Metabolic but not transcriptional regulation by PKM2 is important for Natural Killer cell responses
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Abstract
Natural Killer (NK) cells have an important role in immune responses to viruses and tumours. Integrating changes in signal transduction pathways and cellular metabolism is essential for effective NK cells responses. The PKM2 isoform of the glycolytic enzyme Pyruvate Kinase Muscle has described roles in regulating glycolytic flux and signal transduction, especially gene transcription. While PKM2 expression is robustly induced in activated NK cells, mice lacking PKM2 in NK cells showed no defect in NK cell metabolism or anti-viral responses to MCMV infection. This maintenance of function is explained by compensatory PKM1 expression in PKM2-null NK cell cells demonstrating that PKM2 is not a signalling molecule in this immune cell type. To further investigate the role of PKM2 we forced the tetramerization of the protein with TEPP-46, which increases its catalytic activity while inhibiting any signalling functions mediated by mono/dimeric conformations. NK cells activated with TEPP-46 had reduced effector function due to TEPP-46-induced increases in oxidative stress. Overall, PKM2-regulated glycolytic metabolism and redox status, not transcriptional control, facilitate optimal NK cells responses.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on June 12, 2020, follows.
Summary
In this study, Walls et al examine the role of PKM2 in NK cells. PKM2, the glycolytic enzyme that converts PEP to pyruvate, is expressed in NK cells and further upregulated during NK cell activation by Il-2/IL-12 stimulation. However, NK cells lacking PKM2 are activated normally in vitro and in vivo during MCMV infection, as indicated by proliferation, production of IFNg and TNF, expression of Granzyme B, and viral clearance. The authors attribute the lack of phenotype to compensatory induction of PKM1. The authors' findings also suggest that while in other cell types PKM2 may "moonlight" in a transcriptional role, any such role for PKM2 in NK cells seems not to influence NK cell activation, at least …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on June 12, 2020, follows.
Summary
In this study, Walls et al examine the role of PKM2 in NK cells. PKM2, the glycolytic enzyme that converts PEP to pyruvate, is expressed in NK cells and further upregulated during NK cell activation by Il-2/IL-12 stimulation. However, NK cells lacking PKM2 are activated normally in vitro and in vivo during MCMV infection, as indicated by proliferation, production of IFNg and TNF, expression of Granzyme B, and viral clearance. The authors attribute the lack of phenotype to compensatory induction of PKM1. The authors' findings also suggest that while in other cell types PKM2 may "moonlight" in a transcriptional role, any such role for PKM2 in NK cells seems not to influence NK cell activation, at least in the contexts studied.
PKM2, unlike PKM1, can form a tetramer with increased enzyme activity. In other contexts, such tetramerization is thought to enhance flux through glycolysis which disfavors glycolytic intermediates from being diverted to biosynthetic shunts like the PPP. The authors next asked how such tetramerization of PKM2 may influence NK cell activation, using a small molecule TEPP-46 that enhances PKM2 tetramerization. The authors found that TEPP-46 treatment during NK cell activation led to reduced cellular growth, reduced production of the PPP metabolites R5P and NADPH, increased cellular ROS, and reduced oxidative metabolism, as well as reduced production of IFNg and TNF and reduced expression of Granzyme B.
Essential Revisions
The authors should provide some mechanistic insight into how PKM2 tetramerization leads to reduced NK cell activation. Does treatment with ROS scavengers like NAC or cell permeable glutathione rescue the effects of TEPP-46 on NK cell activation?
Does PKM2 undergo tetramerization in a physiological context? Given the lack of a phenotype in the PKM2 KO in the in vitro or in vivo conditions that the authors analyzed, it seems like tetramerization may not occur (because PKM1, which is upregulated, is thought to not tetramerize). At the very least, the authors should discuss under what conditions PKM2 tetramerization can occur to suppress NK cell activation.
The authors should confirm that TEPP-46 has no effect in PKM2 KO cells.
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