Genomics of AML: Contribution of Cytokine Signaling Pathways to Leukemogenesis

项目4的长期目标是验证已发现的突变的生物学意义，以便 特异性分子靶点可用于指导急性髓系白血病患者的治疗。 参与细胞因子信号转导的基因(如PL73、KRAS、NRAS、KIT)的突变可以在几乎 50%的AML病例。我们假设每个AML肿瘤都含有至少一个细胞因子突变 信号基因，这些突变协同作用导致疾病进展。集中的高吞吐量 表达优先受体酪氨酸激酶(RTK)、胞浆酪氨酸激酶的外显子重测序 (CTK)和RAS-MAPK通路基因对94例初治AML患者进行了检测 样本。已经对序列数据进行了分析，并在 Janus激酶(JAK)家族中的多个基因，包括JAK1、JAK3和TYK2。我们提出以下建议 特定目标：特定目标1：我们将验证体细胞Janus激酶的生物学意义 通过评估原代造血细胞的生长和分化来检测(JAK)家族突变 表达突变的JAK1和TYK2，我们将描述这些 体细胞突变有助于白血病的转化。我们的实验室已经验证了它的功能 生化、细胞培养和小鼠检测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小鼠骨中表达JAK1、TYK2和JAK3突变 骨髓检测以确定这些突变对造血细胞生长和存活的影响。这个 这些突变对突变蛋白自身和相关的亚细胞定位的影响 蛋白质将使用共聚焦显微镜、生化分析和定位标记突变体进行检测。 具体目标2：我们将验证高优先级种系单核苷酸的生物学意义 JAK家族基因TYK2和JAK3的SNPs分析 细胞培养和动物模型系统。我们发现了已知的和之前未确定的非同义词 我们的AML发现集中的SNPs。为了确定这些SNPs的优先顺序以供进一步研究，我们将合作 项目5，以确定这些SNPs在对照人群中的频率。我们将表示高度优先 细胞系和小鼠骨髓转导-移植试验中的非同义SNPs 描述他们在转型中的角色。我们还将进行生物验证实验， 在我们的患者中发现的突变和SNPs合作，以严格评估 发现突变对疾病发病机制的影响。

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.