NEDDylation Regulates CD8+ T-cell Metabolism and Antitumor Immunity

Abstract

NEDDylation; Metabolism; Antitumor immunity

NEDDilación; Metabolismo; Inmunidad antitumoral

NEDDilació; Metabolisme; Immunitat antitumoral

NEDDylation is a posttranslational 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. In this study, we examine 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 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 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 increased the in vitro cytotoxic capability of CD8+ CAR T cells. In addition, LC MS/MS proteomic analyses of NAE1-deficient 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.

This research was supported by the following: Ministerio de Ciencia, Innovación y Cultura (MICIU)/Agencia Española de Investigación (AEI; 10.13039/501100011033) financed “PID2019-107956RA-I00” project (A.P); MICIU/AEI/10.13039/501100011033 and European Union Next Generation EU/PRTR financed “PDC2022-133300-I00” (A. Palazon), “TED2021-129433B-C21” (A. Palazon) and “JDC2022-048612-I” (L. Pérez-Gutiérrez) projects; MICIU/AEI (10.13039/501100011033) and “Fondo Europeo de Desarrollo Regional (FEDER)”/UR financed “PID2022-139344OB-I00” project (A. Palazon); MICIU/AEI/10.13039/501100011033 and “El Fondo Social Europeo (FSE) invierte en tu futuro” financed “RYC2018-024183-I” (A. Palazon) and “PRE2020-092342” (P. Velasco-Beltrán) grants; AEI financed “RYC2021-031213-I” grant (E. Prieto-Fernández), FSE+ financed “PRE2022-104817” (J. Etxaniz-Díaz de Durana) predoctoral grant; Ministerio de Ciencia, Innovación y Universidades MICINN: PID2023-146933OB-100 funded by MCIU /AEI /10.13039/501100011033/FEDER, UE, as part of Plan Estatal de Investigación Científica y Técnica e Innovación (M.L. Martínez-Chantar); MCIN/AEI/10.13039/501100011033 financed the grant CEX2021-001136-S (M.L. Martínez-Chantar); the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program financed grant agreement “no 804236” (A. Palazon); the ERC awarded to G. Baier with “ERC-2018-ADG 786462-HOPE” grant; the Provincial Section of Bizkaia and of the Scientific Foundation of the Spanish Association against cancer (AECC) financed “PRDVZ21640DEBL” (A. de Blas) and “PRDVZ1900BOSCH” (A. Bosch) predoctoral grants; the Scientific Foundation of AECC financed “LABAE211744PALA” project (A. Palazon); “La Caixa” Foundation financed the “HR21-00925” (A. Palazon); “La Caixa” Foundation under the program of INPhINIT fellowships financed Doctoral INPhINIT Fellowship – Retaining under the code “LCF/BQ/DR20/11790022” (A. Antoñana-Vildosola); “XVI” BECA FERO (A. Palazon); the Basque Government financed “PRE_2019_1_0320” predoctoral grant (B. Jiménez-Lasheras) through the “Programa Predoctoral de Formación de Personal Investigador No doctor del Departamento de Educación,” “2023333027” project (A. Palazon) through the “Programa Ayudas a proyectos de investigación y desarrollo en salud del Departamento de Salud,” and “AF-W1-2019-00012” through Programa Bikaintek (A. García-del Río).