The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation

“The metabolic changes associated with T cell activation are reminiscent of the characteristic metabolic activities of tumor cells and may represent a general metabolic reprogramming during cell growth and proliferation (Kim and Dang, 2006; Vander Heiden et al., 2009; Warburg, 1956).”

“The metabolic changes associated with T cell activation are reminiscent of the characteristic metabolic activities of tumor cells and may represent a general metabolic reprogramming during cell growth and proliferation (Kim and Dang, 2006; Vander Heiden et al., 2009; Warburg, 1956).”

“The comparison of cytokine gene expression in resting and active T cells was visualized as heat maps with Cluster (Eisen and Brown, 1999) and TreeView (Eisen et al., 1998).”

“Whereas prokaryotes and unicellular eukaryotes rely primarily on homeostatic regulation of cell metabolism, metabolic programs in multicellular eukaryotes have evolved to be actively controlled by extracellular signals (Deberardinis et al., 2008).”

“Metabolic flux analysis in a glioblastoma cell line further revealed that a major portion of glutamine-derived nitrogen and carbon are incorporated into amino acids and lactate, a fraction of which are secreted as metabolic by-products (DeBerardinis et al., 2007).”

“Recent studies in human cell lines have suggested that enforced expression of Myc regulates the rate-limiting glutaminolyic enzyme glutaminase 1 (Gls1) through both transcription and posttranscriptional mechanisms (Gao et al., 2009; Wise et al., 2008), and p53 regulates the transcription of glutaminase 2 (Gls2) (Hu et…”

“Animals Hif1a and p53-null mice on the C57BL/6 background were purchased from the Jackson Laboratory (Jacks et al., 1994; Ryan et al., 2000) and Hif1a mice were crossed with ROSA26CreERT2 (de Luca et al.”

“Because a-KG is required to maintain cell viability and minimal cell proliferation in the absence of glutamine (Wise et al., 2008), addition of a-KG alone was sufficient to drive only one cell division in the absence of glutamine.”

“To further test the dependency of T cell proliferation on glucose and glutamine catabolism in vivo, we applied 2DG and DON in an adoptive transfer model by using ovalbumin (OVA)specific, TCR-transgenic OT-II T cells (Figure 1G; Shi et al., 2011).”

“Because glutamine flux might regulate cell growth through the mTOR pathway (Nicklin et al., 2009), glutamine-dependent modulation of mTOR activation might partially account for the regulation of T cell growth by Myc.”

“, 2008), and the mammalian target of rapamycin (mTOR), an essential component of PI3K-Akt pathway, has recently been implicated in regulating fatty acid metabolism in memory T cells (Pearce et al., 2009; Araki et al., 2009).”

“, 2008), and the mammalian target of rapamycin (mTOR), an essential component of PI3K-Akt pathway, has recently been implicated in regulating fatty acid metabolism in memory T cells (Pearce et al., 2009; Araki et al., 2009).”

“Signaling via ERK and AKT pathways has been implicated in the uptake of glutamine and glucose, respectively (Carr et al., 2010; Frauwirth et al., 2002; Wofford et al., 2008), and the mammalian target of rapamycin (mTOR), an essential component of PI3K-Akt pathway, has recently been implicated in regulating fatty acid…”

“The metabolic changes associated with T cell activation are reminiscent of the characteristic metabolic activities of tumor cells and may represent a general metabolic reprogramming during cell growth and proliferation (Kim and Dang, 2006; Vander Heiden et al., 2009; Warburg, 1956).”

“Consistent with a direct role ofMyc in regulating cell proliferation (Dang et al., 2006; Eilers and Eisenman, 2008), addition of a-KG with HT and PAs failed to promote cell proliferation in Myc-deficient T cells (Figure S7C).”

“Notably, ectopic overexpression of Myc not only alters the transcription of several glycolytic genes but also preferentially switches the expression of PKM1 to PKM2 by modulating the alternative splicing of the PKM transcript (Dang and Semenza, 1999; David et al., 2010).”

“T cell activation-induced glycolysis and glutaminolysis is reminiscent of the metabolic changes in tumor cells, where both aerobic glycolysis (the Warburg effect) and glutaminoly- sis are driven by aberrant oncogenic signals (Dang and Semenza, 1999; Vander Heiden et al., 2009).”

“The comparison of cytokine gene expression in resting and active T cells was visualized as heat maps with Cluster (Eisen and Brown, 1999) and TreeView (Eisen et al.”

“Whereas prokaryotes and unicellular eukaryotes rely primarily on homeostatic regulation of cell metabolism, metabolic programs in multicellular eukaryotes have evolved to be actively controlled by extracellular signals (Deberardinis et al., 2008).”

“Consistent with a direct role ofMyc in regulating cell proliferation (Dang et al., 2006; Eilers and Eisenman, 2008), addition of a-KG with HT and PAs failed to promote cell proliferation in Myc-deficient T cells (Figure S7C).”

“Primer sequences were obtained from PrimerBank (Spandidos et al., 2010) except for Gls1 and Gls2, which were manually designed.”

“Although recent studies indicate that the tumor suppressor p53 is required for the induction of GLS2 in a human tumor cell line (Hu et al., 2010; Suzuki et al., 2010),”

“TCR- and costimulator-mediated signals drive T cell growth and clonal expansion, and coordinately reprogram metabolic cascades to fulfill the ensuing bioenergetic and biosynthetic demands (Fox et al., 2005; Krauss et al., 2001).”

“Early studies have implicated the involvement of ERK-, AKT-, and mTOR-mediated signaling pathways in regulating T cell metabolism (Carr et al., 2010; Frauwirth et al., 2002; Pearce et al., 2009).”

“We observed that acute depletion of Myc, in vivo, did not compromise the health of the animals over a period of 1 week, consistent with a recent report (Soucek et al., 2008).”

“…and Myc were recently suggested to regulate 874 Immunity 35, 871–882, December 23, 2011 ª2011 Elsevier Inc. cell metabolism in transformed cells (Dang and Semenza, 1999; Gordan et al., 2007) and were highly induced at both the transcription and protein levels upon T cell activation (Figures 2D–2G).”

“Although recent studies indicate that the tumor suppressor p53 is required for the induction of GLS2 in a human tumor cell line (Hu et al., 2010; Suzuki et al., 2010),”

“Arg1flox/flox; Tie2Cre mice that were backcrossed to the C57BL/6 background were obtained from P. Murray (SJCRH, Memphis) (El Kasmi et al., 2008).”

“Consequently, one of the fundamental problems in animal cells is coordination of the metabolic program with cell growth- and proliferation-associated bioenergetic demand (Jorgensen and Tyers, 2004).”

“Early studies showed that polyclonal mitogens such as concanavalin A and phytohaemagglutinin drive the upregulation of glycolysis and glutamine oxidation in thymocytes and mature lymphocytes (Brand et al., 1984; Newsholme et al., 1985b).”

“Although Myc was suggested to be required for T cell proliferation and growth under most experimental conditions (Dose et al., 2006; Iritani et al., 2002), one study indicated that it is essential for cell proliferation but not cell growth (Trumpp et al., 2001).”

“…via ERK and AKT pathways has been implicated in the uptake of glutamine and glucose, respectively (Carr et al., 2010; Frauwirth et al., 2002; Wofford et al., 2008), and the mammalian target of rapamycin (mTOR), an essential component of PI3K-Akt pathway, has recently been implicated in…”

“During proliferation, T cells can undergo a cell division every 4–6 hr (van Stipdonk et al., 2003).”

“Mycflox/flox mice were obtained from F. Alt (Harvard) (de Alboran et al., 2001) and were backcrossed five generations onto the C56BL/6 background before breeding to ROSA26CreER(Tam) (Badea et al., 2003).”

“We generated a mouse model carrying a conditional HIF1a allele (Hif1aflox/flox) and a tamoxifen-inducible Cre recombinase (CreERT2) transgene (de Luca et al., 2005; Ryan et al., 2000) to delete HIF1a flox alleles in an acute manner.”

“Glutamine has long been known to be an indispensible nutrient for proliferating cells (Kovacevic and McGivan, 1983).”

“Early studies showed that polyclonal mitogens such as concanavalin A and phytohaemagglutinin drive the upregulation of glycolysis and glutamine oxidation in thymocytes and mature lymphocytes (Brand et al., 1984; Newsholme et al., 1985b).”

“Freshly isolated murine primary T cells were either maintained in interleukin 7 (IL-7) (hereafter ‘‘resting T cells’’) (Vella et al., 1997) or were stimulated with anti-CD3 plus anti-CD28 (hereafter ‘‘active T cells’’).”

“, 2001) and were backcrossed five generations onto the C56BL/6 background before breeding to ROSA26CreER(Tam) (Badea et al., 2003).”

“Antigen activation of T lymphocytes drives distinct transcriptional programs including upregulation of cell cycle and cytokine genes (Lu et al., 2004; Teague et al., 1999).”

“Intriguingly, the metabolic enzymes Aldh18A1, Prodh, and OAT that link glutamine and proline to ornithine synthesis were induced in a Mycdependent manner upon T cell activation (Figures 7C and S5F), as were the metabolic enzymes, ODC, spermine synthase (SRM), and spermidine synthase (SMS), that convert ornithine to po…”

“I Myc, but Not HIF1a, Is Required for Activation-Induced T Cell Metabolic Reprogramming With an in silico approach (Roider et al., 2009), transcription factors possibly involved in activation-induced T cell metabolic mmunity 35, 871–882, December 23, 2011 ª2011 Elsevier Inc. 873 (A andB) The…”

“TCR- and costimulator-mediated signals drive T cell growth and clonal expansion, and coordinately reprogram metabolic cascades to fulfill the ensuing bioenergetic and biosynthetic demands (Fox et al., 2005; Krauss et al., 2001).”

“…studies clearly demonstrate that Myc directly regulates T cell proliferation mainly through transcriptional control of cell cycle regulators (Dose et al., 2006; Iritani et al., 2002), our studies implicate Myc as an essential coordinator of T cell activation-induced cell growth,…”

“We compared our metabolic gene expression data of active mouse T lymphocytes with metabolic gene expression data from a recently published microarray study of active human T lymphocytes (Wang et al., 2008).”

“…demonstrate that Myc directly regulates T cell proliferation mainly through transcriptional control of cell cycle regulators (Dose et al., 2006; Iritani et al., 2002), our studies implicate Myc as an essential coordinator of T cell activation-induced cell growth, proliferation, and several…”

“…action of arginase as part of the urea cycle (Morris, 2004), urea cycle enzymes including arginase were not induced upon activation (Figure S7D) nor were any defects in proliferation observed in T cells lacking arginase I (Figures 7A and 7B), the dominant isoform in the thymus (Yu et al., 2003).”

“Early studies showed that polyclonal mitogens such as concanavalin A and phytohaemagglutinin drive the upregulation of glycolysis and glutamine oxidation in thymocytes and mature lymphocytes (Brand et al., 1984; Newsholme et al., 1985b).”

“Coinciding with the clearance of antigen, effector T cells undergo an abrupt contraction phase in which the majority of T cells are eliminated by apoptosis (Green, 2003; Schumacher et al., 2010).”

“Previous studies in bovine endothelial and intestinal epithelial cells suggested the possible presence of a noncanonical metabolic pathway, which generates ornithine from glutamine (Wu et al., 2000; Wu and Morris, 1998).”

“Antigen activation of T lymphocytes drives distinct transcriptional programs including upregulation of cell cycle and cytokine genes (Lu et al., 2004; Teague et al., 1999).”

“Consistent with a previous study (Bowlin et al., 1987), difluoromethylornithine (DFMO), a potent inhibitor of ornithine decarboxylase (ODC) and polyamine biosynthesis, inhibited activation-induced T cell proliferation in vitro and in vivo (Figure 6F and 1H), and the addition of exogenous polyamines completely restored…”

“Consistent with previous work (Rowell and Wells, 2006), T cell activation was associated with downregulation of the cell cycle inhibitor p27, the upregulation of cyclins and cyclin-dependent kinases (CDKs), and the appearance of markers of S phase, such as the phosphorylation of RB and Cdc6 (Figure…”

“Coinciding with the clearance of antigen, effector T cells undergo an abrupt contraction phase in which the majority of T cells are eliminated by apoptosis (Green, 2003; Schumacher et al., 2010).”

“We assessed the protein expression of glutamate dehydrogenase (Glud1), which catalyzes the oxidative deamination of glutamate to a-ketoglutarate, an important process for energy homeostasis in T cells (Wang et al., 2011).”

“Loss of Myc was associated with profoundly diminished expression of genes involved in metabolism and the inability for normal primary T cells to grow and proliferate (Wang et al., 2011a).”

“…of in-vitro-and in-vivoactivated T cells and found that both had high ECAR and OCR in comparison to naive cells (Figure 1A), indicating that activated T cells use both aerobic glycolysis and OXPHOS (Gatza et al., 2011; Michalek et al., 2011; van der Windt and Pearce, 2012; Wang et al., 2011).”

“BPTES inhibition of hGA(124-551), a truncated form of human kidneytype glutaminase.”

“Additionally, treating TH1 CD4+ T cells with PKM2activatorDASS,whichwill decrease intracellularPEP levels (data not shown) (Anastasiou et al., 2012), similarly suppressed the production of IFNg and CD40L (Figure S5C).”

“Intriguingly, with nutrient starvation, diminished TOR activity attenuates cell growth through diminished expression of dMyc.”

“It is important to note that OXPHOS is still engaged in TEFF cells (9); however, the production of lactate from pyruvate by aerobic glycolysis is the dominant pathway of glucosemetabolism in TEFF cells.”

“The most dramatic change in T cell metabolism upon activation is a marked increase in glucose metabolism, which appears to be regulated by the PI3K and Akt pathway and the transcription factorsMyc and ERRa (Frauwirth et al., 2002;Michalek et al., 2011; Wang et al., 2011).”

“Activation of PI3K, Akt, and mTOR regulates the switch to anabolic metabolism and subsequent aerobic glycolysis via transcription factors including Myc and HIF-1 (MacIver et al., 2013; Wang et al., 2011).”

“Recently, a role for c-Myc in T cell metabolism was found using an in silico approach to identify transcription factors whose gene targets correlated with changes in metabolic gene expression upon T cell stimulation (36).”

“Myc is a transcription factor that has important roles in upregulating glycolysis and glutaminolysis in activated T cells (Wang et al., 2011).”

“Increases in HIF-1α activity by culturing T cells in physiologic normoxia (~3–5% O2), genetic deletion of von Hippel-Lindau (VHL) factor, or inhibiting activity of the oxygen-sensing prolyl-hydroxylase (PHD) family of proteins, enhances CD8 T cell glycolysis and effector functions and promotes antitumor immunity (Clev…”

“In addition to the cMyc (Wang et al., 2011) and estrogenrelated receptor a (ERRa) (Michalek et al., 2011b) transcription factors that regulate T cell metabolism, mTOR and the PI3KAkt-mTORC1 signaling pathway can promote Glut1 cell-surface trafficking, glycolysis, and lipid synthesis (Düvel et al.,…”

“To confirm that Myc regulates total cellular RNA and mRNA levels, c-Myc null naive B cells were recovered from mice carrying a conditional Myc allele (Mycflox/flox) and a tamoxifen-inducible Cre recombinase CreERTam) (Wang et al., 2011) that were treated with tamoxifen for 3 days.”

“…including dendritic cells (Krawczyk et al. 2010), classically activated M1 macrophages (Michl et al. 1976), neutrophils (Sbarra & Karnovsky, 1959), lymphocytes (T cells (Frauwirth et al. 2002; Wang et al. 2011) and B cells (Doughty et al. 2006)) adopt a high-glycolytic phenotype once activated.”

“Quiescent primary human T cells and resting human B cells use FAO, but upon growth stimulation, these cells switch to glycolysis and glutaminolysis (Wang et al. 2011; Le et al. 2012).”

“The immune quiescence of naïve T cells is accompanied by a catabolic metabolism, characterized by the breakdown of glucose, fatty acids, and amino acids to generate intermediate metabolites, which enter the mitochondrial TCA cycle (17).”

“A fundamental change among T cells is enhanced aerobic glycolysis in response to increasing expression of glucose transporters (GLUTs) on the cell surface induced by the upregulation of glycolysis-related genes in a c-Myc-dependent manner.(124) Concomitant with increased glucose metabolism, multiple signaling pathways…”

“Thus, we observed metabolic reprogramming from lipid oxidation to glycolysis to be one of the early metabolic switches during CD8 + T cell activation and differentiation, a finding consistent with recent observations in CD4 + T cells (17).”

“utilized a combination of mass-spectrum-based metabolomic and metabolic tracer studies to identify the transcriptional factor Myc as a critical regulator of glycolysis and glutaminolysis (97) (Table 1).”

“F or p er so na l u se o nl y. lncRNA: long noncoding RNA PCGEM1: prostate cancer gene expression marker 1 RNF5: ubiquitin ligase ring finger protein ER: endoplasmic reticulum IL-4: interleukin-4 AMPK: AMP-activated protein kinase HIF1: hypoxia-inducible factor 1 OGDH: oxoglutarate dehydrogenase CPSI: carbamoyl phosph…”

“In addition, TCR signals induce c-Myc, which enacts transcription of metabolic genes critical for T cell activation (16).”

“Activation of T cells also enhances mitochondrial biogenesis, uptake of amino acids (AAs), and glutaminolysis (66, 67).”

“S1B), likely due to hypoxia-inducible factor 1a (HIF-1a)– and c-Myc–induced transcription of Ldha (10, 11).”

“…with the cell’s proliferative potential. c-Myc has been shown to be essential for activationinduced metabolic reprogramming and antigen-driven proliferation of naive CD8 T cells (Wang et al., 2011), and our data revealed that the Myc locus acquires an exhaustion-associated de novo DMR (Figure 1I).”

“Importantly, although the data showing that memory T and Tn cells acquire more 2NBDG than Teff are surprising, they do not argue against a role for glucose and glycolysis in Teff cell proliferation or effector function (Chang et al., 2013; Fox et al., 2005; Jacobs et al., 2008; Wang et al., 2011).”

“In activated T cells, c-Myc controlled metabolic reprogramming by regulating their metabolic transcriptome and thereby impacting glycolysis, glutaminolysis, and polyamine synthesis (Wang et al., 2011).”

“In addition to increased use of glucose in activated effector T cells, glutamine uptake andglutaminolysis are also upregulated (Carr et al., 2010; Wang et al., 2011).”

“Transcription factors including Myc (Wang et al., 2011), estrogen-related receptor a (ERRa) (Michalek et al., 2011b), and the liver X receptors (LXRs) (Bensinger et al., 2008) facilitate T cell metabolic reprogramming by enhancing the expression of metabolic genes downstream of TCR signaling, which…”

“CD4+ T cells from O compared Y subjects produced less lactate 46 Cell Metabolism 32, 44–55, July 7, 2020 and had lower ECAR (Figures 2A–2D and S2A–S2D).”

“The up-regulation of glycolysis results from a transcriptional reprogramming regulated by c-MYC, HIF1a (hypoxia-inducible factor 1a), and ERRa (estrogen-related receptor a) (12, 15, 16).”

“In certain cell models, resting cells use fatty acid oxidation as a means to generate ATP, maintain membrane potentials, and turn over organelles (Wang et al. 2011).”

“In this respect, a previous study has implicated c-myc as a major regulator of glucose metabolism in T cells (Wang et al., 2011).”

“It occurs in an environment of extreme inflammation and also plays a dominant role in neutrophil autophagy and survival via FAO (2011) [222].”

“Despite its quick induction, HIF-1a appeared dispensable for the initiation of metabolic changes and activation-associated proliferation in these T cells (21).”

“MYC-dependent cancer cells can utilize either GLUD or aminotransferases to convert glutamine to α-KG for the TCA cycle (Wise et al., 2008; Wang et al., 2011).”

“Myc upregulates the rate-limiting enzymes of the PPP, including G6PD and TKT [87].”

“Metabolic reprogramming in T cells is mediated through the induction of cMyc (5), a well-described regulator of glycolysis, glutaminolysis, and cell growth, together with the PI3K/Akt pathway (2).”

“Oxidative phosphorylation is the main source of energy for naive T cells, but upon activation, naïve T cells undergo metabolic reprogramming to aerobic glycolysis, also known as the Warburg effect, while simultaneously augmenting glutamine uptake and catabolism to maintain effector T cell fitness and drive differentia…”

“For example, citrate is a TCA cycle intermediate that can be used as a precursor for lipid synthesis (95).”

“c-Myc regulates proliferation and metabolism (173) and increases transcription by interactions with RNA polymerases (174–176).”

“Indeed, the metabolic changes described in this study to accompany NK cell activation are similar to those that occur in proinflammatory effector T cell subsets (2, 3, 26, 27).”

“The latter showed that MYC-driven glutamine catabolism couples with multiple biosynthetic pathways, especially ornithine and polyamine biosynthesis (28).”

“Additionally, the flux through glycolysis, PPP and the exosamine pathway also increases [15].”