Oxidation of protein-tyrosine phosphatases in oncogenic cell transformation

不同实体的癌细胞可以产生高水平的活性氧（ROS）。我们之前已经证明，这种情况发生在急性髓性白血病（AML）的细胞中，其中含有FLT3ITD， FLT3ITD是一种在约30%的AML患者中发现的癌蛋白，是白血病细胞转化的既定驱动因素。ROS形成的后果之一是FLT3的一个重要负调节因子，蛋白酪氨酸磷酸酶（PTP） PTPRJ/DEP-1的可逆氧化，从而失活。这一过程导致白血病细胞转化。我们现在已经发现了FLT3ITD下游的一条途径，该途径导致ROS形成和PTP氧化。STAT5在FLT3ITD下游被强烈激活，是转化的关键介质，直接结合nadph氧化酶4 （NOX4）启动子，并驱动其表达升高、ROS形成和PTP氧化。因此，干扰NOX4的表达或活性可以抑制白血病细胞在体内和体外的转化（Jayavelu et al., Leukemia 2016）。此外，FLT3ITD驱动ROS形成的两个新靶点被确定：PTP SHP-1/PTPN6和FLT3ITD激酶分子本身。在AML细胞系中的进一步实验表明，SHP-1作为细胞粘附的负调节因子发挥作用。因此，通过氧化抑制SHP-1有望调节整合素的功能，从而改变体内白血病细胞的命运，例如，归巢到骨髓。我们打算进一步分析AML细胞中SHP-1失活的生物学后果。将评估野生型flt3或FLT3ITD表达细胞，无论是否存在SHP-1缺失，对整合素配体以及基质和内皮细胞的粘附性。将确定整合素信号传导中SHP-1的底物。将在两种白血病样疾病的小鼠模型中分析体内转化和白血病细胞命运的改变。FLT3ITD分子的氧化可以通过特定的亚磺酸残基标记检测到，并导致激酶激活，可能是通过分子间二硫桥形成二聚体。我们打算通过确认内源性氧化水平和鉴定受影响的半胱氨酸，进一步确定FLT3ITD激酶分子的氧化是一个相关的调控过程。FLT3ITD氧化促进二聚体形成作为激活机制的可能性有待进一步研究。此外，半胱氨酸修饰对细胞转化的影响将在完整细胞和已建立的小鼠模型中进行评估。

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.