The role of CaMKK2 in tumor-infiltrating natural killer cells

摘要： 钙/钙调蛋白依赖的蛋白激酶2(CAMKK2)是一种重要的细胞外信号调节因子 新陈代谢。尽管CAMKK2与神经元和巨噬细胞有经典联系，但我们的数据表明，CAMKK2也是 在肿瘤浸润性自然杀伤(NK)细胞中表达，可能调节这些细胞的抗肿瘤活性。 以前的文献表明，CAMKK2的表达通过以下方式促进小鼠乳腺肿瘤的生长 使肿瘤相关巨噬细胞向亲肿瘤的M2表型分化。然而，我们的初步数据 提示CAMKK2在NK细胞中起着相反的作用：CAMKK2信号似乎提高了NK细胞的存活率， 增殖、迁移和抗转移免疫监测。我们推测CAMKK2增强NK细胞 通过激活AKT1、AMPK和mTORC1通路发挥作用。 利用CAMKK2基因敲除小鼠模型和常见的体外检测，我们打算坚定地 阐明CAMKK2对多种NK细胞功能的影响。使用小分子抑制剂或 激活剂，我们将确定AKT1、AMPK和mTORC1通路在CAMKK2介导的 表型。由于CAMKK2在肿瘤条件下选择性地在NK细胞中表达，我们计划使用ATAC 测序以确定导致CAMKK2上调的途径和肿瘤因素。最后，我们将使用 LOX-Cre系统选择性消融小鼠NK细胞中的CAMKK2并确定其整体效果 关于肿瘤转移的研究。如果CAMKK2的丢失被证明是有害的，我们将改造NK细胞系NK-92来 CAMKK2基因的高效表达及对转移瘤小鼠体内抗肿瘤活性的检测。 各种CAMKK2抑制剂和降解剂正在被开发用于癌症治疗，其中一些已经 已经在小鼠乳腺癌模型中被证明是有效的。如果我们证明CAMKK2的丢失或抑制 如假设的那样抑制NK细胞的功能，那么我们的研究将有助于确定靶点 这些新药的患者群体。NK细胞敏感型肿瘤患者(如MHC I类阴性)或 接受NK细胞介导的治疗(例如，单抗治疗)的患者应避免使用CAMKK2抑制剂。 我们的研究还可能促使我们制定策略，使CAMKK2抑制剂对NK细胞和其他细胞的影响脱钩 单元类型。我们认为，CAMKK2抑制剂的大部分治疗效果来自于它们对 因此，应优先考虑针对巨噬细胞的CAMKK2抑制剂的特定递送策略。 此外，我们的研究提出了一种提高NK细胞输注活性的新策略。 永生化的人类NK细胞系，如NK-92，正在作为癌症疗法进行积极的研究。如果CAMKK2 表达增强NK细胞抗肿瘤活性，然后NK细胞株可以被基因修饰为人工 过表达CAMKK2，增强NK细胞输注效果。

Abstract: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is an important regulator of cellular metabolism. Though classically associated with neurons and macrophages, our data show that CaMKK2 is also expressed in tumor-infiltrating natural killer (NK) cells and may regulate the anti-tumor activity of these cells. Previous publications have shown that CaMKK2 expression enhances breast tumor growth in mice by polarizing tumor-associated macrophages toward a pro-tumor M2 phenotype. However, our preliminary data suggest that CaMKK2 plays the opposite role in NK cells: CaMKK2 signaling seems to enhance NK cell survival, proliferation, migration, and anti-metastatic immune surveillance. We postulate that CaMKK2 enhances NK cell functions by activating the AKT1, AMPK, and mTORC1 pathways. Using a genetic knock-out mouse model of CaMKK2 and common in vitro assays, we intend to firmly delineate the effects of CaMKK2 on a variety of NK cellular functions. Using small-molecule inhibitors or activators, we will determine the role of the AKT1, AMPK, and mTORC1 pathways in CaMKK2-mediated phenotypes. Because CaMKK2 is selectively expressed in NK cells under tumor conditions, we plan to use ATAC sequencing to identify pathways and tumor factors responsible for CaMKK2 upregulation. Finally, we will use the Lox-Cre system to selectively ablate CaMKK2 in murine NK cells and determine the overall effect of this ablation on tumor metastasis. If the loss of CaMKK2 proves detrimental, we will engineer the NK cell line NK-92 to overexpress CaMKK2 and assay its anti-tumor efficacy when transfused into mice with metastatic lesions. A variety of CaMKK2 inhibitors and degraders are being developed for cancer treatment, and some have already proven efficacious in murine breast cancer models. If we demonstrate that loss or inhibition of CaMKK2 suppresses NK cell function as hypothesized, then our research will be instrumental in determining the target patient population for these new drugs. Patients with NK-cell sensitive tumors (e.g. MHC Class I-negative) or patients taking NK-cell mediated therapies (e.g. monoclonal antibody therapies) should avoid CaMKK2 inhibitors. Our research may also prompt strategies for decoupling the effects of CaMKK2 inhibitors on NK cells and other cell types. We believe that much of the therapeutic efficacy of CaMKK2 inhibitors comes from their effects on macrophages, so macrophage-specific delivery strategies for CaMKK2 inhibitors should be prioritized. Furthermore, our research suggests a novel strategy for enhancing the activity of NK cell transfusions. Immortalized human NK cell lines such as NK-92 are under active investigation as cancer therapies. If CaMKK2 expression enhances NK cell anti-tumor activity, then an NK cell line could be genetically modified to artificially overexpress CaMKK2, enhancing the efficacy of the NK cell transfusion.