Activating PTPN11 and c-kit Mutations in Myeloproliferative Disorder

激活骨髓增殖性疾病中的 PTPN11 和 c-kit 突变

• 批准号: 7279271
• 负责人: REBECCA J. CHAN
• 金额: $ 35.91万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7279271
• 关键词: Acute Myelocytic Leukemia; Arts; Biochemical; Biochemical Genetics; Biological Assay; Biological Markers; Blood; CSF2 gene; Cell Lineage; Cell Proliferation; Cells; Characteristics; Chromosomal translocation; Chronic; Chronic Myeloid Leukemia; Class; Data; Disease; Disruption; Engineering; Etiology; FLT3 gene; Genetic; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; Hematological Disease; Hematopoietic; Human; Hypersensitivity; In Vitro; Interleukin-3; Juvenile Myelomonocytic Leukemia; LY294002; Ligands; Lipids; Maps; Mast-Cell Leukemia; Mediating; Modeling; Molecular Target; Mus; Mutation; Myelogenous; Myeloid Cells; Myeloproliferative disease; PTPN11 gene; Pathogenesis; Pathologic; Pathway interactions; Pattern; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteome; Proteomics; Proto-Oncogene Protein c-kit; Range; Receptor Protein-Tyrosine Kinases; Research Personnel; Role; Signal Pathway; Signal Transduction; Stem Cell Factor; Stem cells; Systemic Mastocytosis; Techniques; Testing; Transgenes; Work; abl Oncogene; abstracting; base; cell type; cellular engineering; clinical Diagnosis; expectation; gain of function mutation; genetic manipulation; in vivo; innovation; kinase inhibitor; mast cell; mastocytosis; migration; mutant; novel; novel therapeutics; progenitor; protein expression; research study; response; src-Family Kinases; stem; therapeutic target

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.

骨髓增殖性疾病（MPD）是一组异质性血液疾病，其共同特征是骨髓细胞过度生成。我们一直在研究PTPN11（编码蛋白酪氨酸磷酸酶Shp-2）和c-kit（编码干细胞因子（SCF）受体蛋白酪氨酸激酶）的激活突变分别在幼年型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 过度激活的 GM-CSF 信号传导是 JMML 发病机制的核心；然而，我们的初步研究表明，通过脂质激酶磷酸肌醇-3-激酶 (PI3K) 抑制剂 LY294002 激活 PTPN11 突变，可纠正骨髓祖细胞 GM-CSF 超敏反应；因此，我们假设 PI3K 活性过度激活也有助于 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 p85a（IA 类 PI3K 的调节亚基）的基因破坏会消除激活 c-kit 突变诱导的肥大细胞增殖，这使我们推测表达激活 c-kit 突变的肥大细胞的增殖、存活和迁移增强部分是通过过度激活 PI3K。因此，根据我们的初步数据制定的本申请的中心假设是，通过激活 PTPN11 和 c-kit 突变诱导的 IA 类 PI3K 过度激活分别导致 JMML 和系统性肥大细胞增多症的病因学。为了检验这一假设，我们将转导缺乏 PI3K、p85α 调节亚基表达的小鼠造血细胞，并激活 PTPN11 突变体，以进行体外和体内造血祖细胞、存活和增殖测定以及响应 GMCSF 刺激的生化分析，并将利用遗传和生化分析 该方法涉及直接比较缺乏 p85a 或经过改造以逆转录病毒方式表达激活 c-Kit (D814V) 突变的肥大细胞，以寻找体外下游信号通路的生长、存活和激活以及体内 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 D814V) 和 PTPN11 激活突变的小鼠肥大细胞和干/祖细胞的蛋白质组和磷酸蛋白质组。总的来说，这种遗传、生化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PI3K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标，这两种疾病目前都没有好的治疗选择。