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Differentiation of cardiac fibroblasts to myofibroblasts is necessary for matrix remodeling and fibrosis in heart failure. We previously reported that mitochondrial calcium signaling drives α-ketoglutarate-dependent histone demethylation, promoting myofibroblast formation. Here we investigate the role of ATP-citrate lyase (ACLY), a key enzyme for acetyl-CoA biosynthesis, in histone acetylation regulating myofibroblast fate and persistence in cardiac fibrosis. We show that inactivation of ACLY prevents myofibroblast differentiation and reverses myofibroblasts towards quiescence. Genetic deletion of Acly in post-activated myofibroblasts prevents fibrosis and preserves cardiac function in pressure-overload heart failure. TGFβ stimulation enhances ACLY nuclear localization and ACLY-SMAD2/3 interaction, and increases H3K27ac at fibrotic gene loci. Pharmacological inhibition of ACLY or forced nuclear expression of a dominant-negative ACLY mutant prevents myofibroblast formation and H3K27ac. Our data indicate that nuclear ACLY activity is necessary for myofibroblast differentiation and persistence by maintaining histone acetylation at TGFβ-induced myofibroblast genes. These findings provide targets to prevent and reverse pathological fibrosis., (© 2024. The Author(s).)

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within an intrinsically disordered region and includes at least one dynamically phosphorylated residue. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a hematological malignancy in need of novel therapeutic approaches. Here, we identify the ATP-citrate lyase ACLY as overexpressed and as a novel therapeutic target in T-ALL. To test the effects of ACLY in leukemia progression, we developed an isogenic model of NOTCH1-induced Acly conditional knockout leukemia. Importantly, we observed intrinsic antileukemic effects upon loss of ACLY, which further synergized with NOTCH1 inhibition in vivo. Metabolomic profiling upon ACLY loss revealed a metabolic crisis with reduced acetyl-CoA levels, as well as a decreased oxygen consumption rate. Gene expression profiling analyses showed that the transcriptional signature of ACLY loss very significantly correlates with the signature of MYC loss in vivo . Mechanistically, the decrease in acetyl-CoA led to reduced H3K27ac levels in Myc , resulting in transcriptional downregulation of Myc and drastically reduced MYC protein levels. Interestingly, our analyses also revealed a reciprocal relationship whereby ACLY itself is a direct transcriptional target of MYC, thus establishing a feedforward loop that is important for leukemia progression. Overall, our results identified a relevant ACLY-MYC axis and unveiled ACLY as a novel promising target for T-ALL treatment.

Metabolic reprogramming in cancer allows cells to survive in harsh environments and sustain macromolecular biosynthesis to support proliferation. In addition, metabolites play crucial roles as signaling molecules. Metabolite fluctuations are detected by various sensors in the cell to regulate gene expression, metabolism, and signal transduction. Metabolic signaling mechanisms contribute to tumorigenesis by altering the physiology of cancer cells themselves, as well as that of neighboring cells in the tumor microenvironment. In this review, we discuss principles of metabolic signaling and provide examples of how cancer cells take advantage of metabolic signals to promote cell proliferation and evade the immune system, thereby contributing to tumor growth and progression., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Obesity is a leading risk factor for progression and metastasis of many cancers 1,2 , yet can in some cases enhance survival 3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells 6-8 . Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-1 9-12 . Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8 + T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we used kidney-specific expression of quantitative traits and single-nucleus open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2-KO mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulated de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes., Competing Interests: Declaration of interests E.J.W. is an advisor for Danger Bio, Janssen, New Limit, Marengo, Pluto Immunotherapeutics Related Sciences, Rubius Therapeutics, Santa Ana Bio, Synthekine, and Surface Oncology. E.J.W. is a founder of and holds stock in Surface Oncology, Danger Bio, and Arsenal Biosciences. N.J.M. reports consulting fees from Endpoint Health Inc and AstraZeneca and receives funding from Quantum Leap Healthcare Collaborative outside of the published work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1 hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Introduction: The mitochondrial uniporter (MCU) Ca 2+ ion channel represents the primary means for Ca 2+ uptake by mitochondria. Mitochondrial matrix Ca 2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca 2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo . Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro . The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca 2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca 2+ uptake, altered mitochondrial matrix Ca 2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro , both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca 2+ ], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca 2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca 2+ dynamics necessary for cell-cycle progression and cell proliferation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fernandez Garcia, Paudel, Noji, Bowman, Rustgi, Pitarresi, Wellen, Arany, Weissenrieder and Foskett.)

The ability of cells to store and rapidly mobilize energy reserves in response to nutrient availability is essential for survival. Breakdown of carbon stores produces acetyl-CoA (AcCoA), which fuels essential metabolic pathways and is also the acyl donor for protein lysine acetylation. Histones are abundant and highly acetylated proteins, accounting for 40% to 75% of cellular protein acetylation. Notably, histone acetylation is sensitive to AcCoA availability, and nutrient replete conditions induce a substantial accumulation of acetylation on histones. Deacetylation releases acetate, which can be recycled to AcCoA, suggesting that deacetylation could be mobilized as an AcCoA source to feed downstream metabolic processes under nutrient depletion. While the notion of histones as a metabolic reservoir has been frequently proposed, experimental evidence has been lacking. Therefore, to test this concept directly, we used acetate-dependent, ATP citrate lyase-deficient mouse embryonic fibroblasts (Acly -/- MEFs), and designed a pulse-chase experimental system to trace deacetylation-derived acetate and its incorporation into AcCoA. We found that dynamic protein deacetylation in Acly -/- MEFs contributed carbons to AcCoA and proximal downstream metabolites. However, deacetylation had no significant effect on acyl-CoA pool sizes, and even at maximal acetylation, deacetylation transiently supplied less than 10% of cellular AcCoA. Together, our data reveal that although histone acetylation is dynamic and nutrient-sensitive, its potential for maintaining cellular AcCoA-dependent metabolic pathways is limited compared to cellular demand., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.

The efficacy of immunotherapy is limited by the paucity of T cells delivered and infiltrated into the tumors through aberrant tumor vasculature. Here, we report that phosphoglycerate dehydrogenase (PHGDH)-mediated endothelial cell (EC) metabolism fuels the formation of a hypoxic and immune-hostile vascular microenvironment, driving glioblastoma (GBM) resistance to chimeric antigen receptor (CAR)-T cell immunotherapy. Our metabolome and transcriptome analyses of human and mouse GBM tumors identify that PHGDH expression and serine metabolism are preferentially altered in tumor ECs. Tumor microenvironmental cues induce ATF4-mediated PHGDH expression in ECs, triggering a redox-dependent mechanism that regulates endothelial glycolysis and leads to EC overgrowth. Genetic PHGDH ablation in ECs prunes over-sprouting vasculature, abrogates intratumoral hypoxia, and improves T cell infiltration into the tumors. PHGDH inhibition activates anti-tumor T cell immunity and sensitizes GBM to CAR T therapy. Thus, reprogramming endothelial metabolism by targeting PHGDH may offer a unique opportunity to improve T cell-based immunotherapy., Competing Interests: Declaration of interests Y.F. and D.Z. are inventors on a patent application related to PHGDH inhibition for cancer immunotherapy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Few metabolites can claim a more central and versatile role in cell metabolism than acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is produced during nutrient catabolism to fuel the tricarboxylic acid cycle and is the essential building block for fatty acid and isoprenoid biosynthesis. It also functions as a signalling metabolite as the substrate for lysine acetylation reactions, enabling the modulation of protein functions in response to acetyl-CoA availability. Recent years have seen exciting advances in our understanding of acetyl-CoA metabolism in normal physiology and in cancer, buoyed by new mouse models, in vivo stable-isotope tracing approaches and improved methods for measuring acetyl-CoA, including in specific subcellular compartments. Efforts to target acetyl-CoA metabolic enzymes are also advancing, with one therapeutic agent targeting acetyl-CoA synthesis receiving approval from the US Food and Drug Administration. In this Review, we give an overview of the regulation and cancer relevance of major metabolic pathways in which acetyl-CoA participates. We further discuss recent advances in understanding acetyl-CoA metabolism in normal tissues and tumours and the potential for targeting these pathways therapeutically. We conclude with a commentary on emerging nodes of acetyl-CoA metabolism that may impact cancer biology., (© 2023. Springer Nature Limited.)

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation., (Copyright © 2022 The Authors.)

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA and lipid production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA and lipids. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Insulin stimulates adipose tissue to extract fatty acids from circulation and sequester them inside adipose cells. How fatty acids are transported across the capillary endothelial barrier, and how this process is regulated, remains unclear. We modeled the relationship of adipocytes and endothelial cells in vitro to test the role of insulin in fatty acid transport. Treatment of endothelial cells with insulin did not affect endothelial fatty acid uptake, but endothelial cells took up more fatty acids when exposed to medium conditioned by adipocytes treated with insulin. Manipulations of this conditioned medium indicated that the secreted factor is a small, hydrophilic, non-proteinaceous metabolite. Factor activity was correlated with lactate concentration, and inhibition of lactate production in adipocytes abolished the activity. Finally, lactate alone was sufficient to increase endothelial uptake of both free fatty acids and lipids liberated from chylomicrons, and to promote transendothelial transport, at physiologically relevant concentrations. Taken together, these data suggest that insulin drives adipocytes to secrete lactate, which then acts in a paracrine fashion to promote fatty acid uptake and transport across the neighboring endothelial barrier., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

There is an increasing appreciation for the role of metabolism in cell signaling and cell decision making. Precise metabolic control is essential in development, as evident by the disorders caused by mutations in metabolic enzymes. The metabolic profile of cells is often cell-type specific, changing as cells differentiate or during tumorigenesis. Recent evidence has shown that changes in metabolism are not merely a consequence of changes in cell state but that metabolites can serve to promote and/or inhibit these changes. Metabolites can link metabolic pathways with cell signaling pathways via several mechanisms, for example, by serving as substrates for protein post-translational modifications, by affecting enzyme activity via allosteric mechanisms, or by altering epigenetic markers. Unraveling the complex interactions governing metabolism, gene expression, and protein activity that ultimately govern a cell's fate will require new tools and interactions across disciplines. On March 24 and 25, 2021, experts in cell metabolism, developmental biology, and human disease met virtually for the Keystone eSymposium, "Metabolic Decisions in Development and Disease." The discussions explored how metabolites impact cellular and developmental decisions in a diverse range of model systems used to investigate normal development, developmental disorders, dietary effects, and cancer-mediated changes in metabolism., (© 2021 New York Academy of Sciences.)

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth., Competing Interests: SC, CM, LI, HA, MN, BS, TT, KS, ST, SK, JB, KW No competing interests declared, IB IAB is a founder of Proteoform Bio and a paid consultant for Calico, Chimerix, PTC Therapeutics, Takeda Pharmaceuticals, and Vivo Capital., (© 2021, Campbell et al.)

Lactate accumulation is a hallmark of solid cancers and is linked to the immune suppressive phenotypes of tumor-infiltrating immune cells. We report herein that interleukin-4 (IL-4)–induced M0 → M2 macrophage polarization is accompanied by interchangeable glucose- or lactate-dependent tricarboxylic acid (TCA) cycle metabolism that directly drives histone acetylation, M2 gene transcription, and functional immune suppression. Lactate-dependent M0 → M2 polarization requires both mitochondrial pyruvate uptake and adenosine triphosphate–citrate lyase (ACLY) enzymatic activity. Notably, exogenous acetate rescues defective M2 polarization and histone acetylation following mitochondrial pyruvate carrier 1 (MPC1) inhibition or ACLY deficiency. Lastly, M2 macrophage–dependent tumor progression is impaired by conditional macrophage ACLY deficiency, further supporting a dominant role for glucose/lactate mitochondrial metabolism and histone acetylation in driving immune evasion. This work adds to our understanding of how mitochondrial metabolism affects macrophage functional phenotypes and identifies a unique tumor microenvironment (TME)–driven metabolic-epigenetic link in M2 macrophages.

Background: Precision medicine approaches for managing patients with metastatic castrate-resistant prostate cancer (mCRPC) are lacking. Non-invasive approaches for molecular monitoring of disease are urgently needed, especially for patients suffering from bone metastases for whom tissue biopsy is challenging. Here we utilized baseline blood samples to identify mCRPC patients most likely to benefit from abiraterone plus prednisone (AAP) or enzalutamide., Methods: Baseline blood samples were collected for circulating tumor cell (CTC) enumeration and qPCR-based gene expression analysis from 51 men with mCRPC beginning treatment with abiraterone or enzalutamide., Results: Of 51 patients (median age 68 years [51-82]), 22 received AAP (abiraterone 1000 mg/day plus prednisone 10 mg/day) and 29 received enzalutamide (160 mg/day). The cohort was randomly divided into training (n = 37) and test (n = 14) sets. Baseline clinical variables (Gleason score, PSA, testosterone, and hemoglobin), CTC count, and qPCR-based gene expression data for 141 genes/isoforms in CTC-enriched blood were analyzed with respect to overall survival (OS). Genes with expression most associated with OS included MSLN, ARG2, FGF8, KLK3, ESRP2, NPR3, CCND1, and WNT5A. Using a Cox-elastic net model for our test set, the 8-gene expression signature had a c-index of 0.87 (95% CI [0.80, 0.94]) and was more strongly associated with OS than clinical variables or CTC count alone, or a combination of the three variables. For patients with a low-risk vs. high-risk gene expression signature, median OS was not reached vs. 18 months, respectively (HR 5.32 [1.91-14.80], p = 0.001). For the subset of 41 patients for whom progression-free survival (PFS) data was available, the median PFS for patients with a low-risk vs high-risk gene expression signature was 20 vs. 5 months, respectively (HR 2.95 [1.46-5.98], p = 0.003)., Conclusions: If validated in a larger prospective study, this test may predict patients most likely to benefit from second-generation antiandrogen therapy.

Imbalance between fat production and consumption causes various metabolic disorders. Nonalcoholic fatty liver disease (NAFLD), one such pathology, is characterized by abnormally increased fat synthesis and subsequent fat accumulation in hepatocytes 1,2 . While often comorbid with obesity and insulin resistance, this disease can also be found in lean individuals, suggesting specific metabolic dysfunction 2 . NAFLD has become one of the most prevalent liver diseases in adults worldwide, but its incidence in both children and adolescents has also markedly increased in developed nations 3,4 . Progression of this disease into nonalcoholic steatohepatitis (NASH), cirrhosis, liver failure, and hepatocellular carcinoma in combination with its widespread incidence thus makes NAFLD and its related pathologies a significant public health concern. Here, we review our understanding of the roles of dietary carbohydrates (glucose, fructose, and fibers) and the gut microbiota, which provides essential carbon sources for hepatic fat synthesis during the development of NAFLD.

Metabolic and epigenetic reprogramming are characteristics of cancer cells that, in many cases, are linked. Oncogenic signaling, diet, and tumor microenvironment each influence the availability of metabolites that are substrates or inhibitors of epigenetic enzymes. Reciprocally, altered expression or activity of chromatin-modifying enzymes can exert direct and indirect effects on cellular metabolism. In this article, we discuss the bidirectional relationship between epigenetics and metabolism in cancer. First, we focus on epigenetic control of metabolism, highlighting evidence that alterations in histone modifications, chromatin remodeling, or the enhancer landscape can drive metabolic features that support growth and proliferation. We then discuss metabolic regulation of chromatin-modifying enzymes and roles in tumor growth and progression. Throughout, we highlight proposed therapeutic and dietary interventions that leverage metabolic-epigenetic cross talk and have the potential to improve cancer therapy., Competing Interests: DISCLOSURE STATEMENT The authors declare no financial interests, memberships, affiliations, or funding that would affect the objectivity of their review.

Macrophages represent a major immune cell population in atherosclerotic plaques and play central role in the progression of this lipid-driven chronic inflammatory disease. Targeting immunometabolism is proposed as a strategy to revert aberrant macrophage activation to improve disease outcome. Here, we show ATP citrate lyase (Acly) to be activated in inflammatory macrophages and human atherosclerotic plaques. We demonstrate that myeloid Acly deficiency induces a stable plaque phenotype characterized by increased collagen deposition and fibrous cap thickness, along with a smaller necrotic core. In-depth functional, lipidomic, and transcriptional characterization indicate deregulated fatty acid and cholesterol biosynthesis and reduced liver X receptor activation within the macrophages in vitro. This results in macrophages that are more prone to undergo apoptosis, whilst maintaining their capacity to phagocytose apoptotic cells. Together, our results indicate that targeting macrophage metabolism improves atherosclerosis outcome and we reveal Acly as a promising therapeutic target to stabilize atherosclerotic plaques.

The Pro47Ser variant of p53 (S47) exists in African-descent populations and is associated with increased cancer risk in humans and mice. Due to impaired repression of the cystine importer Slc7a11 , S47 cells show increased glutathione (GSH) accumulation compared to cells with wild -type p53. We show that mice containing the S47 variant display increased mTOR activity and oxidative metabolism, as well as larger size, improved metabolic efficiency, and signs of superior fitness. Mechanistically, we show that mTOR and its positive regulator Rheb display increased association in S47 cells; this is due to an altered redox state of GAPDH in S47 cells that inhibits its ability to bind and sequester Rheb. Compounds that decrease glutathione normalize GAPDH-Rheb complexes and mTOR activity in S47 cells. This study reveals a novel layer of regulation of mTOR by p53, and raises the possibility that this variant may have been selected for in early Africa., Competing Interests: KG, JL, SB, TB, MG, JL, WQ, CK, RA, JB, KW, QL, ZS, DG No competing interests declared, MM Senior Editor, eLife, (© 2020, Gnanapradeepan et al.)

Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARγ and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalysed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here, we use liquid chromatography-high-resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13 C 3 15 N 1 -isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10 -8 pmol per cell in cell culture and 0.0172 pmol mg -1 tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 350 times lower than predominant acyl-CoAs such as acetyl-, propionyl- and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.

Background: Many metabolites serve as important signalling molecules to adjust cellular activities and functions based on nutrient availability. Links between acetyl-CoA metabolism, histone lysine acetylation, and gene expression have been documented and studied over the past decade. In recent years, several additional acyl modifications to histone lysine residues have been identified, which depend on acyl-coenzyme A thioesters (acyl-CoAs) as acyl donors. Acyl-CoAs are intermediates of multiple distinct metabolic pathways, and substantial evidence has emerged that histone acylation is metabolically sensitive. Nevertheless, the metabolic sources of acyl-CoAs used for chromatin modification in most cases remain poorly understood. Elucidating how these diverse chemical modifications are coupled to and regulated by cellular metabolism is important in deciphering their functional significance., Scope of Review: In this article, we review the metabolic pathways that produce acyl-CoAs, as well as emerging evidence for functional roles of diverse acyl-CoAs in chromatin regulation. Because acetyl-CoA has been extensively reviewed elsewhere, we will focus on four other acyl-CoA metabolites integral to major metabolic pathways that are also known to modify histones: succinyl-CoA, propionyl-CoA, crotonoyl-CoA, and butyryl-CoA. We also briefly mention several other acyl-CoA species, which present opportunities for further research; malonyl-CoA, glutaryl-CoA, 3-hydroxybutyryl-CoA, 2-hydroxyisobutyryl-CoA, and lactyl-CoA. Each acyl-CoA species has distinct roles in metabolism, indicating the potential to report shifts in the metabolic status of the cell. For each metabolite, we consider the metabolic pathways in which it participates and the nutrient sources from which it is derived, the compartmentalisation of its metabolism, and the factors reported to influence its abundance and potential nuclear availability. We also highlight reported biological functions of these metabolically-linked acylation marks. Finally, we aim to illuminate key questions in acyl-CoA metabolism as they relate to the control of chromatin modification., Major Conclusions: A majority of acyl-CoA species are annotated to mitochondrial metabolic processes. Since acyl-CoAs are not known to be directly transported across mitochondrial membranes, they must be synthesized outside of mitochondria and potentially within the nucleus to participate in chromatin regulation. Thus, subcellular metabolic compartmentalisation likely plays a key role in the regulation of histone acylation. Metabolite tracing in combination with targeting of relevant enzymes and transporters will help to map the metabolic pathways that connect acyl-CoA metabolism to chromatin modification. The specific function of each acyl-CoA may be determined in part by biochemical properties that affect its propensity for enzymatic versus non-enzymatic protein modification, as well as the various enzymes that can add, remove and bind each modification. Further, competitive and inhibitory effects of different acyl-CoA species on these enzymes make determining the relative abundance of acyl-CoA species in specific contexts important to understand the regulation of chromatin acylation. An improved and more nuanced understanding of metabolic regulation of chromatin and its roles in physiological and disease-related processes will emerge as these questions are answered., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Mature B-cell neoplasms are the fifth most common neoplasm. Due to significant heterogeneity at the clinical and genetic levels, current therapies for these cancers fail to provide long-term cures. The clinical success of proteasome inhibition for the treatment of multiple myeloma and B-cell lymphomas has made the ubiquitin pathway an important emerging therapeutic target. In this study, we assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. FBXW7 targeted the frequently inactivated tumor suppressor KMT2D for protein degradation, subsequently regulating gene expression signatures related to oxidative phosphorylation (OxPhos). Loss of FBXW7 inhibited diffuse large B-cell lymphoma cell growth and further sensitized cells to OxPhos inhibition. These data elucidate a novel mechanism of regulation of KMT2D levels by the ubiquitin pathway and uncover a role of FBXW7 in regulating oxidative phosphorylation in B-cell malignancies. SIGNIFICANCE: These findings characterize FBXW7 as a prosurvival factor in B-cell lymphoma via degradation of the chromatin modifier KMT2D., (©2020 American Association for Cancer Research.)

Recent years have seen a great expansion in our knowledge of the roles that metabolites play in cellular signaling. Structural data have provided crucial insights into mechanisms through which amino acids are sensed. New nutrient-coupled protein and RNA modifications have been identified and characterized. A growing list of functions has been ascribed to metabolic regulation of modifications such as acetylation, methylation, and glycosylation. A current challenge lies in developing an integrated understanding of the roles that metabolic signaling mechanisms play in physiology and disease, which will inform the design of strategies to target such mechanisms. In this brief article, we review recent advances in metabolic signaling through post-translational modification during cancer progression, to provide a framework for understanding signaling roles of metabolites in the context of cancer biology and illuminate areas for future investigation., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods 1 , and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease 2-4 . Fructose intake triggers de novo lipogenesis in the liver 4-6 , in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates 7 . Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases 8 . However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota 9 , and this supplies lipogenic acetyl-CoA independently of ACLY 10 . Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.

Objective: The dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13 C-labeled substrates targeting central metabolic pathways., Methods: We designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS)., Results: We show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic and reflects the metabolic capacity of the sub-cellular compartment analyzed, but it is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses., Conclusions: Our data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment., (Copyright © 2019 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Purpose of Review: To examine the consequences of metabolism compartmentalized at the subcellular level, provide prototypical examples of compartmentalized metabolism, and describe methods to examine compartmentalized metabolism., Recent Findings: Progress in metabolomics and isotope tracing has underscored the importance of subcellular compartments of metabolism. The discovery of biological effects of metabolites as bioenergetic intermediates, anabolic building blocks, signaling mediators, and effectors in posttranslation modifications of proteins and nucleic acids have highlighted the role of compartmentalization in determining metabolic fate. Recent advances in both direct and indirect methods to quantify compartmentalized metabolism have improved upon historical approaches. Genetically encoded metabolite sensors, chemical probes, immunoaffinity purification, and compartment-resolved metabolic modeling have all been recently applied to study compartmentalization., Summary: Accurate measurement of metabolites in distinct subcellular compartments is important for understanding and pharmacologically targeting metabolic pathways in diverse disease contexts, including cancer, diabetes, heart failure, obesity, and regulation of the immune system. Direct and indirect approaches to quantify compartmentalized metabolism are advancing rapidly. Yet, major challenges remain in the generalizability, rigor, and interpretation of data from the available methods to quantify compartmentalized metabolism.

In this issue of Cancer Discovery , Gimple and colleagues examine superenhancers in glioblastoma and glioma stem cells (GSC), identifying one which promotes expression of ELOVL2, an enzyme in polyunsaturated fatty acid (PUFA) synthesis. They find that ELOVL2 products help maintain cell membrane organization and EGFR signaling in GSCs, and that targeting PUFA metabolism along with EGFR offers a potential novel therapeutic strategy for glioblastoma. See related article by Gimple et al., p. 1248 ., (©2019 American Association for Cancer Research.)

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNA Leu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD + /NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD + /NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, Acly FAT-/- females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas Acly FAT-/- males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use., (© 2019 Bazilevsky et al.)

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring Kras mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in KRAS -mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA-producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT-ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA-dependent processes exerts anticancer effects. SIGNIFICANCE: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways. See related commentary by Halbrook et al., p. 326 . This article is highlighted in the In This Issue feature, p. 305 ., (©2019 American Association for Cancer Research.)