Characterizing and Targeting the Novel IL-15- AKT-XBP1s Pathway in NK Cells

NK细胞是对抗肿瘤细胞的第一道防线。我们最近开发了CAR NK细胞，用于治疗 多发性骨髓瘤(MM)，表现出更好的抗MM活性。然而，NK细胞仍然存在挑战- 基于癌症的免疫治疗：一些肿瘤细胞对寿命较短的NK细胞的细胞毒作用具有抵抗力 在给病人输液后。因此，进一步了解自然杀伤细胞对于更好地利用这一群体至关重要。 用于免疫治疗。我们最近在《自然免疫学》上发表的数据表明，一种重要的转录因子， XBP1s由非常规剪接的X-box结合蛋白(XBP1)的mRNA编码，正向调节NK 细胞存活和效应器功能。XBP1S通过直接调节NK细胞杀伤活性和GZMB基因表达 启动子结合。XBP1s在物理上与T-BET相互作用，T-BET是NK细胞的主要调节因子，这表明XBP1s 可以将T-BET招募到靶基因的启动子。这一发现填补了T-BET领域目前的一个空白 在GZMB启动子上缺乏直接的T-BET结合位点的情况下，对GZMB的表达进行正向调控。 此外，IL-15是调节NK细胞生存、发育和效应的重要细胞因子之一 功能，激活AKT信号，通过去泛素化增加XBP1s蛋白的稳定性，导致XBP1s 核聚积。与这些人类数据一致，小鼠XBP1的条件性基因敲除显示出减少 NK细胞数量及NK细胞抗肿瘤活性受损。我们相信我们已经确定了一种新的IL-15-AKT- XBP1s信号通路在调节NK细胞生物学的多个方面发挥关键作用。因此，我们的整体 假设本课题组新发现的IL-15-AKT-XBP1s信号通路为阳性 调节NK细胞存活和效应功能，可用于改善NK细胞或CAR-NK细胞- 基于癌症的免疫疗法。我们提出关于XBP1s如何调节NK细胞存活的机制研究 和效应器功能。我们还建议IL-15/IL-15Rα与CS1-CAR NK细胞或与 B-I09，一种靶向XBP1与XBP1剪接的新药，对多种癌症具有很强的治疗活性，包括 IL-15/IL-15Rα可选择性稳定XBP1S蛋白并阻止B-I09介导的下调 XBP1s及其相关的NK细胞功能抑制。我们将改造CS1-CAR NK细胞来表达IL-1 15/IL-15Rα或XBP1增强了这些细胞在体内的持久性，以持续根除肿瘤。三 提出了目标。目的1：研究增强型XBP1s蛋白的作用机制和功能后果 通过IL-15-AKT信号在NK细胞中的表达水平。目的2：研究XBP1s是否和/或如何调节NK细胞 生存、扩散和贩运。目的3：研究XBP 1在IL-15/IL-15Rα联合治疗中的作用 通过靶向XPB1、B-I09和/或CS1-CAR NK细胞的新药治疗MM。我们的项目是 重要的和及时的，因为IL-15和IL-15/IL-15Rα是癌症免疫治疗的顶级药物，并正在积极 在诊所里做了测试。我们的研究将导致对NK细胞生物学的新见解，改善以NK细胞为基础的结果 免疫疗法，并促进开发创新的癌症免疫疗法。

NK cells are the first line of defense against tumor cells. We recently developed CAR NK cells for the treatment of multiple myeloma (MM), which demonstrates improved anti-MM activity. However, challenges still exist for NK cell- based cancer immunotherapy: some tumor cells are resistant to cytotoxicity of NK cells that have a short life span after infusion into patients. Thus, further understanding of NK cells will be critical to better harness this population for immunotherapy. Our data recently published in Nature Immunology show that an important transcription factor, XBP1s, encoded by an unconventionally spliced mRNA of X-box binding protein (XBP1), positively regulates NK cell survival and effector functions. XBP1s regulates NK cell cytotoxicity and GZMB gene expression via direct promoter binding. XBP1s physically interacts with T-BET, a master regulator in NK cells, suggesting that XBP1s can recruit T-BET to the promoters of target genes. This discovery fills a current gap in the field, as T-BET is known to positively regulate the expression of GZMB despite lacking direct T-BET binding sites on the GZMB promoter. Moreover, IL-15, one of the most important cytokines regulating NK cell survival, development, and effector functions, activates AKT signaling to increase stability of XBP1s protein via deubiquitination, leading to XBP1s nuclear accumulation. Consistent with these human data, conditional knockout of Xbp1 in mice shows a decreased number of NK cells and impaired NK cell anti-tumor activity. We believe that we have identified a novel IL-15-AKT- XBP1s signaling pathway that plays key roles in regulating multiple aspects of NK cell biology. Thus, our overall hypothesis is that this IL-15-AKT-XBP1s signaling pathway newly identified by our group positively regulates NK cell survival and effector functions and can be utilized to improve NK cell or CAR NK cell- based cancer immunotherapy. We propose mechanistic studies regarding how XBP1s regulates NK cell survival and effector functions. We also propose combinational therapies of IL-15/IL-15Rα with CS1-CAR NK cells or with B-I09, a novel drug targeting XBP1 splicing to XBP1s that has strong activity in treating various cancers including MM. IL-15/IL-15Rα can selectively stabilize the XBP1s protein and prevent B-I09-mediated downregulation of XBP1s and the associated inhibition of functions in NK cells. We will engineer CS1-CAR NK cells to express IL- 15/IL-15Rα or XBP1s to have enhanced in vivo persistence of these cells for continuous tumor eradication. Three Aims are proposed. Aim 1: Study the mechanism and functional consequences of enhanced XBP1s protein expression levels via IL-15-AKT signaling in NK cells. Aim 2: Study whether and/or how XBP1s regulates NK cell survival, proliferation, and trafficking. Aim 3: Study the role of XBP1s in combination therapies of IL-15/IL-15Rα with a novel drug targeting XPB1s, B-I09, and/or with CS1-CAR NK cells for the treatment of MM. Our project is significant and timely as IL-15 and IL-15/IL-15Rα are top agents for cancer immunotherapy and are being actively tested in the clinic. Our studies will lead to new insights into NK cell biology, improve outcomes for NK cell-based immunotherapy, and facilitate developing innovative cancer immunotherapeutics.