Post-transcriptional control of metabolic programs that improve T cell responses against tumors
Immunotherapies including adoptive T cell transfer and checkpoint blockade have proven very successful in the treatment of cancer. However, to date only a fraction of malignancies can be addressed with such treatments, and within the responsive cancer types only a fraction of patients respond. The current limitations of these therapies include the identification of mutated neoantigens and engineered antigen receptors.
The Roquin and Regnase RNA-binding proteins (RBPs) control T cell activation and differentiation, and they prevent the development of humoral autoimmunity. In our studies of these factors we will now analyze how these proteins control CD8+ T cell activation, metabolism and cytotoxicity, and find out how we can make use of this post-transcriptional system to enhance anti-tumor responses. Roquin as well as Regnase-1 proteins are inactivated through proteolytic cleavage by MALT1 in a TCR strength-dependent manner. Here, we propose that the inactivation of Roquin- and Regnase-1 by MALT1 may represent an important aspect for the success of engineered T cells in anti-tumor responses. In Aim 1 we will test our hypothesis with TCR transgenic CD8+ T cells of gradually different structural affinities and functional avidities. We will then assess cleavage and activity of the system in T cells with TCRs recognizing viral or tumor antigens as well as in T cells bearing chimeric antigen receptors. As a proof-of-concept, we will determine anti-tumor responses of wild type cytotoxic T cells compared to mutant counterparts, which cannot induce cleavage of Roquin-1 by MALT1. These experiments shall define a threshold of TCR structural affinity and MALT1 signaling that enables optimal CD8+ anti-tumor responses. Studying CD4+ T cells with inducible inactivation of Roquin and Regnase-1 encoding alleles we uncovered profound alterations of T cell metabolism including spontaneous T cell activation, elevated protein biosynthesis and increased PI3K/Akt as well as mTOR signaling. These changes were associated with induced proliferation and Tfh differentiation, but also with survival, memory and exhaustion phenotypes, which varied among T cells of different genotypes. Our hypothesis is that productive T cell activation by antigen recognition with sufficient TCR strength will involve inactivation of Roquin and Regnase via MALT1 cleavage to impose phenotypic changes that we observe in the knockout situation. In Aim 2 of this project we will now investigate the contribution of the system to the metabolic state of CD8+ T cells. Specifically, we will address how TCR-dependent regulation of Roquin and Regnase-1 proteins impacts on glycolysis, oxidative phosphorylation, mitochondrial respiration and lipolysis. Very recently, we have been able to establish a cross- linking and immunoprecipitation (iCLIP) approach for Roquin in mouse CD4+ T cells, enabling us to identify directly bound targets at near-nucleotide resolution. This assay allows a discrimination of the very upstream and direct targets from downstream and indirect gene regulation. In Aim 3 we will transfer this assay to mouse and human CD8+ T cells to comprehensively determine targets and binding sites. In a systematic approach we will then test by adoptive transfer of tumor-specific T cells into tumor bearing hosts how and when the inactivation of Roquin and Regnase-1 or the interference with their cooperation in T cells will impact on tumor responses. Focusing on their identified direct targets we will overexpress or inactivate those genes that contribute to metabolic reprogramming and determine how to manipulate metabolic switches to improve the function of engineered T cells. Together, these experiments will unravel how the Roquin- and Regnase-mediated gene regulation contributes to anti-tumor responses of CD8+ T cells and evaluate how it can be utilized immune cell therapies.
Tavernier SJ, Athanasopoulos V, Verloo P, Behrens G, Staal J, Bogaert DJ, Naesens L, De Bruyne M, Van Gassen S, Parthoens E, Ellyard J, Cappello J, Morris LX, Van Gorp H, Van Isterdael G, Saeys Y, Lamkanfi M, Schelstraete P, Dehoorne J, Bordon V, Van Coster R, Lambrecht BN, Menten B, Beyaert R, Vinuesa CG, Heissmeyer V, Dullaers M, Haerynck F. 2019. A human immune dysregulation syndrome characterized by severe hyperinflammation with a homozygous nonsense Roquin-1 mutation. Nat Commun 10: 4779
Essig K, Kronbeck N, Guimaraes JC, Lohs C, Schlundt A, Hoffmann A, Behrens G, Brenner S, Kowalska J, Lopez- Rodriguez C, Jemielity J, Holtmann H, Reiche K, Hackermuller J, Sattler M, Zavolan M, Heissmeyer V. 2018. Roquin targets mRNAs in a 3′-UTR-specific manner by different modes of regulation. Nat Commun 9: 3810
Rehage N, Davydova E, Conrad C, Behrens G, Maiser A, Stehklein JE, Brenner S, Klein J, Jeridi A, Hoffmann A, Lee E, Dianzani U, Willemsen R, Feederle R, Reiche K, Hackermuller J, Leonhardt H, Sharma S, Niessing D, Heissmeyer V. 2018. Binding of NUFIP2 to Roquin promotes recognition and regulation of ICOS mRNA. Nat Commun 9: 299
Essig K, Hu D, Guimaraes JC, Alterauge D, Edelmann S, Raj T, Kranich J, Behrens G, Heiseke A, Floess S, Klein J, Maiser A, Marschall S, Hrabe de Angelis M, Leonhardt H, Calkhoven CF, Noessner E, Brocker T, Huehn J, Krug AB, Zavolan M, Baumjohann D, Heissmeyer V. 2017. Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells. Immunity 47: 1067-82 e12
Janowski R, Heinz GA, Schlundt A, Wommelsdorf N, Brenner S, Gruber AR, Blank M, Buch T, Buhmann R, Zavolan M, Niessing D, Heissmeyer V, Sattler M. 2016. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat Commun 7: 11032
Gewies A, Gorka O, Bergmann H, Pechloff K, Petermann F, Jeltsch KM, Rudelius M, Kriegsmann M, Weichert W, Horsch M, Beckers J, Wurst W, Heikenwalder M, Korn T, Heissmeyer V, Ruland J. 2014. Uncoupling Malt1 threshold function from paracaspase activity results in destructive autoimmune inflammation. Cell Rep 9: 1292-305
Jeltsch KM, Hu D, Brenner S, Zoller J, Heinz GA, Nagel D, Vogel KU, Rehage N, Warth SC, Edelmann SL, Gloury R, Martin N, Lohs C, Lech M, Stehklein JE, Geerlof A, Kremmer E, Weber A, Anders HJ, Schmitz I, Schmidt-Supprian M, Fu M, Holtmann H, Krappmann D, Ruland J, Kallies A, Heikenwalder M, Heissmeyer V. 2014. Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote T(H)17 differentiation. Nat Immunol 15: 1079-89
Schlundt A, Heinz GA, Janowski R, Geerlof A, Stehle R, Heissmeyer V, Niessing D, Sattler M. 2014. Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation. Nat Struct Mol Biol 21: 671-8
Vogel KU, Edelmann SL, Jeltsch KM, Bertossi A, Heger K, Heinz GA, Zoller J, Warth SC, Hoefig KP, Lohs C, Neff F, Kremmer E, Schick J, Repsilber D, Geerlof A, Blum H, Wurst W, Heikenwalder M, Schmidt-Supprian M, Heissmeyer V. 2013. Roquin paralogs 1 and 2 redundantly repress the Icos and Ox40 costimulator mRNAs and control follicular helper T cell differentiation. Immunity 38: 655-68
Glasmacher E, Hoefig KP, Vogel KU, Rath N, Du L, Wolf C, Kremmer E, Wang X, Heissmeyer V. 2010. Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression. Nat Immunol 11: 725-33