Transcriptomic Analysis of Human Primary T Cells After Short-term Leucine-deprivation and Evaluation of Kinase GCN2's Role in Regulating Differential Gene Expression

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2025 Feb 18

PMID 39965008

Authors

Aurore Douge

Gwendal Cueff

Celine Keime

Valerie Carraro

Celine Jousse

Paul Rouzaire

Alain Bruhat

Affiliations

Soon will be listed here.

Abstract

Chimeric Antigen Receptor T (CAR-T) cells offer a promising strategy for cancer treatment. These CAR-T cells are either autologous or allogeneic T cells that are genetically modified to express a chimeric antigen receptor targeting a specific tumor antigen. Ongoing research aims to optimize the CAR-T cell efficacy, including strategies to modulate their metabolism. One such approach involves inducing transgene expression by activating the GCN2 kinase signaling pathway through dietary deprivation of an essential amino acid. In this study, we investigated the general impact of a 6-hour leucine deprivation on primary activated human T cells using RNA-seq technology. Our analysis identified 3,431 differentially expressed genes between T cells cultured in regular medium and those cultured in leucine-deprived medium. Gene Set Enrichment Analysis revealed that "TNFα signaling via NFκB", "interferon-γ response", and "unfolded protein response" gene sets were positively enriched, while "mTORC1 signaling", "Myc targets", and "oxidative phosphorylation" gene sets were negatively enriched. To further evaluate the involvement of GCN2 kinase in regulating the differential gene expression during the 6-hour leucine deprivation, T cells were cultured with or without a GCN2 inhibitor. We found that 59% of the differentially expressed genes in our dataset were dependent on the kinase GCN2 (n = 2028), with 1,140 up-regulated and 888 down-regulated genes. These findings suggest a promising strategy to enhance CAR-T cell efficacy by combining short amino acid starvation with transient overexpression of a target gene.

References

Anders S, Pyl P, Huber W . HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2014; 31(2):166-9. PMC: 4287950. DOI: 10.1093/bioinformatics/btu638. View

Gauthier-Coles G, Rahimi F, Broer A, Broer S . Inhibition of GCN2 Reveals Synergy with Cell-Cycle Regulation and Proteostasis. Metabolites. 2023; 13(10). PMC: 10609202. DOI: 10.3390/metabo13101064. View

Bhattacharya S, HuangFu W, Dong G, Qian J, Baker D, Karar J . Anti-tumorigenic effects of Type 1 interferon are subdued by integrated stress responses. Oncogene. 2012; 32(36):4214-21. PMC: 3766494. DOI: 10.1038/onc.2012.439. View

Xu D, Dai W, Kutzler L, Lacko H, Jefferson L, Dennis M . ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation. J Nutr. 2019; 150(5):1022-1030. PMC: 7198311. DOI: 10.1093/jn/nxz309. View

St Paul M, Saibil S, Kates M, Han S, Lien S, Laister R . Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy. Cell Rep Med. 2024; 5(3):101465. PMC: 10983112. DOI: 10.1016/j.xcrm.2024.101465. View

Eleftheriadis T, Pissas G, Antoniadi G, Liakopoulos V, Tsogka K, Sounidaki M . Differential effects of the two amino acid sensing systems, the GCN2 kinase and the mTOR complex 1, on primary human alloreactive CD4⁺ T-cells. Int J Mol Med. 2016; 37(5):1412-20. DOI: 10.3892/ijmm.2016.2547. View

Langmead B, Salzberg S . Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4):357-9. PMC: 3322381. DOI: 10.1038/nmeth.1923. View

Chaveroux C, Bruhat A, Carraro V, Jousse C, Averous J, Maurin A . Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids. Nat Biotechnol. 2016; 34(7):746-51. DOI: 10.1038/nbt.3582. View

Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S . STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2012; 29(1):15-21. PMC: 3530905. DOI: 10.1093/bioinformatics/bts635. View

10.

Rial Saborido J, Volkl S, Aigner M, Mackensen A, Mougiakakos D . Role of CAR T Cell Metabolism for Therapeutic Efficacy. Cancers (Basel). 2022; 14(21). PMC: 9658570. DOI: 10.3390/cancers14215442. View

11.

Sterner R, Sterner R . CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021; 11(4):69. PMC: 8024391. DOI: 10.1038/s41408-021-00459-7. View

12.

Douge A, El Ghazzi N, Lemal R, Rouzaire P . Adoptive T Cell Therapy in Solid Tumors: State-of-the Art, Current Challenges, and Upcoming Improvements. Mol Cancer Ther. 2023; 23(3):272-284. DOI: 10.1158/1535-7163.MCT-23-0310. View

13.

Jenkins Y, Zabkiewicz J, Ottmann O, Jones N . Tinkering under the Hood: Metabolic Optimisation of CAR-T Cell Therapy. Antibodies (Basel). 2021; 10(2). PMC: 8167549. DOI: 10.3390/antib10020017. View

14.

Hou Y, Wei D, Zhang Z, Lei T, Li S, Bao J . Downregulation of nutrition sensor GCN2 in macrophages contributes to poor wound healing in diabetes. Cell Rep. 2024; 43(1):113658. DOI: 10.1016/j.celrep.2023.113658. View

15.

Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov J, Tamayo P . The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2016; 1(6):417-425. PMC: 4707969. DOI: 10.1016/j.cels.2015.12.004. View

16.

Broer A, Gauthier-Coles G, Rahimi F, van Geldermalsen M, Dorsch D, Wegener A . Ablation of the () gene encoding a neutral amino acid transporter reveals transporter plasticity and redundancy in cancer cells. J Biol Chem. 2019; 294(11):4012-4026. PMC: 6422075. DOI: 10.1074/jbc.RA118.006378. View

17.

Rostamian H, Fallah-Mehrjardi K, Khakpoor-Koosheh M, M Pawelek J, Hadjati J, Brown C . A metabolic switch to memory CAR T cells: Implications for cancer treatment. Cancer Lett. 2020; 500:107-118. DOI: 10.1016/j.canlet.2020.12.004. View

18.

Kemp K, Poe C . Stressed: The Unfolded Protein Response in T Cell Development, Activation, and Function. Int J Mol Sci. 2019; 20(7). PMC: 6479341. DOI: 10.3390/ijms20071792. View

19.

Bchir W, Maurin A, Carraro V, Averous J, Jousse C, Muranishi Y . The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Res. 2013; 41(16):7683-99. PMC: 3763548. DOI: 10.1093/nar/gkt563. View

20.

Wang P, Xu Y, Zhang J, Shi L, Lei T, Hou Y . The amino acid sensor general control nonderepressible 2 (GCN2) controls T9 cells and allergic airway inflammation. J Allergy Clin Immunol. 2019; 144(4):1091-1105. DOI: 10.1016/j.jaci.2019.04.028. View