Activating PTPN11 and c-kit Mutations in Myeloproliferative Disorder

骨髓增生性疾病（MPD）是一组异质性血液病，其共同特征是骨髓细胞过度生成。我们一直在研究PTPN 11（编码蛋白酪氨酸磷酸酶Shp-2）和c-kit（编码干细胞因子（SCF）的受体蛋白酪氨酸激酶）的激活突变分别在青少年粒单核细胞白血病和系统性肥大细胞增多症中的作用。通过Ras超活化的GM-CSF信号传导是JMML发病机制的核心;然而，我们有初步研究表明，脂质激酶磷酸肌醇-3-激酶（PI 3 K）抑制剂LY 294002可纠正通过激活PTPN 11突变诱导的髓系祖细胞GM-CSF超敏反应;因此，我们假设PI 3 K活性的超活化也有助于JMML的发病机制。 此外，在系统性肥大细胞增多症模型中，我们有证据表明，p85 a（IA类PI 3 K的调节亚基）的遗传破坏消除了活化c-kit突变诱导的肥大细胞增殖，这使我们假设表达活化c-kit突变的肥大细胞的增殖、存活和迁移的增强部分是通过PI 3 K的超活化介导的。因此，根据我们的初步数据，本申请的中心假设是，由激活PTPN 11和c-kit突变诱导的IA类PI 3 K的过度激活分别导致JMML和系统性肥大细胞增多症的病因。为了验证这一假设，我们将用活化PTPN 11突变体来诱导缺乏PI 3 K调节亚基p85 α表达的小鼠造血细胞，以进行体外和体内造血祖细胞存活，和增殖测定以及响应于GMCSF刺激的生物化学分析，并将利用遗传和生物化学方法，包括直接比较缺乏GMCSF的肥大细胞，p85 a或经工程改造以逆转录病毒表达活化c-Kit（D814 V）突变，以寻找体外生长、存活和下游信号传导途径的活化以及体内MPD的调节。为了确定JMML和系统性肥大细胞增多症的其他潜在治疗靶点，我们将分别绘制表达c-Kit（c-Kit D814 V）和PTPN 11激活突变的鼠肥大细胞和干/祖细胞的蛋白质组和磷酸化蛋白质组。总的来说，这种遗传、生物化学和蛋白质组学实验的组合方法将鉴定由Shp-2和c-kit通过IA类PI 3 K的p85亚基控制的全方位功能，并将为治疗JMML和系统性肥大细胞增多症的分子疗法提供新的靶点，这两种疾病目前都没有良好的治疗选择。

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.