NPM ALK mediated transformation of T lymphocytes

描述：（由申请人提供）本申请的总体目标是 研究NPM-ALK融合产物的分子和生物学作用， 从易位1（2;5）特征的间变性大细胞淋巴瘤 （ALCL）。我们最近还证明，在人类和小鼠中， NPM-ALK激活Jak 3、Stat 3和Erk 1 -2分子。更重要的是， 使用条件性Stat 3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Stat 3。此外，使用NI 7 DNRasxNPM-ALK Tg小鼠，我们已经证明Ras是产生 的NPM-ALK T细胞淋巴瘤和Stat 3中丝氨酸727的磷酸化。 然而，导致Stat 3和Ras活化的机制是 丝氨酸727的磷酸化在肿瘤中的致病作用 第3章还不清楚 在第一个目标中，我们提出了一系列旨在识别 导致Stat 3激活的分子机制。我们将测试 NPM-ALK能够直接磷酸化Stat 3，或者如果Stat 3 需要一个衔接蛋白，使其能够对接到ALK或未知的 这反过来又会激活Stat 3。我们还建议确定 Stat 3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，我们产生了Stat 3条件性T细胞特异性Tg 小鼠，其与CD 4-NPM-ALK Tg.因为100%的NPM-ALK 小鼠发展淋巴瘤，有可能研究是否遗传损失， Stat 3可以预防或延缓这些肿瘤的发生。最后 将研究Stat 3在ALK T细胞淋巴瘤维持中的作用 在转导诱导型Crc后使用FIox/-Stat 3/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Stat 3调控的基因，将在 在Stat 3删除之后。这些研究不仅应确定 Stat 3在NPM-ALK转化中的作用，但更重要的是，它们可能揭示更多 携带失调的Stat 3的肿瘤的一般机制。第二个目标，我们 建议确定导致Ras激活的分子机制， 确定Stat 3的丝氨酸727磷酸化在NPM-ALK介导的 转型IRS-1、She和Grb 2的作用将使用DN和 几种NPM-ALK突变构建体。为了研究假定的致瘤作用， 丝氨酸727，我们将利用来源于S727 ASat 3敲入的MEF 小鼠这将是第一个遗传学方法来剖析致病作用 的差异磷酸化状态。在第三个目标中，我们将 奋进证明NPM-ALK在NPM-ALK维护中的要求 使用新的条件性ploxNPM-ALK Tg的阳性淋巴瘤。这种方法将 明确显示了NPM-ALK在转化细胞中的作用，并将为我们提供 测试新治疗方法功效的原理，旨在 抑制ALK嵌合体的功能。

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.