Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals
The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα−/− cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.
KeywordsDiacylglycerol kinase Cancer immunotherapy Lck T-cell activation R59949 Serotonin receptors
Centro Nacional de Biotecnología
Consejo Superior de Investigaciones Científicas
Extracellular signal-regulated kinase
Human muscarinic receptor-1
Lymphocyte-specific protein tyrosine kinase
Nuclear factor of activated T cells
We thank Rosa Liébana for maintenance of the mouse colonies and technical assistance in the isolation of mouse cells, Alejandra Cordero for technical assistance, Carmen Moreno for technical assistance in cytometry data acquisition, and Catherine Mark for excellent editorial assistance.
Javier Arranz-Nicolás and Antonia Ávila-Flores performed mouse and Jurkat cell experiments, acquired, analyzed and interpreted data, and prepared the figures. Jesús Ogando performed the PBMC experiments and acquired the data. Jesús Ogando and Santos Mañes interpreted the PBMC data. Denise Soutar performed mouse experiments. Daniel Meraviglia-Crivelli and Raquel Arcos-Pérez performed Jurkat and mouse cells RT-PCR experiments. Raquel Arcos-Pérez developed the luciferase constructs. Antonia Ávila-Flores and Isabel Mérida designed and supervised the study, interpreted the data and wrote the manuscript. All authors read and gave input on the manuscript.
Javier Arranz-Nicolás and Jesús Ogando hold predoctoral FPI fellowships from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was supported in part by grants from the MINECO/FEDER/EU (BFU2016-77207-R), Spanish Ministry of Health (Instituto de Salud Carlos III; RD12/0036/0059) to Isabel Mérida, MINECO/FEDER/EU (SAF2017-83732-R) to Santos Mañes, and from the Madrid regional government (IMMUNOTHERCAM Consortium CM B2017/BMD3733) to Isabel Mérida and Santos Mañes.
Compliance with ethical standards
Conflict of interest
The authors declare no potential conflicts of interest.
Mice were maintained and handled in accordance with Spanish and European directives. All procedures performed with animals were conducted according the protocols approved by the CNB/CSIC Ethics Committee on Animal Experimentation (RD53/2013). PBMC were from the Blood Transfusion Center, Red Cross (Madrid, Spain), obtained with appropriate informed consent from the donors. No personal data were registered and all procedures performed with these cells were in accordance with the ethical standards of the CNB/CSIC Ethics Committee.
C57BL/6J-DGKα−/− mice were kindly donated by Dr. Xiao-Ping Zhong (Duke University Medical Center, Durham NC). C57BL/6J-DGKζ−/− mice were a gift of Dr. Gary Koretzky (University of Pennsylvania, Philadelphia PA). These mouse lines were used to generate the corresponding OT-I DGK−/− mice. The colonies were maintained in pathogen-free conditions in the CNB animal facility, following institutional guidelines.
Cell line authentication
Human leukemic Jurkat T cells were authenticated by polymorphic short tandem repeat (STR) locus analysis (Genomics Service, Centro de Investigaciones Biomédicas-CSIC).
- 3.Robert C, Ribas A, Wolchok JD, Hodi FS, Hamid O, Kefford R, Weber JS, Joshua AM, Hwu WJ, Gangadhar TC, Patnaik A, Dronca R, Zarour H, Joseph RW, Boasberg P, Chmielowski B, Mateus C, Postow MA, Gergich K, Elassaiss-Schaap J, Li XN, Iannone R, Ebbinghaus SW, Kang SP, Daud A (2014) Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet 384(9948):1109–1117. https://doi.org/10.1016/S0140-6736(14)60958-2 CrossRefPubMedGoogle Scholar
- 4.Weber JS, D’Angelo SP, Minor D, Hodi FS, Gutzmer R, Neyns B, Hoeller C, Khushalani NI, Miller WH Jr, Lao CD, Linette GP, Thomas L, Lorigan P, Grossmann KF, Hassel JC, Maio M, Sznol M, Ascierto PA, Mohr P, Chmielowski B, Bryce A, Svane IM, Grob JJ, Krackhardt AM, Horak C, Lambert A, Yang AS, Larkin J (2015) Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol 16(4):375–384. https://doi.org/10.1016/S1470-2045(15)70076-8 CrossRefPubMedGoogle Scholar
- 5.Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J, Kapoor V, Scholler J, Pure E, Milone MC, June CH, Riley JL, Wherry EJ, Albelda SM (2014) Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 20(16):4262–4273. https://doi.org/10.1158/1078-0432.CCR-13-2627 CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Prinz PU, Mendler AN, Masouris I, Durner L, Oberneder R, Noessner E (2012) High DGK-alpha and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8 + T cells that is reversible by pharmacologic intervention. J Immunol 188(12):5990–6000. https://doi.org/10.4049/jimmunol.1103028 CrossRefPubMedGoogle Scholar
- 11.Sato M, Liu K, Sasaki S, Kunii N, Sakai H, Mizuno H, Saga H, Sakane F (2013) Evaluations of the selectivities of the diacylglycerol kinase inhibitors R59022 and R59949 among diacylglycerol kinase isozymes using a new non-radioactive assay method. Pharmacology 92(1–2):99–107. https://doi.org/10.1159/000351849 CrossRefPubMedGoogle Scholar
- 14.Akhondzadeh S, Mohajari H, Reza Mohammadi M, Amini H (2003) Ritanserin as an adjunct to lithium and haloperidol for the treatment of medication-naive patients with acute mania: a double blind and placebo controlled trial. BMC Psychiatry 3:7. https://doi.org/10.1186/1471-244X-3-7 CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Jiang Y, Sakane F, Kanoh H, Walsh JP (2000) Selectivity of the diacylglycerol kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2, 3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) among diacylglycerol kinase subtypes. Biochem Pharmacol 59(7):763–772CrossRefPubMedGoogle Scholar
- 18.Martinez-Moreno M, Garcia-Lievana J, Soutar D, Torres-Ayuso P, Andrada E, Zhong XP, Koretzky GA, Merida I, Avila-Flores A (2012) FoxO-dependent regulation of diacylglycerol kinase alpha gene expression. Mol Cell Biol 32(20):4168–4180. https://doi.org/10.1128/MCB.00654-12 CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Schaap D, van der Wal J, van Blitterswijk WJ, van der Bend RL, Ploegh HL (1993) Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon. Biochem J 289(Pt 3):875–881CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Dunn GP, Old LJ, Schreiber RD (2004) The three Es of cancer immunoediting. Annu Rev Immunol 22:329–360. https://doi.org/10.1146/annurev.immunol.22.012703.104803 CrossRefPubMedGoogle Scholar
- 29.Torres-Ayuso P, Daza-Martin M, Martin-Perez J, Avila-Flores A, Merida I (2014) Diacylglycerol kinase alpha promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src. Oncotarget 5(20):9710–9726. https://doi.org/10.18632/oncotarget.2344 CrossRefPubMedPubMedCentralGoogle Scholar
- 38.Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL (2004) SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol 173(2):945–954CrossRefPubMedGoogle Scholar
- 39.Patsoukis N, Bardhan K, Chatterjee P, Sari D, Liu B, Bell LN, Karoly ED, Freeman GJ, Petkova V, Seth P, Li L, Boussiotis VA (2015) PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation. Nat Commun 6:6692. https://doi.org/10.1038/ncomms7692 CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Gharbi SI, Rincon E, Avila-Flores A, Torres-Ayuso P, Almena M, Cobos MA, Albar JP, Merida I (2011) Diacylglycerol kinase zeta controls diacylglycerol metabolism at the immunological synapse. Mol Biol Cell 22(22):4406–4414. https://doi.org/10.1091/mbc.E11-03-0247 CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Joshi RP, Schmidt AM, Das J, Pytel D, Riese MJ, Lester M, Diehl JA, Behrens EM, Kambayashi T, Koretzky GA (2013) The zeta isoform of diacylglycerol kinase plays a predominant role in regulatory T cell development and TCR-mediated ras signaling. Sci Signal 6(303):ra102. https://doi.org/10.1126/scisignal.2004373 CrossRefPubMedPubMedCentralGoogle Scholar
- 42.Baldanzi G, Pighini A, Bettio V, Rainero E, Traini S, Chianale F, Porporato PE, Filigheddu N, Mesturini R, Song S, Schweighoffer T, Patrussi L, Baldari CT, Zhong XP, van Blitterswijk WJ, Sinigaglia F, Nichols KE, Rubio I, Parolini O, Graziani A (2011) SAP-mediated inhibition of diacylglycerol kinase alpha regulates TCR-induced diacylglycerol signaling. J Immunol 187(11):5941–5951. https://doi.org/10.4049/jimmunol.1002476 CrossRefPubMedPubMedCentralGoogle Scholar
- 43.Prinz PU, Mendler AN, Brech D, Masouris I, Oberneder R, Noessner E (2014) NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention. Int J Cancer 135(8):1832–1841. https://doi.org/10.1002/ijc.28837 CrossRefPubMedGoogle Scholar
- 44.Dominguez CL, Floyd DH, Xiao A, Mullins GR, Kefas BA, Xin W, Yacur MN, Abounader R, Lee JK, Wilson GM, Harris TE, Purow BW (2013) Diacylglycerol kinase alpha is a critical signaling node and novel therapeutic target in glioblastoma and other cancers. Cancer Discov 3(7):782–797. https://doi.org/10.1158/2159-8290.CD-12-0215 CrossRefPubMedPubMedCentralGoogle Scholar
- 47.Rainero E, Caswell PT, Muller PA, Grindlay J, McCaffrey MW, Zhang Q, Wakelam MJ, Vousden KH, Graziani A, Norman JC (2012) Diacylglycerol kinase alpha controls RCP-dependent integrin trafficking to promote invasive migration. J Cell Biol 196(2):277–295. https://doi.org/10.1083/jcb.201109112 CrossRefPubMedPubMedCentralGoogle Scholar
- 48.Guo R, Wan CK, Carpenter JH, Mousallem T, Boustany RM, Kuan CT, Burks AW, Zhong XP (2008) Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta. Proc Natl Acad Sci USA 105(33):11909–11914. https://doi.org/10.1073/pnas.0711856105 CrossRefPubMedPubMedCentralGoogle Scholar
- 49.Kortum RL, Rouquette-Jazdanian AK, Miyaji M, Merrill RK, Markegard E, Pinski JM, Wesselink A, Nath NN, Alexander CP, Li W, Kedei N, Roose JP, Blumberg PM, Samelson LE, Sommers CL (2013) A phospholipase C-gamma1-independent, RasGRP1-ERK-dependent pathway drives lymphoproliferative disease in linker for activation of T cells-Y136F mutant mice. J Immunol 190(1):147–158. https://doi.org/10.4049/jimmunol.1201458 CrossRefPubMedGoogle Scholar