NEDDylation regulates CD8+ T cell metabolism and anti-tumor immunity.

NEDDylation is a post-translational modification whereby the ubiquitin-like molecule NEDD8 is attached to protein substrates in a process dependent on NEDD8 activating enzyme regulatory subunit (NAE1). NEDDylation is emerging as a regulator of cancer biology, but its precise role in antitumor immunity has not been thoroughly characterized. Here, we study the impact of NEDDylation in CD8+ T cell-mediated antitumor responses. Analysis of publicly available single-cell RNA sequencing databases revealed that CD8+ tumor-infiltrating lymphocytes (TILs) showed increased expression of NEDD8 during their differentiation into effector memory cells. In vitro activation of mouse and human CD8+ T cells drove the upregulation of the NEDDylation enzymatic pathway, resulting in an enrichment of NEDDylated proteins. In vivo tumor challenge assays demonstrated that CD8+ T cells lacking NAE1 (NAE1-KO) exhibited reduced antitumor capability and a less activated phenotype with compromised differentiation into effector cells. Upregulating NEDDylation by knocking out deNEDDylase sentrin-specific protease 8 (SENP8) increased the in vitro cytotoxic capability of CD8+ CAR T cells. In addition, LC MS/MS proteomic analyses of NAE1-KO CD8+ T cells and CD8+ T cells treated with the NEDDylation inhibitor MLN4924, showed a pronounced impairment in metabolic pathways, including glycolysis and oxidative phosphorylation. In this context, we validated lactate dehydrogenase A, α-enolase and hexokinase 1, which are relevant glycolytic enzymes, as NEDD8 targets. In line with this, NEDDylation-deficient CD8+ T cells demonstrated reduced transcription, protein expression and enzymatic activity of lactate dehydrogenase. In summary, we uncover NEDDylation as a critical regulator of CD8+ T cell-mediated antitumor immunity.