The adaptor protein Crk in immune responses

项目总结： 本提案是对以前的R01(AI130197)的续展申请。在上一次的授权期内，私人投资公司 发表同行评议论文30余篇，申请专利10项。其中最重要的一个 上一个资助期的直接发现是，PI已经确定了三个有希望的CRK 并围绕这些抑制剂申请了一项专利(专利号：63/305,354)。在前一个供资周期中， PI试图确定小接头蛋白CT10的激酶样调节蛋白的机制(S) (CrkL)及其磷酸化(PCrkL)通过利用人NK细胞控制NK细胞的激活和抑制 和新的NK细胞特异性条件性基因敲除小鼠。长期目标是利用这些知识和 揭示NK细胞激活和抑制的分子基础并开发新的成像技术 人类原发免疫缺陷疾病以及癌症和病毒等慢性疾病的新疗法 感染。在发现新的pCrkL抑制剂后，我们假设新发现的pCrkL 抑制物决定了Crk与关键的下游信号分子相互作用的能力，并最终形成 将肌动蛋白细胞骨架转化为功能性免疫突触(IS)，可潜在地用于治疗 感染和癌症。在强劲的初步数据的指引下，在这次续签申请中，我们将测试这些 通过三个具体目标的假说：1)通过如何定义精确的分子机制 类Crk(PCrkL)蛋白抑制物的磷酸化影响NK细胞的功能。拟议的工作将 将尖端成像技术引入NK细胞研究领域。实验将确定在哪里和 当Crk在IS被磷酸化时，以及它如何与关键的受体、信号分子和 有无pCrkL抑制剂的肌动蛋白细胞骨架；2)确定CrkL在T细胞中的作用 部分DiGeorge综合征(PDGS)患者。通过研究最常见的一种(3000个新生儿中就有一个) 免疫缺陷疾病，pDGS(主要由CrkL单倍体不足引起)，利用我们以前的 PDGS上显示pDGS中NK细胞整合素激活缺陷的发现，我们将确定如何 CrkL的缺失影响了CD8+T细胞整合素的信号转导和功能，可在pDGS中产生新的 免疫治疗(例如，调节整合素信号)未来治疗pDGS患者；3)发现其作用 PCrkL抑制剂在癌症治疗中的应用。我们发现了几种新的pCrkL抑制剂，它们可以与 并抑制pCrkL蛋白。在体外对几种实体瘤细胞系的增殖有抑制作用，对肿瘤细胞有抑制作用 线源性(CDX)异种移植和患者原位异种移植(PDX)小鼠模型；我们将确定 该抑制物及其类似物在免疫细胞(主要是NK和T细胞)介导的免疫应答中的作用 活体内的实体癌。拟议的工作涉及关键的信号参与者和监管机制，以及 生成一个新的模型系统来确定CrkL作为主调节分子的作用。它是宽泛的 与治疗原发免疫缺陷疾病和癌症的直接临床意义相关。

Project Summary: This proposal is a renewal application of a previous R01 (AI130197). During the last grant period, the PI has published more than 30 peer-reviewed papers and applied for 10 patents. One of the most important discoveries directly resulting from the last funding period is that the PI has identified three promising Crk inhibitors and has filed a patent around these inhibitors (Patent #: 63/305,354). In the previous funding cycle, the PI seeks to identify the mechanism(s) by which the small adaptor protein CT10 regulator of the kinase-like (CrkL) and its phosphorylation (pCrkL) control NK cell activation and inhibition by using both human NK cells and novel NK cell-specific conditional knockout mice. The long-term goal is to use this knowledge and the novel imaging techniques to uncover the molecular basis of NK cell activation and inhibition and to develop new treatments for human primary immunodeficiency diseases and chronic diseases such as cancer and viral infection. After discovering the novel pCrkL inhibitors, we hypothesize that the newly discovered pCrkL inhibitor determines Crk’s ability to interact with critical downstream signaling molecules and ultimately shapes the actin cytoskeleton into a functional immunological synapse (IS), which can be potentially used to treat infection and cancer. Guided by strong preliminary data, in this renewal application, we will test these hypotheses via three Specific Aims: 1) Define the precise molecular mechanisms by how phosphorylation of Crk-like (pCrkL) protein inhibitors affects NK cell functions. The proposed work will bring cutting-edge imaging technology to the field of NK cell research. Experiments will determine where and when Crk is phosphorylated at the IS, as well as how it interacts with key receptors, signaling molecules, and the actin cytoskeleton with and without pCrkL inhibitors; 2) Determine the role of CrkL in T cells from patients with partial DiGeorge syndrome (pDGS). By studying one of the most common (1 in 3,000 births) immunodeficiency diseases, pDGS (mainly caused by CrkL haploinsufficiency), leveraging our previous discoveries on pDGS that demonstrate defective integrin activation in NK cells in pDGS, we will determine how the loss of CrkL affects CD8+ T cell integrin signaling and function in pDGS, which can generate novel immunotherapy (e.g., modulating integrin signaling) to treat pDGS patients in the future; 3) Discover the role of pCrkL inhibitors in cancer therapy. We identified several novel pCrkL inhibitors, which can interact with and inhibit the pCrkL protein. It inhibits the proliferation of several solid tumor cell lines in vitro, and tumor cell line-derived (CDX) xenograft and orthotopic patient-derived xenograft (PDX) mouse models; we will determine this inhibitor and its analogs’ roles in immune cell (focusing on NK and T)-mediated immune responses against solid cancers in vivo. The proposed work involves key signaling players and regulatory mechanisms and generates a novel model system to determine the role of CrkL as a master regulatory molecule. It is broadly relevant with direct clinical implications for treating primary immunodeficiency diseases and cancers.