CKAP4调控AML骨髓微环境NK细胞的生物力学及免疫学特性

急性髓系白血病 (AML) 肿瘤微环境中自然杀伤细胞（NK）免疫缺陷、功能降低，但机制不清。前期转录组测序对正常人及初诊AML病人骨髓NK（AML-NK）进行分析，发现细胞骨架相关蛋白CKAP4在AML-NK显著高表达；敲低AML-NK中CKAP4表达后，其生物力学特性改变（变形性减弱、硬度增加），杀伤性增强；均趋于正常骨髓NK特性。推测CKAP4在维持AML-NK生物力学及免疫学特性中起重要作用。为验证这一假设，本课题将借助慢病毒系统及NK细胞特异性Ckap4敲除/转基因小鼠，结合微流控、原子力显微镜等，体内外明确CKAP4对NK生物力学及免疫学特性的调控作用；转录组测序、RNA pulldown等阐明CKAP4对AML-NK功能特性的调控机制；ChIP-Seq/ChIP-qPCR并结合骨髓微环境解析CKAP4在AML-NK高表达的上游调控机制，为基于NK细胞的白血病免疫治疗提供新线索。

Natural killer cells in the tumor microenvironment of acute myeloid leukemia (AML) patients show a dramatic impairment in cytotoxic activity. However, little is known about the underlying mechanisms. In our preliminary data, we used single cell RNA-seq to analyze human NK cells from normal bone marrow (Normal-NK) and bone marrow of AML patients (AML-NK) respectively, and identified CKAP4，a cytoskeleton-associated protein, overexpressed in AML-NK cells but down regulated in Normal-NK cells. In addition, we found that CKAP4 siRNA decreased the deformability and increased the stiffness of AML-NK cells, but promoted the cytotoxicity of AML-NK cells, which are similar with the properties of Normal-NK cells. Based on these data, we hypothesized that CKAP4 plays a critical role in maintaining the biomechanical and immunological properties of NK cells in AML bone marrow microenvironment. To test this hypothesis, we will conduct a series of experiments as follows: we plan to use the lentivirus vector systems, NK cell specific Ckap4 knockout/knock-in mice models, as well as microfluidics, AFM, and 3D cell culture systems to investigate the regulatory roles of CKAP4 in human NK cells. Moreover, we would use RNA-seq/RNA pulldown/ FACS and Western blot to investigate the possible molecular and intracellular signaling mechanisms which were are related to NK cell biomechanics and immuno-function. We would also use ChIP-Seq/ChIP-qPCR and mimic bone marrow microenvironment, to uncover the upstream regulation mechanisms for CKAP4 differential expressions in various human NK cell subsets. The successes of this project will provide a new mechanistic explanation for regulation of NK cell immune function by biomechanics and ultimately implicate a potential application of NK cells in clinical immunotherapy.