Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3β) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3β in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3β-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3β levels, allowing us to study GSK3β function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3β. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3β-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3β in regulating NK cell metabolism.