Elucidate and Modulate Cell Signaling in NK Cells for Glioma Treatment

项目摘要 自然杀伤(NK)细胞是先天免疫的重要组成部分，对肿瘤细胞和病毒具有杀伤作用。 被感染的细胞。溶瘤单纯疱疹病毒1型(OHSV)，最近已被FDA批准用于 癌症的治疗，在治疗胶质母细胞瘤(GBM)这一高度致命的脑肿瘤方面具有巨大的潜力。 然而，根据我们之前的研究，宿主NK细胞在中枢神经系统中提供了快速和强大的反应 神经系统感染OHSV，从而为有效的溶瘤病毒治疗提供了障碍 GBM。细胞毒性基因，包括颗粒酶B(GZMB)，在这些环境中决定NK细胞的杀伤活性； 然而，调控Gzmb表达的分子机制在很大程度上是未知的。在这 应用方面，我们的初步数据表明，Smad4是转化生长因子-β超家族信号转导中的一种共Smad蛋白 途径，以不依赖于转化生长因子-β的方式正向调节Gzmb的表达，这与 NK特异性Smad4缺乏小鼠的抗肿瘤活性降低。使用这个老鼠模型，我们还 发现Smad4通过上调Blimp1，a正向调节NK细胞的动态平衡和成熟 NK细胞成熟的正性调节因子。Smad4也是骨形态发生蛋白(BMP)中的一种共同Smad 发信号。我们的初步数据支持这样的假设，即转化生长因子-β不依赖于Smad4在调控中的作用 NK细胞的功能位于BMP信号的下游，BMP信号已被报道对NK细胞有正向调节作用 功能。在这里，我们建议探索这一概念上的新发现的机制，并调节 骨巨噬细胞溶瘤病毒治疗中NK细胞BMP-Smad4信号转导的研究我们的总体假设是 Smad4正向调节NK细胞的发育和对靶细胞的杀伤作用，包括 OHSV感染的GBM细胞，这可以通过暂时抑制BMP-Smad4信号转导来调节 提高OHSV治疗的疗效。我们建议对分子机制进行深入研究。 因此，Smad4正向调节Gzmb的表达、细胞毒性和小鼠NK细胞的发育 我们的上述动物模型和人类使用家族性幼年性息肉病患者的样本 (FJP)，在Smad4中有生殖系功能丧失突变。此外，我们还建议调节BMP- Smad4信号通路暂时抑制NK细胞对OHSV的反应以增强其治疗OHSV的疗效 GBM。为了检验我们的假说，我们从三个方面概述了这些问题。目标1是剖析 Smad4正向调节NK细胞对靶细胞的杀伤作用。目标2是描述这些机制的特征 通过Smad4正向调节NK细胞的发育。目标3是暂时抑制BMP-Smad4 NK细胞中的信号转导以改善体内外对基底膜的溶瘤病毒治疗。我们相信，结果是 这些研究将为细胞毒细胞治疗的基本机制和探索提供新的见解 临床前模型中的新疗法将促进GBM的治疗。

Project Summary Natural killer (NK) cells are a critical component of innate immunity and are cytolytic to tumor cells and viral- infected cells. Oncolytic herpes simplex virus 1 (oHSV), which has recently been approved by the FDA for the treatment of cancer, holds great potential in the treatment of glioblastoma (GBM), a highly lethal brain tumor. However, based on our previous studies, host NK cells provide a rapid and robust response following central nervous system (CNS) infection with oHSV, thus presenting a barrier for effective oncolytic virotherapy for GBM. Cytotoxic genes, including granzyme B (Gzmb), determine NK cell cytolytic activity in these settings; however, the molecular mechanisms responsible for regulating Gzmb expression are largely unknown. In this application, our preliminary data show that Smad4, a co-Smad protein in the TGF-beta superfamily signaling pathway, positively regulates Gzmb expression in a TGF-beta-independent manner, which correlates with decreased anti-tumor activity in mice with an NK-specific Smad4 deficiency. Using this mouse model, we also discovered that Smad4 positively regulates NK cell homeostasis and maturation by upregulating Blimp1, a positive regulator of NK cell maturation. Smad4 is also a co-Smad in bone morphogenetic protein (BMP) signaling. Our preliminary data support the hypothesis that the TGF-β-independent role of Smad4 in regulating NK cell function is downstream of BMP signaling, which has been reported to positively regulate NK cell function. Here, we propose to explore the mechanisms for this conceptually novel discovery and to modulate BMP-Smad4 signaling in NK cells in the setting of oncolytic virotherapy for GBM. Our overall hypothesis is that Smad4 positively regulates NK cell development and cytotoxicity against target cells including oHSV-infected GBM cells, and this can be modulated by temporarily inhibiting BMP-Smad4 signaling to enhance the efficacy of oHSV therapy. We propose an in-depth investigation into the molecular mechanisms whereby smad4 positively regulates Gzmb expression, cytotoxicity, and development of NK cells in mice using our aforementioned animal model and in humans using samples of patients with familial juvenile polyposis (FJP), having a germline loss-of-function mutation in Smad4. Additionally, we also propose to modulate BMP- Smad4 signaling to temporarily inhibit NK cell responses to oHSV to enhance its efficacy for the treatment of GBM. These are outlined in three Aims to test our hypothesis. Aim 1 is to dissect the mechanisms by which Smad4 positively regulates NK cell cytotoxicity against target cells. Aim 2 is to characterize the mechanisms by which Smad4 positively regulates NK cell development. Aim 3 is to temporarily inhibit BMP-Smad4 signaling in NK cells to improve oncolytic viral therapy for GBM in vitro and in vivo. We believe that the results of these studies will lend new insights into basic mechanisms of cytotoxic cell therapy and that exploration of novel therapeutics in preclinical models will advance the treatment of GBM.