NPM-ALK AND ALK IN LYMPHOMA AND NORMAL DEVELOPMENT

描述：(改编自调查人员的摘要) 恶性肿瘤特有的染色体易位的特征 疾病导致了许多起作用的基因的识别 在正常细胞发育或增殖中的关键作用，以及 当改变时会产生异常的生长。约10%的非 霍奇金S淋巴瘤具有t(2；5)(p23；q35)染色体重排。 在申请人实验室进行的位置克隆研究 发现t(2；5)产生了一个融合基因，它编码一个嵌合体 由核仁的氨基末端部分组成的蛋白质 核磷蛋白(NPM)连接到a的催化域 胰岛素受体亚家族的新受体酪氨酸激酶， 间变性淋巴瘤激酶(ALK)。与碱性磷酸酶相互作用的配体 该受体的正常功能(S)尚不清楚。结果就是 在NPM-ALK融合中，ALK激酶被结构性激活并 在淋巴样细胞中异位表达。此外，国家经贸委 嵌合体的残基与野生型NPM结合，靶向部分 融合蛋白进入细胞核。目前尚不清楚 完整的表达需要NPM-ALK的核定位 它的生物活性。 这项提议的长期目标是通过以下方式确定机制 哪些NPM-ALK参与了淋巴瘤的发生，并确定了其作用 ALK受体激酶在正常细胞生长发育中的作用。这个 实验计划是确定所需的结构主题 ALK催化结构域的激活和NPM-ALK的核靶向性 以确定这些模体改变的生物学后果 通过使用体外转化试验，以及基于 NPM-ALK嵌合体促进造血细胞生长的能力 不依赖于因子，以分析包含 融合的NPM部分。嵌合蛋白的致瘤性研究 在生理条件下将通过产生转基因进行测试 启动子控制下表达NPM-ALK基因的小鼠 针对淋巴样细胞发育的不同阶段。的作用 碱性磷酸酶在胚胎发育中的作用及其在特定组织中的正常功能 将通过三种相辅相成的办法加以解决。首先，模式 在不同时间的小鼠胚胎中将测定ALK的表达 发育阶段，并在选定的成人组织中进行原位观察 杂交和免疫细胞化学染色。第二，体内 将评估ALK受体失活的表型效应 通过有针对性地破坏小鼠的基因。第三，表达克隆 将使用策略来识别和表征同源配体 ALK的开关，用于确定ALK信令何时正常发送 发生。综上所述，这些研究应该提供一种机械性的 由t(2；5)激活的ALK如何有助于 非霍奇金S淋巴瘤的发病机制

DESCRIPTION: (adapted from the investigator's abstract) The characterization of chromosomal translocations specific to malignant disease has led to the identification of a number of genes that play critical roles in normal cellular development or proliferation, and that produce abnormal growth when altered. Approximately 10% of all non- Hodgkin s lymphomas possess a t(2;5)(p23;q35) chromosomal rearrangement. Positional cloning studies performed in the applicants laboratory have revealed that the t(2;5) produces a fusion gene which encodes a chimeric protein consisting of the amino-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) linked to the catalytic domain of a novel receptor tyrosine kinase of the insulin receptor subfamily, anaplastic lymphoma kinase (ALK). The ligand that interacts with ALK and the normal function(s) of this receptor are unknown. As a consequence of NPM-ALK fusion, the ALK kinase becomes constitutively activated and is expressed ectopically in lymphoid cells. In addition, the NPM residues of the chimera bind to wild-type NPM, targeting a portion of the fusion protein to the nucleus. It is presently unknown whether the nuclear localization of NPM-ALK is required for the complete expression of its biological activity. The long-range goals of this proposal are to determine the mechanisms by which NPM-ALK contributes to lymphomagenesis, and to determine the role of the ALK receptor kinase in normal cell growth and development. The experimental plan is to identify the structural motifs required for activation of the ALK catalytic domain and NPM-ALK nuclear targeting and to determine the biological consequences of alterations of these motifs by using in vitro transformation assays, as well as assays based on the ability of the NPM-ALK chimera to render hematopoietic cells growth factor-independent, to analyze constructs containing mutations of the NPM portion of the fusion. The oncogenicity of the chimeric protein under physiological conditions will be tested by generating transgenic mice that express the NPM-ALK gene under the control of promoters specific for different stages of lymphoid cell development. The role of ALK in embryonic development and its normal function in specific tissues will be addressed by three complementary approaches. First, the pattern of ALK expression will be determined in murine embryos at different developmental stages, and in selected adult tissues, by in situ hybridization and immunocytochemical staining. Second, the in vivo phenotypic effects of inactivation of the ALK receptor will be assessed by targeted disruption of the gene in mice. Third, expression cloning strategies will be used to identify and characterize the cognate ligand of ALK -- the on-off switch that determines when ALK signaling normally occurs. Taken together, these studies should provide a mechanistic framework for how activation of ALK by the t(2;5) contributes to the genesis of non-Hodgkin s lymphoma.