Receptor protein-tyrosine phosphatases controlling activity of the oncoprotein FLT3 ITD

控制癌蛋白Flt3活性的受体蛋白酪氨酸磷酸酶ITDA急性髓系白血病(AML)是一组由多种基因损伤相结合引起的异质性疾病。在25%-30%的患者中，导致癌蛋白Flt3ITD(具有内部串联复制的FMS样酪氨酸激酶)的突变是重要的驱动突变类别之一。Flt3是一种III型受体酪氨酸激酶(RTK)，在淋系和髓系造血祖细胞的存活、增殖和分化中发挥作用。突变的Flt3ITD蛋白表现出信号和运输性质的改变，这是导致细胞转化的原因。Flt3 ITD还促进了活性氧物种(ROS)的产生。我们先前已经证明，两个跨膜(受体样)蛋白酪氨酸磷酸酶(RPTP)PTPRJ/DEP-1和PTPRC/CD45 RPTP在体外对野生型Flt3起负调控作用。这两种RPTP在体内的重叠活性以前也在另一种情况下观察到。在Flt3ITD转化的细胞中，PTPRJ/DEP-1的活性被ROS介导的可逆氧化部分折衷。该项目的目的是利用小鼠模型在体内评估PTPRJ/DEP-1和PTPRC/CD45在调节AML相关癌蛋白Flt3ITD转化髓系细胞中的作用。还应探讨通过增强RPTP活性来减弱转化的可能性。为此，我们将致力于以下具体目标：i)通过利用先前建立的Flt3 ITD基因敲除-Ptprj基因敲除小鼠品系，研究PTPRJ缺乏对Flt3 ITD依赖转化和骨髓增殖性疾病发展的影响。ii)在相同的策略和技术下，也将探索相关的跨膜PTP RPTPC/CD45的影响。我们将使用不同的技术来增强DEP-1的PTP活性，包括自然存在的配体TSP1，使用ROS灭活剂猝灭PTP氧化，以及CRISPR/Cas9介导的基因组编辑的遗传工具，以探索PTP激活作为对抗Flt3 ITD介导的转化的新策略。

Receptor protein-tyrosine phosphatases controlling activity of the oncoprotein FLT3 ITDAcute Myeloid Leukemia (AML) is a heterogeneous group of diseases caused by combination of several genetic lesions. Mutations giving rise to the oncoprotein FLT3 ITD (Fms-like tyrosine kinase with internal tandem duplications) represent one of the important classes of driver mutations in a subset of 25-30% of patients. FLT3 is a class III receptor tyrosine kinase (RTK) which plays a role in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Mutant FLT3 ITD proteins exhibit an altered signalling and trafficking quality, which causally contribute to cell transformation. FLT3 ITD also drives production of reactive oxygen species (ROS). We have previously shown that the two transmembrane (receptor-like) protein-tyrosine phosphatases (RPTP) PTPRJ/DEP-1 and PTPRC/CD45 RPTP act as negative regulators of wild type FLT3 in vitro. Overlapping activity of both RPTP in vivo has previously also been observed in another context. In FLT3 ITD-transformed cells, PTPRJ/DEP-1 activity is partially compromised by ROS-mediated reversible oxidation. The aim of the proposed project is to assess in vivo the role of PTPRJ/DEP-1 and PTPRC/CD45 in regulating the transformation of myeloid cells by the AML-related oncoprotein FLT3 ITD using mouse models. The possibility to attenuate transformation by enhancing RPTP activity shall also be explored. For these aims, we will address the following specific goals:I) By using a previously established FLT3 ITD knock in- Ptprj knock out mouse strain, the effect of PTPRJ deficiency on FLT3 ITD-dependent transformation and the development of myeloproliferative disease shall be investigated.II) With the same strategy and technology, the effect of the relevant transmembrane PTP RPTPC/CD45, shall also be explored. Moreover, the effect of combined loss of both RPTP PTPRJ and PTPRC will be analyzed.III) We will use different techniques to enhance PTP activity of DEP-1, including the naturally occurring ligand TSP1, quenching PTP oxidation using ROS inactivating agents, and the genetic tool of CRISPR/Cas9-mediated genomic editing to explore PTP activation as novel strategy to combat FLT3 ITD-mediated transformation.Taken together, we expect a better understanding of the mechanism of negative control of FLT3 ITD-mediated transformation and novel insights into possible therapeutic approaches by enhancing PTP activity.