Genomics of AML: Contribution of Cytokine Signaling Pathways to Leukemogenesis

项目4的长期目标是验证发现的突变的生物学意义， 特异性分子靶点可用于指导急性髓性白血病（AMI）患者的治疗。 参与细胞因子信号传导的基因（例如PL 73、KRAS、NRAS、KIT）的突变可以在几乎所有的细胞中鉴定。 50%的AML病例。我们假设每一个急性髓细胞白血病肿瘤都至少有一个细胞因子突变 信号基因，这些突变合作，导致疾病的进展。聚焦高通量 表达优先受体酪氨酸激酶（RTK）、胞质酪氨酸激酶 (CTK)对94例初治AML患者进行了Ras-MAPK通路基因检测 样品已经分析了序列数据，并且已经鉴定了新的非同义突变， Janus激酶（JAK）家族中的多个基因，包括JAK 1、JAK 3和TYK 2。我们提议下列 具体目标：具体目标1：我们将验证体细胞Janus激酶的生物学意义 (JAK)通过评估原代造血细胞的生长和分化， 表达突变体JAK 1和TYK 2，我们将描述这些突变体的机制。 体细胞突变有助于白血病转化。我们的实验室已经验证了功能 利用生化、细胞培养和小鼠实验研究几种髓系白血病相关癌基因的意义 模型分析。我们将在细胞系和原代鼠骨中表达JAK 1、TYK 2和JAK 3突变 骨髓测定以确定这些突变对造血细胞生长和存活的影响。的 这些突变对突变蛋白本身的亚细胞定位的影响，以及相关的 将使用共聚焦显微镜、生化分析和定位标记的突变体来检查蛋白质。 具体目标2：我们将验证高优先级生殖系单核苷酸的生物学意义 JAK家族基因TYK 2和JAK 3的多态性（SNP），通过使用 细胞培养和动物模型系统。我们发现了已知的和以前未确认的非同义词 我们AML发现集中的SNP。为了优先考虑这些SNP进行进一步研究，我们将合作 与项目5一起确定这些SNP在对照人群中的频率。我们会优先考虑 细胞系和鼠骨髓转导-移植测定中的非同义SNP， 他们在转型中的角色。我们还将进行生物验证实验， 突变和SNP的合作，因为他们发现在我们的病人，严格评估的贡献， 发现了疾病发病机制的突变。

The long-term goal of Project 4 is to validate the biological significance of discovered mutations so that specific molecular targets can be used to guide the treatment of patients with acute myeloid leukemia (AMI). Mutations in genes involved in cytokine signaling (e.g. PL73, KRAS, NRAS, KIT) can be identified in nearly 50% of AML cases. We hypothesized that every AML tumor harbors at least one mutation in a cytokine signaling gene, and that these mutations cooperate to cause disease progression. Focused high-throughput exonic re-sequencing of expression-prioritized receptor tyrosine kinase (RTK), cytoplasmic tyrosine kinase (CTK), and Ras-MAPK pathway genes has been performed on 94 selected primary de novo AML patient samples. Sequence data has been analyzed and novel, non-synonymous mutations have been identified in multiple genes in the Janus kinase (JAK) family, including JAK1, JAK3, and TYK2. We propose the following Specific Aims: Specific Aim 1: We will validate the biological significance of somatic Janus kinase (JAK) family mutations by assessing the growth and differentiation of primary hematopoietic cells expressing mutant JAK1 and TYK2, and we will characterize the mechanisms by which these somatic mutations contribute to leukemic transformation. Our laboratory has validated the functional significance of several myeloid leukemia-associated oncogenes using biochemical, cell culture, and mouse model assays. We will express JAK1, TYK2, and JAK3 mutations in cell lines and primary murine bone marrow assays to determine the effect of these mutations on growth and survival of hematopoietic cells. The effects of these mutations on the subcellular localization of the mutant proteins themselves, and associated proteins will be examined using confocal microscopy, biochemical analysis, and localization-tagged mutants. Specific Aim 2: We will validate the biological significance of high-priority germline single nucleotide polymorphisms (SNPs) in JAK family genes TYK2 and JAK3 by characterizing their functions using cell culture and animal model systems. We have found known and previously unidentified nonsynonymous SNPs in our AML discovery set. To prioritize these SNPs for further study, we will collaborate with Project 5 to determine the frequency of these SNPs in control populations. We will express high-priority non-synonymous SNPs in cell lines and in murine bone marrow transduction-transplantation assays to characterize their role in transformation. We will also perform biological validation experiments with mutations and SNPs in cooperation as they are found in our patients to rigorously assess the contribution of discovered mutations to disease pathogenesis.