Survival Mechanisms in Leukemic NK Cells

描述(由申请人提供)：这项建议的广泛长期目标是了解NK白血病的发病机制。白血病NK细胞扩张的机制尚不清楚。这一建议的中心假设是NK白血病是细胞凋亡失调的结果。尽管白血病NK细胞同时表达高水平的Fas受体和Fas配体，但对Fas介导的死亡具有相对的抵抗力。IL-2激活后Fas抵抗的逆转提示白血病NK细胞中Fas信号的抑制。特定目的1的实验旨在确定白血病NK细胞中Fas耐药的机制。最初的实验将集中在我们从白血病NK细胞克隆的新型抑制性诱骗Fas受体的作用上。MAPK信号通路参与Fas耐药的假设也将在特定的目的中得到验证：1.白血病NK细胞显示MAPK的结构性激活；此外，抑制MEK/MAPK可逆转Fas耐药。在痘苗病毒中表达的MEK/MAPK和显性负性MEK蛋白的药理抑制将被用来研究MAPK下游信号转导导致Fas耐药的机制。我们假设白血病NK细胞是激活的细胞毒细胞，依赖于体内靶点识别产生的生存信号。初步数据显示，连接未知的NK激活受体可诱导NK细胞系中MAPK的激活并调节Fas的敏感性。在特定目的2中的实验将研究MAPK激活的NK受体信号上游的机制。负责保护白血病NK细胞免于Fas诱导死亡的特定NK受体将被确定。特定目标3的实验将检验构成STAT信号调节细胞凋亡抵抗的假设。初步数据显示，白血病NK细胞中STAT的结构性激活；此外，抑制STAT激活直接诱导白血病NK细胞的凋亡，并增强Fas的敏感性。该实验策略将利用调节STAT激活的特定酪氨酸激酶抑制剂以及DN-STAT蛋白。这些研究对于了解NK白血病的发病机制具有重要意义。这些研究的结果应该确定MAPK和STAT信号通路中的分子靶点，这些信号通路对血液系统恶性肿瘤的治疗发展至关重要。

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.