Survival Mechanisms in Leukemic NK Cells

描述（由申请人提供）：本提案的广泛长期目标是了解NK白血病的发病机制。负责白血病NK细胞扩增的机制尚不清楚。该建议的中心假设是NK白血病由失调的细胞凋亡引起。尽管表达高水平的Fas受体和Fas配体，但白血病NK细胞对Fas介导的死亡具有相对抗性。IL-2激活后Fas抗性的恢复表明白血病NK细胞中Fas信号传导的抑制。具体目标1中的实验针对确定白血病NK细胞中Fas抗性的机制。最初的实验将集中在新的，抑制性诱饵Fas受体，我们已经从白血病NK细胞克隆的作用。MAPK信号传导有助于Fas抗性的假设也将在具体目标1中进行检查。白血病NK细胞显示MAPK的组成性激活;此外，MEK/MAPK的抑制逆转Fas抗性。药理学抑制的MEK/MAPK和显性阴性（DN）的MEK蛋白表达的牛痘病毒将被用来检查MAPK下游信号传导的机制，导致Fas抗性。我们假设白血病NK细胞是依赖于体内靶识别产生的存活信号的活化细胞毒性细胞。初步数据表明，连接一个未知的NK激活受体诱导MAPK激活和调节Fas敏感性的NK细胞系。具体目标2中的实验将检查MAPK活化上游的NK受体信号传导机制。将鉴定负责保护白血病NK细胞免于Fas诱导死亡的特异性NK受体。具体目标3中的实验将检验组成性STAT信号传导调节凋亡抗性的假设。初步数据显示，在白血病NK细胞中存在组成性STAT活化;此外，抑制STAT活化直接诱导白血病NK细胞凋亡以及赋予Fas敏感性。实验策略将利用调节STAT活化以及DN-STAT蛋白的酪氨酸激酶的特异性抑制剂。这些研究对了解NK白血病的发病机制具有重要意义。这些研究的结果应确定MAPK和STAT信号通路中的分子靶点，这些靶点对血液系统恶性肿瘤的治疗开发很重要。

DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to understand the pathogenesis of NK leukemia. The mechanisms responsible for expansion of leukemic NK cells are not known. The central hypothesis of this proposal is that NK leukemia results from dysregulated apoptosis. Leukemic NK cells are relatively resistant to Fas-mediated death despite expressing high levels of both Fas receptor and Fas ligand. Reversal of Fas resistance after IL-2 activation suggests inhibition of Fas signaling in leukemic NK cells. Experiments in specific aim 1 are directed at determining mechanisms of Fas-resistance in leukemic NK cells. Initial experiments will focus on the role of novel, inhibitory decoy Fas receptors, which we have cloned from leukemic NK cells. The hypothesis that MAPK signaling contributes to Fas resistance will also be examined in specific aim 1. Leukemic NK cells show constitutive activation of MAPK; furthermore inhibition of MEK/MAPK reverses Fas resistance. Pharmacological inhibition of MEK/MAPK and dominant negative (DN) MEK proteins expressed in vaccinia virus will be utilized to examine mechanisms of MAPK downstream signaling leading to Fas resistance. We hypothesize that leukemic NK cells are activated cytotoxic cells dependent on survival signals resulting from target recognition in vivo. Preliminary data show that ligation of an unknown NK activating receptor induces MAPK activation and modulates Fas sensitivity in an NK cell line. Experiments in specific aim 2 will examine mechanisms of NK receptor signaling upstream of MAPK activation. Specific NK receptors responsible for protecting leukemic NK cells from Fas-induced death will be identified. Experiments in specific aim 3 will examine the hypothesis that constitutive STAT signaling regulates apoptotic resistance. Preliminary data show constitutive STAT activation in leukemic NK cells; furthermore, inhibition of STAT activation directly induces apoptosis in leukemic NK cells as well as conferring Fas-sensitivity. The experimental strategy will utilize specific inhibitors of tyrosine kinases that regulate STAT activation as well as DN- STAT proteins. These studies are important for understanding the pathogenesis of NK leukemia. Results of these studies should identify molecular targets in MAPK and STAT signaling pathways important for therapeutic development in hematologic malignancies.