Exhausted T cells (TEX) in cancer and chronic viral infections undergo metabolic and epigenetic remodeling, impairing their protective capabilities. However, the impact of nutrient metabolism on epigenetic modifications that control TEX differentiation remains unclear. We showed that TEX cells shifted from acetate to citrate metabolism by downregulating acetyl-CoA synthetase 2 (ACSS2) while maintaining ATP-citrate lyase (ACLY) activity. This metabolic switch increased citrate-dependent histone acetylation, mediated by histone acetyltransferase KAT2A-ACLY interactions, at TEX signature-genes while reducing acetate-dependent histone acetylation, dependent on p300-ACSS2 complexes, at effector and memory T cell genes. Nuclear ACSS2 overexpression or ACLY inhibition prevented TEX differentiation and enhanced tumor-specific T cell responses. These findings unveiled a nutrient-instructed histone code governing CD8 + T cell differentiation, with implications for metabolic- and epigenetic-based T cell therapies.

Glucose is essential for T cell proliferation and function, yet its specific metabolic roles in vivo remain poorly defined. Here, we identify glycosphingolipid (GSL) biosynthesis as a key pathway fueled by glucose that enables CD8+ T cell expansion and cytotoxic function in vivo. Using 13C-based stable isotope tracing, we demonstrate that CD8+ effector T cells use glucose to synthesize uridine diphosphate-glucose (UDP-Glc), a precursor for glycogen, glycan, and GSL biosynthesis. Inhibiting GSL production by targeting the enzymes UDP-Glc pyrophosphorylase 2 (UGP2), UDP-Gal-4-epimerase (GALE), or UDP-Glc ceramide glucosyltransferase (UGCG) impairs CD8+ T cell expansion upon pathogen challenge. Mechanistically, we show that glucose-dependent GSL biosynthesis is required for plasma membrane lipid raft integrity and optimal T cell receptor (TCR) signaling. Moreover, UGCG-deficient CD8+ T cells display reduced granzyme expression, cytolytic activity, and tumor control in vivo. Together, our data establish GSL biosynthesis as a critical metabolic fate of glucose-beyond energy production-that is required for CD8+ T cell responses in vivo.

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

The progressive decline of CD8 T cell effector function-also known as terminal exhaustion-is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-γ and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states.

Infusion of 13 C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13 C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes –infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)—which regulates de novo aspartate synthesis—for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Reducing calorie intake without malnutrition limits tumor progression but the underlying mechanisms are poorly understood. Here we show that dietary restriction (DR) suppresses tumor growth by enhancing CD8+ T cell-mediated anti-tumor immunity. DR reshapes CD8+ T cell differentiation within the tumor microenvironment (TME), promoting the development of effector T cell subsets while limiting the accumulation of exhausted T (Tex) cells, and synergizes with anti-PD1 immunotherapy to restrict tumor growth. Mechanistically, DR enhances CD8+ T cell metabolic fitness through increased ketone body oxidation (ketolysis), which boosts mitochondrial membrane potential and fuels tricarboxylic acid (TCA) cycle-dependent pathways essential for T cell function. T cells deficient for ketolysis exhibit reduced mitochondrial function, increased exhaustion, and fail to control tumor growth under DR conditions. Our findings reveal a critical role for the immune system in mediating the anti-tumor effects of DR, highlighting nutritional modulation of CD8+ T cell fate in the TME as a critical determinant of anti-tumor immunity.

Primary screening data, Lead candidates and GO analysis

Supplementary Table S2 and Supplementary Figures S1 to S10

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including j3-hydroxybutyrate (j3OHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. j3OHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, j3OHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.

How environmental nutrient availability impacts T cell metabolism and function remains poorly understood. Here, we report that the presence of physiologic carbon sources (PCSs) in cell culture medium broadly im-pacts glucose utilization by CD8(+) T cells, independent of transcriptional changes in metabolic reprogramming. The presence of PCSs reduced glucose contribution to the TCA cycle and increased effector function of CD8(+) T cells, with lactate directly fueling the TCA cycle. In fact, CD8(+) T cells responding to Listeria infection preferentially consumed lactate over glucose as a TCA cycle substrate in vitro, with lactate enhancing T cell bioenergetic and biosynthetic capacity. Inhibiting lactate-dependent metabolism in CD8(+) T cells by silencing lactate dehydrogenase A (Ldha) impaired both T cell metabolic homeostasis and proliferative expansion in vivo. Together, our data indicate that carbon source availability shapes T cell glucose metabolism and identifies lactate as a bioenergetic and biosynthetic fuel for CD8(+)effector T cells.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) – including β-hydroxybutyrate (βOHB) and acetoacetate (AcAc) – as essential fuels supporting CD8 + T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8 + T effector (Teff) cells and βOHB directly increases CD8 + Teff cell IFN-γ production and cytolytic activity. Using metabolic tracers, we establish that CD8 + Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo . KBs directly boost the respiratory capacity of CD8 + T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that βOHB is a major substrate for acetyl-CoA production in CD8 + T cells and regulates effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8 + T cell effector responses. One Sentence summary Ketone bodies promote CD8 + T cell metabolism and effector function through regulation of epigenetic programming

AbstractThere is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor–targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1–targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic “class” effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1–targeted combination therapy for Ewing sarcoma.

Background: Ewing sarcoma (ES) is a primary bone tumor of adolescence with poor prognosis. ES is characterized by the presence of EWS-FLI1 which acts as an oncogenic transcription factor to promote and maintain a tumorigenic phenotype. All ES cells express EWS-FLI1, however, other actionable mutations are exceedingly rare. We previously identified mithramycin (MMA) as a potent and specific EWS-FLI1 inhibitor. MMA effectively decreases ES tumor cell viability and tumor volume in vivo by reversing the EWS-FLI1 transcriptional signature. Structural and biochemical data suggest that sub-micromolar MMA increases the stability of the DNA binding domain of EWS-FLI1 at GGAA repeats. These data suggest that MMA may prevent EWS-FLI1 from successfully promoting Pol II transcriptional elongation.Methods: We used ChIP-qPCR to detect EWS-FLI1 binding at the promoter of well characterized targets. We used a Pol II processivity assay to measure the efficiency of transcriptional elongation at EWS-FLI1 targets. We use RT-qPCR, western blotting, and RNAseq to confirm that MMA sensitizes ES cells to elongation inhibitors. Finally, we use GROseq to identify the changes to Pol II dynamics at EWS-FLI1 targets following MMA treatment.Results: Our ChIP-qPCR data demonstrate an increased fraction of EWS-FLI1 DNA binding at the promoters of target genes with GGAA microsatellite regions. MMA significantly inhibits the processivity of Pol II at EWS-FLI1 targets when added both into a run-on reaction or following ES cell pretreatment. Combining MMA with elongation inhibitors induces strong synergy as measured by Bliss-Independence, renders multiple ES cell lines unviable by inhibiting the effects of EWS-FLI1 at the mRNA and protein level, and is effective in vivo. MMA biases GROseq reads toward the 5' end of genes, a result consistent with a promoter trapping mechanism. We are currently correlating GROseq data to global EWS-FLI1 DNA binding using ChIPseq.Conclusions: We characterized a mechanism of MMA-induced EWS-FLI1 inhibition that involves increased stabilization of the fusion protein at target promoter regions. This mechanism is supported by changes to EWS-FLI1 DNA-binding, EWS-FLI1 target transcriptional processivity, and a reversal of the EWS-FLI1 transcriptional signature by combining MMA with downstream elongation inhibition. We hope that this work will inform future endeavors to develop a targeted therapy for ES that inhibits the main driver of disease, EWS-FLI1.Citation Format: Guillermo Flores, Susan Kitchen-Goosen, Brandon Oswald, Elissa Boguslawski, Marie Adams, Ian Beddows, Zachary Madaj, Patrick Grohar. Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2882.

Background: Ewing sarcoma (ES) is a pediatric soft tissue tumor with a poor prognosis. ES is dependent on the presence and activity of the oncogenic transcription factor, EWS-FLI1, that dysregulates gene expression at hundreds of targets. We have previously identified mithramycin (MMA) as a potent inhibitor of EWS-FLI1 driven transcription. However, only sub-therapeutic concentrations have been achieved in pediatric patients. To maximize inhibition, we conducted an siRNA screen to identify gene targets that potentiated the effects of MMA in ES. Knock-down of several transcriptionally related genes significantly sensitized ES cells to the effects of MMA. We next developed a drug matrix screening platform to identify commercially available transcriptional inhibitors that also potentiated the effects of MMA on EWS-FLI1 driven transcription and ES cell viability. PHA-767491, a cyclin-dependent kinase 9 inhibitor (CDK9i), reverses the effect on EWS-FLI1 on several targets at both the mRNA and protein level across multiple ES cell lines when combined with MMA. Methods: We utilize matrix drug screening to identify that PHA-767491 synergizes with MMA in terms of a reduction in cell viability as measured by MTS and cell growth as measured by time-lapse microscopy. We use RT-qPCR to measure changes in gene expression across multiple well characterized targets of EWS-FLI1 driven transcription. We use western blot analysis to measure changes at the protein level for these targets as well. We then use an orthotopic xenograft mouse model to measure changes in tumor size after administration of our MMA-CDK9i combination therapy. Results: Our combination of MMA and PHA-767491 displays strong synergy as measured by Bliss-Independence. Multiple ES cell lines become unviable, with minimal effect on non-ES cells, and this effect is stable after removal of the compounds. This synergy is recapitulated as a reversal of EWS-FLI1 driven transcription across multiple targets at both the mRNA and protein levels. We are currently evaluating the combination therapy in our animal model comparing MMA-CDK9i to control or either agent alone. Importantly our MMA-CDK9i combination uses drug concentrations that are clinically achievable. Conclusions: We describe an MMA-CDK9i combination that displays excellent activity against EWS-FLI1 driven transcription. We confirmed this using multiple independent assays in both in vitro and in vivo models. We complete this work with the hope that it can eventually be translated to patients. Citation Format: Guillermo Flores, Joel Everett, Brandon Oswald, Natasha Caplen, Lee Helman, Zachary Madaj, Patrick Grohar. Suppression of EWS-FLI1 transcription using a combination therapy of mithramycin and cyclin-dependent kinase 9 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1632.