Activating PTPN11 and c-kit Mutations in Myeloproliferative Disorder

骨髓增生性疾病(MPD)是一组异质性的血液病，具有髓系细胞过度增殖的共同特征。我们一直在研究PTPN11和c-kit的激活突变在幼年粒单核细胞白血病和系统性肥大细胞增多症中的作用。PTPN11编码蛋白酪氨酸磷酸酶SHP-2，c-kit编码干细胞因子(SCF)的受体蛋白酪氨酸激酶。通过RAS过度激活的GM-CSF信号在JMML的发病机制中处于核心地位；然而，我们的初步研究表明，通过脂肪激酶磷酸肌醇-3-激酶(PI3K)抑制剂LY294002激活PTPN11突变而诱导的髓系祖细胞GM-CSF超敏反应得到纠正；因此，我们假设PI3K活性的过度激活也参与了JMML的发病。此外，在系统性肥大细胞增多症的模型中，我们有证据表明，p85a是IA类PI3K的一个调节亚基，它的基因破坏可以阻止由激活c-kit突变诱导的肥大细胞增殖，这导致我们假设，表达激活的c-kit突变的肥大细胞的增殖、存活和迁移的促进部分是通过PI3K的过度激活来调节的。因此，根据我们的初步数据提出的这一应用的中心假设是，通过激活PTPN11和c-kit突变而诱导的IA类PI3K的过度激活分别与JMML和系统性肥大细胞增多症的病因有关。为了验证这一假说，我们将转导PI3K调节亚基p85pha表达缺失的小鼠造血细胞，并激活PTPN11突变体，进行体外和体内造血祖细胞、存活和增殖分析以及对GMCSF刺激做出反应的生化分析，并将利用遗传学和生化方法，直接比较p85a缺陷的肥大细胞或逆转录病毒表达激活c-Kit(D814V)突变的肥大细胞，以寻找在体外和体内对下游信号通路的生长、存活和激活的调节作用。为了确定JMML和系统性肥大细胞增多症的潜在治疗靶点，我们将分别绘制表达c-Kit(c-Kit D814V)和PTPN11激活突变的小鼠肥大细胞和干细胞/祖细胞的蛋白质组和磷蛋白质组。总的来说，这种结合了遗传学、生化和蛋白质组学实验的方法，将确定由SHP-2和c-kit通过IA类PI3K的P85亚基控制的一系列功能，并将为JMML和系统性肥大细胞增多症的分子治疗提供新的靶点，这两种疾病目前都没有很好的治疗选择。

Myeloproliferative disorder (MPD) is a heterogeneous group of hematologic diseases which share the common characteristic of myeloid cell overproduction. We have been examining the role of activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp-2, and of c-kit, which encodes the receptor protein tyrosine kinase for stem cell factor (SCF), in juvenile myelomonocytic leukemia and systemic mastocytosis, respectively. GM-CSF signaling via Ras hyperactivation is central to the pathogenesis of JMML; however, we have preliminary studies demonstrating correction of myeloid progenitor GM-CSF hypersensitivity induced by activating PTPN11 mutations by the lipid kinase phosphoinsositol-3-kinase (PI3K) inhibitor, LY294002; therefore, we hypothesize hyperactivation of PI3K activity also contributes to the pathogenesis of JMML. Additionally, in a model of systemic mastocytosis, we have evidence demonstrating that genetic disruption of p85a, a regulatory subunit of class IA PI3K, abrogates mast cell proliferation induced by activating c-kit mutations, leading us to hypothesize that the enhanced proliferation, survival, and migration of mast cells expressing activating c-kit mutations is mediated in part via hyperactivation of PI3K. Therefore, the central hypothesis of this application, formulated on the basis of our preliminary data, is that hyperactivation of class IA PI3K induced by activating PTPN11 and c-kit mutations contributes to the etiology of JMML and systemic mastocytosis, respectively. To examine this hypothesis, we will transduce murine hematopoietic cells lacking expression of the regulatory subunit of PI3K, p85alpha, with activating PTPN11 mutants to conduct in vitro and in vivo hematopoietic progenitor, survival, and proliferation assays as well as biochemical analysis in response to GMCSF stimulation and will utilize a genetic and a biochemical approach involving a direct comparison of the mast cells deficient in p85a or engineered to retrovirally express the activating c-Kit (D814V) mutation to look for modulation of growth, survival and activation of downstream signaling pathways in vitro and MPD in vivo. To define additional potential therapeutic targets in JMML and systemic mastocytosis, we will map the proteome and the phosphoproteome of murine mast cells and stem/progenitor cells expressing the activating mutations of c-Kit (c-Kit D814V) and PTPN11, respectively. Collectively, this combined approach of genetic, biochemical, and proteomic experiments will identify a full range of functions that are controlled by Shp-2 and c-kit via p85 subunits of class IA PI3K and will provide novel targets for molecular therapies in the treatment of JMML and systemic mastocytosis, both of which currently have no good treatment options.