1 inhibition by oxidation is therefore expected to modulate integrin functions and thereby to alter the fate of leukemic cells in vivo, e.g. the homing to the bone marrow. We intend to further analyze the biological consequences of SHP-1 inactivation in AML cells. The adhesion of wildtype-FLT3 or FLT3ITD expressing cells, with or without SHP-1 depletion, to integrin ligands as well as to stroma and endothelial cells will be assessed. Substrates of SHP-1 in integrin signaling will be identified. Alterations in transformation and leukemic cell fates in vivo will be analyzed in two mouse models of leukemia-like disease. Oxidation of the FLT3ITD molecule was detectable by specific labeling of sulfenic acid residues and led to kinase activation, possibly by dimer formation through intermolecular disulfide bridges. We intend to further establish the oxidation of the FLT3ITD kinase molecule as a relevant regulatory process by confirming oxidation at endogenous levels and identifying the affected cysteine(s). The possibility that FLT3ITD oxidation promotes dimer formation as an activating mechanism shall be further investigated. Furthermore, the consequences of cysteine modification for cell transformation will be assessed in intact cells and in established mouse models.