NPM ALK mediated transformation of T lymphocytes

NPM ALK 介导的 T 淋巴细胞转化

• 批准号: 6991316
• 负责人: David E Levy
• 金额: $ 29.38万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-21 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6991316
• 关键词: T lymphocyte; chimeric proteins; gene induction /repression; genetically modified animals; immunoprecipitation; laboratory mouse; lymphoma; matrix assisted laser desorption ionization; neoplasm /cancer genetics; neoplastic transformation; oncoproteins; phosphoproteins; phosphorylation; transcription factor

DESCRIPTION: (provided by applicant) The overall goal of this application is to study the molecular and biological role of the NPM-ALK fusion product derived from the translocation 1(2;5) characteristic of anaplastic large cell lymphomas (ALCL). We have also recently demonstrated that in human as well as in murine cells, NPM-ALK activates Jak3, Stat3, and Erk1-2 molecules. More importantly, using conditional Stat3 mouse embryonal fibroblasts (MEF) we have also proven that Stat3 is required for NPM-ALK mediated transformation. Moreover, using NI 7DNRasxNPM-ALK Tg mice we have shown that Ras is necessary for the generation of NPM-ALK T cell lymphomas and for the phosphorylation of serine 727 in Stat3. Nevertheless, the mechanisms leading to the activation of Stat3 and Ras are still elusive and the pathogenetic role of the phosphorylation ofserine 727 in Stat3 remains unclear. In the first Aim, we propose a series of experiments designed to identify the molecular mechanisms leading to the activation of Stat3. We will test whether NPM-ALK is able to directly phosphorylate Stat3 or alternatively if Stat3 requires an adaptor protein which allows it to dock to ALK or to an unknown kinase which in turn will activate Stat3. We also propose to determine the pathogenetic role of Stat3 in NPM-ALK mediated transformation of T lymphocytes. To accomplish this goal we have generated Stat3 conditional T cell specific Tg mice, which were crossed with CD4-NPM-ALK Tg. Because 100 percent of NPM-ALK mice develop lymphomas it is possible to study whether the genetic loss of Stat3 will prevent or delay the occurrence of these neoplasms. Finally, the role of Stat3 in the maintenance of ALK T cell lymphomas will be investigated using FIox/-Stat3/NPM-ALK lymphoma lines after transduction of an inducible Crc retrovirus (CreER-IRES-GFP). Survival of tumor cells and the expression of Bclx and Survivin, genes known to be regulated by Stat3, will be evaluated before and after Stat3 deletion. These studies should not only establish the role of Stat3 in NPM-ALK transformation but more importantly they may reveal more general mechanisms in tumors carrying deregulated Stat3. In the second Aim, we propose to determine the molecular mechanisms leading the Ras activation and to define the role of serine 727 phosphorylation of Stat3 in NPM-ALK mediated transformation. The role of IRS-1, She and Grb2 will be tested using DN and several NPM-ALK mutated constructs. To study the putative tumorigenic role of serine 727, we will take advantage of MEF derived from S727AStat3 knock-in mice. This will be the first genetic approach to dissect the pathogenetic role of the differential phosphorylation status of Stat3. In our third Aim, we will endeavor to prove the requirement of NPM-ALK in the maintenance of NPM-ALK positive lymphomas using a new conditional ploxNPM-ALK Tg. This approach will unequivocally show the role of NPM-ALK in transformed cells and will give us the rationale to test the efficacy of new therapeutic approaches designed to inhibit the function of ALK chimeras.

描述：(申请人提供)本申请的总体目标是 NPM-ALK融合产物的分子生物学功能研究 间变性大细胞淋巴瘤的1(2；5)易位 (ALCL)。我们最近在人类和小鼠身上也证明了这一点。 NPM-ALK激活JAK3、STAT3和ERK1-2分子。更重要的是， 利用条件STAT3小鼠胚胎成纤维细胞(MEF)，我们也证明了 NPM-ALK介导的转化需要STAT3。此外，使用NI 7DNRasxNPM-ALK TG小鼠我们已经证明RAS是世代所必需的 NPM-ALK T细胞淋巴瘤和STAT3中丝氨酸727的磷酸化。 然而，导致STAT3和RAS激活的机制是 丝氨酸727的磷酸化及其致病作用仍然难以捉摸 STAT3仍不清楚。 在第一个目标中，我们提出了一系列实验，旨在识别 STAT3激活的分子机制。我们将测试一下 NPM-ALK能够直接磷酸化STAT3，或者如果STAT3 需要一种接头蛋白，使其能够对接到ALK或未知的分子上 该信号转而激活STAT3。我们亦建议厘定 STAT3在NPM-ALK介导的T淋巴细胞转化中的致病作用 为了实现这一目标，我们生成了STAT3条件T细胞特异性TG 小鼠，与CD4-NPM-ALK TG杂交。因为100%的NPM-ALK 小鼠患上淋巴瘤是有可能研究是否存在基因缺失 STAT3将预防或延缓这些肿瘤的发生。最后， 将研究STAT3在ALK T细胞淋巴瘤维持中的作用 转导可诱导结直肠癌后的FIox/-STAT3/NPM-ALK淋巴瘤细胞系 逆转录病毒(CRER-IRES-GFP)。肿瘤细胞存活与Bclx的表达 而Survivin，已知受STAT3调控的基因，将在 和STAT3缺失后。这些研究不仅应该确定 在NPM-ALK转化中的STAT3，但更重要的是，它们可能揭示更多 携带非调控STAT3的肿瘤的一般机制。在第二个目标中，我们 建议确定导致RAS激活的分子机制，并 确定STAT3丝氨酸727磷酸化在NPM-ALK介导中的作用 转型。IRS-1、SHE和Grb2的作用将使用Dn和 几个NPM-ALK突变的结构。为了研究其可能的致癌作用 丝氨酸727，我们将利用从S727AStat3敲入而来的MEF 老鼠。这将是第一个剖析致病作用的遗传学方法。 STAT3的差异磷酸化状态。在第三个目标中，我们将 努力证明NPM-ALK在维护中的要求 使用新的条件倍增NPM-ALK TG检测阳性淋巴瘤。这一方法将 明确地展示了NPM-ALK在转化细胞中的作用，并将给我们 测试新的治疗方法的有效性的理由是 抑制ALK嵌合体的功能。