NPM ALK mediated transformation of T lymphocytes

NPM ALK 介导的 T 淋巴细胞转化

• 批准号: 7197729
• 负责人: David E Levy
• 金额: $ 4.13万
• 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 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197729
• 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：（由申请人提供）本申请的总体目标是 研究衍生的 NPM-ALK 融合产物的分子和生物学作用 来自间变性大细胞淋巴瘤的易位 1(2;5) 特征 （ALCL）。我们最近还证明，在人类和小鼠中 在细胞中，NPM-ALK 激活 Jak3、Stat3 和 Erk1-2 分子。更重要的是， 使用条件 Stat3 小鼠胚胎成纤维细胞 (MEF)，我们还证明了 NPM-ALK 介导的转化需要 Stat3。此外，使用NI 我们已经证明Ras对于7DNRasxNPM-ALK Tg小鼠的产生是必需的 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 磷酸化的致病作用仍然难以捉摸 Stat3 仍不清楚。 在第一个目标中，我们提出了一系列旨在识别 导致 Stat3 激活的分子机制。我们将测试是否 NPM-ALK 能够直接磷酸化 Stat3，或者如果 Stat3 需要一个接头蛋白，使其能够对接 ALK 或未知蛋白 激酶反过来会激活 Stat3。我们还建议确定 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，可以研究是否基因缺失 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 转导诱导型 Crc 后使用 FIox/-Stat3/NPM-ALK 淋巴瘤系 逆转录病毒（CreER-IRES-GFP）。肿瘤细胞的存活和Bclx的表达 和 Survivin，已知受 Stat3 调节的基因，将在之前进行评估 以及 Stat3 删除后。这些研究不仅应该确立 NPM-ALK 转换中的 Stat3 但更重要的是它们可能会揭示更多 携带失调的 Stat3 的肿瘤的一般机制。在第二个目标中，我们 提议确定导致 Ras 激活的分子机制并 定义 Stat3 丝氨酸 727 磷酸化在 NPM-ALK 介导中的作用 转变。 IRS-1、She 和 Grb2 的作用将使用 DN 和 几个 NPM-ALK 突变结构。研究假定的致瘤作用 丝氨酸 727，我们将利用源自 S727AStat3 敲入的 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。在我们的第三个目标中，我们将 努力证明NPM-ALK维护NPM-ALK的要求 使用新的条件 ploxNPM-ALK Tg 检测阳性淋巴瘤。这种方法将 明确显示 NPM-ALK 在转化细胞中的作用，并将为我们提供 测试新治疗方法有效性的基本原理 抑制 ALK 嵌合体的功能。

项目成果

NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

• DOI: 10.1158/0008-5472.can-09-2655
• 发表时间: 2009-11-15
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Ambrogio C;Martinengo C;Voena C;Tondat F;Riera L;di Celle PF;Inghirami G;Chiarle R
• 通讯作者: Chiarle R