Elucidate and Modulate Cell Signaling in NK Cells for Glioma Treatment

项目摘要 自然杀伤（NK）细胞是先天免疫的关键组成部分，对肿瘤细胞和病毒具有细胞溶解性。 被感染的细胞溶瘤性单纯疱疹病毒1（oHSV），其最近已被FDA批准用于 在治疗恶性胶质瘤（GBM）（一种高致死性脑肿瘤）方面具有巨大的潜力。 然而，根据我们以前的研究，宿主NK细胞在中枢免疫后提供了快速和强大的应答。 神经系统（CNS）感染oHSV，从而为有效的溶瘤病毒疗法提供屏障， GBM。细胞毒性基因，包括颗粒酶B（Gzmb），决定了NK细胞在这些环境中的细胞溶解活性; 然而，负责调节Gzmb表达的分子机制在很大程度上是未知的。在这 应用，我们的初步数据表明，Smad 4，一个在TGF-β超家族信号传导中的co-Smad蛋白， Gzmb信号通路以TGF-β非依赖性方式正向调节Gzmb表达， 在具有NK特异性Smad 4缺陷的小鼠中降低的抗肿瘤活性。使用这种小鼠模型，我们还 发现Smad 4通过上调Blimp 1（一种免疫调节因子）来积极调节NK细胞的稳态和成熟。 NK细胞成熟的正调节因子。Smad 4也是骨形态发生蛋白（BMP）中的一种辅助Smad。 发信号。我们的初步数据支持这一假设，即Smad 4在调节TGF-β依赖性的作用， NK细胞功能是BMP信号传导的下游，已报道BMP信号传导正调节NK细胞功能。 功能在这里，我们建议探索这一概念上新颖的发现的机制，并调节 在用于GBM的溶瘤病毒疗法的背景下NK细胞中的BMP-Smad 4信号传导。我们的总体假设是 Smad 4积极调节NK细胞发育和对靶细胞的细胞毒性，包括 oHSV感染的GBM细胞，这可以通过暂时抑制BMP-Smad 4信号传导来调节， 增强oHSV治疗的功效。我们建议深入研究分子机制 其中smad 4正调节Gzmb表达、细胞毒性和NK细胞在小鼠中的发育， 我们前述的动物模型和使用家族性幼年型息肉病患者样品的人类模型 (FJP)，在Smad 4中具有生殖系功能丧失突变。此外，我们还建议调节BMP- Smad 4信号传导以暂时抑制NK细胞对oHSV的应答以增强其治疗oHSV的功效。 GBM。这些都概述了在三个目标，以测试我们的假设。目的1是剖析机制， Smad 4正调节NK细胞对靶细胞的细胞毒性。目的2是描述机制 Smad 4通过其正向调节NK细胞发育。目的3是暂时抑制BMP-Smad 4 在NK细胞中的信号传导以改善体外和体内用于GBM的溶瘤病毒疗法。我们认为， 这些研究将为细胞毒性细胞治疗的基本机制提供新的见解， 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.