The role of CaMKK2 in natural killer cell anti-tumor immunity

Abstract

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a calcium-activated regulator of energy homeostasis in many cell types, including neurons and hepatocytes. In these cells, CaMKK2 connects calcium signaling to several ubiquitous energy and metabolism pathways such as AMPK, CaMKI, and CaMKIV. Tumor cells often express CaMKK2 ectopically, commandeering this enzyme to promote survival, proliferation, and metastasis across a wide variety of cancers. Recent work has established additional roles for CaMKK2 in tumor immunity. Notably, expression of this enzyme promotes M2 polarization of tumor-associated macrophages and facilitates the development of myeloid-derived suppressor cells, indirectly promoting tumor growth. Thus, there has been considerable interest in developing CaMKK2 inhibitors, including competitive kinase antagonists and ligand-directed degraders (LDDs), as potential cancer therapeutics. Indeed, the classic competitive inhibitor STO609 has been shown to reduce primary breast tumor growth in mice. However, the role of CaMKK2 in most cellular compartments within the tumor immune environment remains unknown, which is a significant impediment to the clinical development of these agents. To develop a complete understanding of the effects of CaMKK2 inhibition on the tumor environment, we set out to systematically delineate which cells express CaMKK2 and for what purpose.Our most significant finding is that natural killer (NK) cells upregulate CaMKK2 expression in the tumor environment, conferring a fitness advantage that improves anti-tumor immune activity. We report that CaMKK2 is expressed at low levels in NK cells isolated from murine spleens but that its expression is dramatically increased in NK cells isolated from tumors or exposed to tumor conditions. A series of in vitro functional assays showed that CaMKK2 expression suppresses apoptosis and promotes proliferation of NK cells without affecting the cytotoxic efficacy of individual cells. These findings were highly intriguing as NK cells play a critical role in the anti-tumor immune response, especially in controlling metastatic disease. As such, we demonstrated that NK cell-intrinsic deletion of CaMKK2 increases metastatic progression across several murine models, establishing a critical role for this enzyme in NK cell tumor immunity. Interestingly, ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK cell survival, a result which reveals a unique scaffold function for this enzyme. Finally, we identified lactic acid as a key driver of CaMKK2 upregulation under tumor conditions, an interesting finding given the immune suppressive nature of lactic acid. Intracellular importation of lactic acid through the MCT channels, and not general acidification, is necessary for CaMKK2 upregulation in NK cells. These results suggest that CaMKK2 upregulation in tumor-infiltrating NK cells is an adaptive mechanism by which these cells mitigate the deleterious effects of a lactate-rich tumor environment. This work will inform strategies to manipulate the CaMKK2 signaling axis as a therapeutic approach in cancer. Because CaMKK2 acts as a scaffold in NK cells, competitive inhibitors and LDDs are likely to have distinct therapeutic utilities. Our work suggests that competitive inhibitors may be broadly preferable in the clinical setting, and we aim to test this hypothesis in mouse tumor models once suitable LDDs become available. We are also working to identify components of the CaMKK2 scaffold complex and elucidate the downstream pathways, which may yield further insights into how this pathway can be manipulated in NK cells. Moreover, because intracellular lactate import is necessary for CaMKK2 upregulation, there may be liabilities associated with the emerging use of MCT1 inhibitors in cancer therapy, which we intend to explore in mouse models. Our findings also raise the possibility that NK cells can be modified to constitutively express CaMKK2 as a proactive shield against suppressive tumor factors, and we are working to replicate our core findings in human NK cells. Overall, our work holds significant implications for the therapeutic manipulation of tumor immunity.