Cellular acetyl-CoA Levels Research Articles

Induction of Fatty Acid Oxidation Underlies DNA Damage-Induced Cell Death and Ameliorates Obesity-Driven Chemoresistance.

The DNA damage response is essential for preserving genome integrity and eliminating damaged cells. Although cellular metabolism plays a central role in cell fate decision between proliferation, survival, or death, the metabolic response to DNA damage remains largely obscure. Here, this work shows that DNA damage induces fatty acid oxidation (FAO), which is required for DNA damage-induced cell death. Mechanistically, FAO induction increases cellular acetyl-CoA levels and promotes N-alpha-acetylation of caspase-2, leading to cell death. Whereas chemotherapy increases FAO related genes through peroxisome proliferator-activated receptor α (PPARα), accelerated hypoxia-inducible factor-1α stabilization by tumor cells in obese mice impedes the upregulation of FAO, which contributes to its chemoresistance. Finally, this work finds that improving FAO by PPARα activation ameliorates obesity-driven chemoresistance and enhances the outcomes of chemotherapy in obese mice. These findings reveal the shift toward FAO induction is an important metabolic response to DNA damage and may provide effective therapeutic strategies for cancer patients with obesity.

BCL11B and the NuRD complex cooperatively guard T-cell fate and inhibit OPA1-mediated mitochondrial fusion in T cells.

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.

Fatty acid oxidation supports melanoma cell migration through autophagy regulation

Metastasis is one of the most malignant characteristics of cancer cells, in which metabolic reprogramming is crucial for promoting and sustaining multi-steps of metastasis, including invasion, migration and infiltration. Recently, it has been shown that melanoma cells undergo a metabolic switching toward the upregulation of fatty acid oxidation (FAO) during metastasis. However, the underlying mechanisms by which FAO contributes to metastasis of melanoma cells remain obscure. Here, we report that FAO contributes to melanoma cell migration and invasion by regulating the formation of autophagosomes. Pharmacological or genetic inhibition of FAO impairs migration of melanoma cells, which seems not to be linked to energy production or redox homeostasis. Importantly, we reveal that acetyl-CoA production by FAO contributes to melanoma cell migration through autophagy regulation. Mechanistically, FAO inhibition results in increased autophagosome formation, which suppresses migration and invasion properties of melanoma cells. Our results underscore the crucial role of FAO in melanoma cell migration and support the potential therapeutic relevance of modulating cellular acetyl-CoA levels to inhibit cancer metastasis.

Suppression of fatty acid oxidation supports pancreatic cancer growth and survival under hypoxic conditions through autophagy induction.

Hypoxia, one of the key features of solid tumors, induces autophagy, which acts as an important adaptive mechanism for tumor progression under hypoxic environment. Cellular metabolic reprogramming has been correlated with hypoxia, but the molecular connection to the induction of autophagy remains obscure. Here, we show that suppression of fatty acid oxidation (FAO) by hypoxia induces autophagy in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for their growth and survival. Reduced cellular acetyl-CoA levels caused by FAO inhibition decreases LC3 acetylation, resulting in autophagosome formation. Importantly, PDAC cells are significantly dependent on this metabolic reprogramming, as improving FAO leads to a reduction in hypoxia-induced autophagy and an increase in cell death after chemotherapy. Thus, our study supports that suppression of FAO is an important metabolic response to hypoxia and indicates that targeting this pathway in PDAC may be an effective therapeutic approach.

Simultaneously targeting hyperinflammation and hyperglycemia following respiratory viral infection

Abstract Individuals with hyperglycemia are at great risk of developing exaggerated pulmonary inflammation following infection with respiratory viruses including SARS-CoV-2. Conversely, SARS-CoV-2 infection has been linked to the development of new onset of hyperglycemia and diabetes. Despite global vaccination efforts, SARS-CoV-2 infection continues to be a great public health challenge, and corticosteroids targeting hyper-inflammation remain the mainstay treatment for severe COVID-19. However, steroidal medicine arises the risk of hyperglycemia, limiting its usage. Here, we showed that a second-generation thiazolidines drug MSDC-0602K (MSDC), a mitochondrial pyruvate carrier inhibitor, simultaneously decreased excessive pulmonary inflammation and hyperglycemia in normal and obese hosts during influenza infection, as such, reducing host morbidity and mortality. scRNA-seq analysis revealed that MSDC treatment reduced lung macrophage inflammation, and myeloid-specific ablation of MPC2 led to dampened lung inflammation. Further, MSDC inhibited human lung macrophage inflammatory responses following SARS-CoV-2 infection, and dampened inflammation in lung autopsies of COVID-19 patients. Mechanistically, MSDC treatment destabilized HIF-1a protein, a transcription factor promoting inflammatory responses of lung macrophages, by repressing cellular Acetyl-CoA levels and HIF-1a acetylation. Our results suggest that MSDC has the great potential to treat severe COVID-19 or other respiratory viral infection, particularly in patients with underlying metabolic conditions, by simultaneously repressing hyper-cytokinemia and hyperglycemia. Supported by NIH AG069264, NIH AI150599

Long Noncoding RNA TINCR-Mediated Regulation of Acetyl-CoA Metabolism Promotes Nasopharyngeal Carcinoma Progression and Chemoresistance

Frontier evidence suggests that dysregulation of long noncoding RNAs (lncRNA) is ubiquitous in all human tumors, indicating that lncRNAs might have essential roles in tumorigenesis. Therefore, an in-depth study of the roles of lncRNA in nasopharyngeal carcinoma (NPC) carcinogenesis might be helpful to provide novel therapeutic targets. Here we report that lncRNA TINCR was significantly upregulated in NPC and was associated positively with poor survival. Silencing TINCR inhibited NPC progression and cisplatin resistance. Mechanistically, TINCR bound ACLY and protected it from ubiquitin degradation to maintain total cellular acetyl-CoA levels. Accumulation of cellular acetyl-CoA promoted de novo lipid biosynthesis and histone H3K27 acetylation, which ultimately regulated the peptidyl arginine deiminase 1 (PADI1)-MAPK-MMP2/9 pathway. In addition, insulin-like growth factor 2 mRNA-binding protein 3 interacted with TINCR and slowed its decay, which partially accounted for TINCR upregulation in NPC. These findings demonstrate that TINCR acts as a crucial driver of NPC progression and chemoresistance and highlights the newly identified TINCR-ACLY-PADI1-MAPK-MMP2/9 axis as a potential therapeutic target in NPC. SIGNIFICANCE: TINCR-mediated regulation of a PADI1-MAPK-MMP2/9 signaling pathway plays a critical role in NPC progression and chemoresistance, marking TINCR as a viable therapeutic target in this disease.

LXR Activation Induces a Proinflammatory Trained Innate Immunity-Phenotype in Human Monocytes.

ObjectivesThe concept of trained innate immunity describes a long-term proinflammatory memory in innate immune cells. Trained innate immunity is regulated through reprogramming of cellular metabolic pathways including cholesterol and fatty acid synthesis. Here, we have analyzed the role of Liver X Receptor (LXR), a key regulator of cholesterol and fatty acid homeostasis, in trained innate immunity.Methods and ResultsHuman monocytes were isolated and incubated with different stimuli for 24 h, including LXR agonists, antagonists and Bacillus Calmette-Guerin (BCG) vaccine. After 5 days resting time, cells were restimulated with the TLR2-agonist Pam3cys. LXR activation did not only increase BCG trained immunity, but also induced a long-term inflammatory activation by itself. This inflammatory activation by LXR agonists was accompanied by characteristic features of trained innate immunity, such as activating histone marks on inflammatory gene promoters and metabolic reprogramming with increased lactate production and decreased oxygen consumption rate. Mechanistically, LXR priming increased cellular acetyl-CoA levels and was dependent on the activation of the mevalonate pathway and IL-1β signaling. In contrast to mevalonate pathway inhibition, blocking fatty acid synthesis further increased proinflammatory priming by LXR.ConclusionWe demonstrate that LXR activation induces a proinflammatory trained immunity phenotype in human monocytes through epigenetic and metabolic reprogramming. Our data reveal important novel aspects of LXR signaling in innate immunity.

Cyanobacterial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from carbon dioxide or a single organic substrate: improved polymer elongation with an extremely high 3-hydroxyvalerate mole proportion

The bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) changes material properties upon altering mole ratio (mol%) of the two monomers: 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Biotechnology for producing PHBV with a wide range of HV mol% using low amount of organic substrate is required. Here, 20 evolutionarily divergent cyanobacteria were examined for PHBV production. Under photoautotrophy with carbon dioxide (CO2) supply, one strain produced PHBV. But under heterotrophy using a medium containing only one type of organic substrate, 4, 15, and 13 strains were found to accumulate PHBV when supplied with acetate (C2H3O2), propionate (C3H5O2), and valerate (C5H9O2), respectively. Hence, these cyanobacterial PHBV productions required only CO2 or a single type of organic substrate. The obtained PHBV showed a broad range of HV mole ratios from 4 to 96 mol%. The cells producing PHBV with high HV mole ratios (31–96 mol%) exhibited low cellular acetyl-CoA levels. The PHBV with a 96.2 mol% HV and 3.8 mol% HB from Nostoc microscopicum elongated at 98 ± 15% (length/length), a 19.6-fold higher elongation than that of commercial poly-3-hydroxybutyrate containing 100 mol% HB. The results demonstrated the economical utility of cyanobacterial conversion of CO2 and a single organic substrate to synthesize PHBV with improved elongation quality.

ITGA2 promotes expression of ACLY and CCND1 in enhancing breast cancer stemness and metastasis

Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Here we report that the integrin receptor CD49b-encoding ITGA2, a direct target of miR-206, promotes breast cancer stemness and metastasis. ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. ITGA2 overexpression reversed miR-206-caused cell cycle arrest in G1. RNA sequencing analyses revealed that ITGA2 knockdown inhibits genes related to cell cycle regulation and lipid metabolism, including CCND1 and ACLY as representative targets, respectively. Knockdown of CCND1 or ACLY inhibits mammosphere formation of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. ACLY-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels. CCND1 knockdown further mimics ITGA2 knockdown in abolishing lung colonization of breast cancer cells. We identified that the low levels of miR-206 as well as high expression levels of ITGA2, ACLY and CCND1 are associated with an unfavorable relapse-free survival of the patients with estrogen receptor-negative or high grade breast cancer, especially basal-like or TNBC, possibly serving as potential biomarkers of cancer stemness and therapeutic targets of breast cancer metastasis.

Acetate supplementation restores chromatin accessibility and promotes tumor cell differentiation under hypoxia

Despite the fact that Otto H. Warburg discovered the Warburg effect almost one hundred years ago, why cancer cells waste most of the glucose carbon as lactate remains an enigma. Warburg proposed a connection between the Warburg effect and cell dedifferentiation. Hypoxia is a common tumor microenvironmental stress that induces the Warburg effect and blocks tumor cell differentiation. The underlying mechanism by which this occurs is poorly understood, and no effective therapeutic strategy has been developed to overcome this resistance to differentiation. Using a neuroblastoma differentiation model, we discovered that hypoxia repressed cell differentiation through reducing cellular acetyl-CoA levels, leading to reduction of global histone acetylation and chromatin accessibility. The metabolic switch triggering this global histone hypoacetylation was the induction of pyruvate dehydrogenase kinases (PDK1 and PDK3). Inhibition of PDKs using dichloroacetate (DCA) restored acetyl-CoA generation and histone acetylation under hypoxia. Knocking down PDK1 induced neuroblastoma cell differentiation, highlighting the critical role of PDK1 in cell fate control. Importantly, acetate or glycerol triacetate (GTA) supplementation restored differentiation markers expression and neuron differentiation under hypoxia. Moreover, ATAC-Seq analysis demonstrated that hypoxia treatment significantly reduced chromatin accessibility at RAR/RXR binding sites, which can be restored by acetate supplementation. In addition, hypoxia-induced histone hypermethylation by increasing 2-hydroxyglutarate (2HG) and reducing α-ketoglutarate (αKG). αKG supplementation reduced histone hypermethylation upon hypoxia, but did not restore histone acetylation or differentiation markers expression. Together, these findings suggest that diverting pyruvate flux away from acetyl-CoA generation to lactate production is the key mechanism that Warburg effect drives dedifferentiation and tumorigenesis. We propose that combining differentiation therapy with acetate/GTA supplementation might represent an effective therapy against neuroblastoma.

The pathobiology of polycystic kidney disease from a metabolic viewpoint.

Autosomal dominant polycystic kidney disease (ADPKD) affects an estimated 1 in 1,000 people and slowly progresses to end-stage renal disease (ESRD) in about half of these individuals. Tolvaptan, a vasopressin 2 receptor blocker, has been approved by regulatory authorities in many countries as a therapy to slow cyst growth, but additional treatments that target dysregulated signalling pathways in cystic kidney and liver are needed. Metabolic reprogramming is a prominent feature of cystic cells and a potentially important contributor to the pathophysiology of ADPKD. A number of pathways previously implicated in the pathogenesis of the disease, such as dysregulated mTOR and primary ciliary signalling, have roles in metabolic regulation and may exert their effects through this mechanism. Some of these pathways are amenable to manipulation through dietary modifications or drug therapies. Studies suggest that polycystin-1 and polycystin-2, which are encoded by PKD1 and PKD2, respectively (the genes that are mutated in >99% of patients with ADPKD), may in part affect cellular metabolism through direct effects on mitochondrial function. Mitochondrial dysfunction could alter the redox state and cellular levels of acetyl-CoA, resulting in altered histone acetylation, gene expression, cytoskeletal architecture and response to cellular stress, and in an immunological response that further promotes cyst growth and fibrosis.

Analysis of global and gene-specific acetylation of histones in the liver of American bullfrog (Rana catesbeiana) tadpoles acclimated to low temperature.

Severe environmental stressors such as low temperatures can affect gene expression by changing epigenetic states. American bullfrog (Rana catesbeiana) can overwinter as tadpoles, which can be active even in winter. However, the molecular mechanisms of epigenetic controls by which the tadpoles acclimate to low temperature are still unclear. In this study, we aimed to clarify the molecular mechanisms of global and gene-specific epigenetic regulations of low-temperature acclimation. We found that the global acetylation was decreased in the liver of bullfrog tadpoles acclimated to low temperature. The amounts of transcripts for two histone acetyltransferases were higher in the liver of tadpoles acclimated to low temperature than in those acclimated to warm temperature, while we observed no significant differences in the amounts of transcripts for histone deacetylases. We also found that the amounts of transcripts and acetylated histones on the specific temperature-responsive genes scd and cyp7a1 whose transcripts were increased and decreased, respectively, in response to low temperature were positively correlated. Cellular acetyl-CoA levels were higher in the liver of tadpoles acclimated to low temperature than in those acclimated to warm temperature. These results contradicted the states of histone acetylation, suggesting that bullfrog tadpoles have different epigenetic mechanisms to modify the histones when compared with those of other organisms such as reptiles and mammals, even though the relationship between the transcript amount and the states of histone acetylation on temperature-responsive genes was similar to that of mammals.

ACOT12-Dependent Alteration of Acetyl-CoA Drives Hepatocellular Carcinoma Metastasis by Epigenetic Induction of Epithelial-Mesenchymal Transition

Metabolic reprogramming plays an important role in supporting tumor growth. However, little is known about the metabolic alterations that promote cancer metastasis. In this study, we identify acyl-CoA thioesterase 12 (ACOT12) as a key player in hepatocellular carcinoma (HCC) metastasis. The expression of ACOT12 is significantly down-regulated in HCC tissues and is closely associated with HCC metastasis and poor survival of HCC patients. Gain- and loss-of-function studies demonstrate that ACOT12 suppresses HCC metastasis both invitro and invivo. Further mechanistic studies reveal that ACOT12 regulates the cellular acetyl-CoA levels and histone acetylation in HCC cells and that down-regulation of ACOT12 promotes HCC metastasis by epigenetically inducing TWIST2 expression and the promotion of epithelial-mesenchymal transition. Taken together, our findings link the alteration of acetyl-CoA with HCC metastasis and imply that ACOT12 could be a prognostic marker and a potential therapeutic target for combating HCC metastasis.

<i>ITGA2</i> Promotes Breast Cancer Stemness and Metastasis Through Enhanced <i>ACLY</i> and <i>CCND1</i> Expression

Background: Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Accumulating evidence demonstrates that integrins are essential for normal development and malignancies; however, the molecular functions and clinical relevance for each integrin subunit in cancer metastasis have yet to be fully elucidated. Methods: Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Findings: Here we report that the integrin receptor CD49b-encoding ITGA2, a direct target of miR-206, promotes breast cancer stemness and metastasis through maintaining ACLY and CCND1 levels. ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. RNA sequencing analyses revealed that ITGA2 knockdown inhibits genes related to cell cycle regulation and lipid metabolism such as CCND1 and ACLY. ACLY-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels and mammosphere formation of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. Interpretation: We identified that the miR-206/ITGA2/ACLY/CCND1 axis is associated with relapse-free survival of the patients with high grade breast cancer, especially basal-like breast cancer, possibly serving as potential biomarkers and therapeutic targets of breast cancer stemness and metastasis. Funding Statement: This manuscript has been partially supported by NIH/NCI grants R00CA160638 (H.L. & V.A.) and R01CA213843 (R.A.K), American Cancer Society grant ACS127951-RSG-15-025-01-CSM (H.L.); the Susan G. Komen Foundation CCR15332826 (H.L.) and CCR18548501 (X.L); the Department of Defense W81XWH-16-1-0021 (H.L.); the Lynn Sage Cancer Research Foundation (X.L. and H. L.); Northwestern University's Endocrinology Training Grant T32DK007169-39 (A.H.). Declaration of Interests: There is no conflict of interest for the authors of this manuscript. Ethics Approval Statement: All animal procedures complied with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the respective Institutional Animal Care and Use Committees.

Integrated Analysis of Acetyl-CoA and Histone Modification via Mass Spectrometry to Investigate Metabolically Driven Acetylation.

Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin-bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, falls within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation, and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt, and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4-20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation.

Seasonal acclimatization and thermal acclimation induce global histone epigenetic changes in liver of bullfrog (Lithobates catesbeianus) tadpole

The American bullfrog (Lithobates catesbeianus) is a eurythermal amphibian that is naturally distributed from subarctic to subtropical areas. The tadpoles of this species overwinter, in water, in cold environments. Therefore, they may have adapted to a wide range of temperatures in an active state. To understand the adaptation mechanisms to cope with low or high temperatures, we investigated global epigenetic modifications, histone variants, transcript levels of related genes, and the cellular acetyl coenzyme A (acetyl-CoA) and free CoA (CoA-SH) levels, in the livers of tadpoles collected in summer and winter and of those acclimated to 4 °C and 21 °C. Among epigenetic marks tested, the levels of acetylated histones and the histone variant H2A.Z were influenced by different temperature conditions. Histone acetylation levels were higher in summer than in winter and increased within 3 days of warm acclimation, whereas histone H2A.Z levels were higher in winter than in summer and decreased within 2 weeks of warm acclimation. Transcript analysis revealed that decreased expression of histone H2A.Z in warm acclimation was regulated at the transcriptional level. Acetyl-CoA levels were not correlated with those of the acetylated histones, indicating that cellular acetyl-CoA levels may not directly influence the state of histone acetylation in the tadpole liver. Such epigenetic and metabolic changes in the tadpole liver may contribute to the maintenance of energy balance during seasonal acclimatization and thermal acclimation.

Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase.

Macrophages exposed to the Th2 cytokines interleukin (IL) IL-4 and IL-13 exhibit a distinct transcriptional response, commonly referred to as M2 polarization. Recently, IL-4-induced polarization of murine bone marrow-derived macrophages (BMDMs) has been linked to acetyl-CoA levels through the activity of the cytosolic acetyl-CoA-generating enzyme ATP-citrate lyase (ACLY). Here, we studied how ACLY regulated IL-4-stimulated gene expression in human monocyte-derived macrophages (MDMs). Although multiple ACLY inhibitors attenuated IL-4-induced target gene expression, this effect could not be recapitulated by silencing ACLY expression. Furthermore, ACLY inhibition failed to alter cellular acetyl-CoA levels and histone acetylation. We generated ACLY knockout human THP-1 macrophages using CRISPR/Cas9 technology. While these cells exhibited reduced histone acetylation levels, IL-4-induced gene expression remained intact. Strikingly, ACLY inhibitors still suppressed induction of target genes by IL-4 in ACLY knockout cells, suggesting off-target effects of these drugs. Our findings suggest that ACLY may not be the major regulator of nucleocytoplasmic acetyl-CoA and IL-4-induced polarization in human macrophages. Furthermore, caution should be warranted in interpreting the impact of pharmacological inhibition of ACLY on gene expression.

Inhibition of CK2 by CX4945 to induce autophagy-mediated cell death through dephosphorylating acetyl-CoA carboxylase in squamous cell carcinoma of head and neck cancer.

e17503 Background: Accumulating evidence has shown that overexpression of casein kinase 2 (CK2) in patients with squamous cell carcinoma of head and neck (SCCHN) correlates with worsened survival. Strong expression of phosphorylated acetyl CoA carboxylase (pACC) was found to be an independent prognostic marker for patients with node-positive SCCHN. Computational analysis showed that ACC was a potential substrate for CK2, and CK2 inhibition can suppress ACC phosphorylation in non-cancer cells. CX-4945, also known as silmitasertib, is an orally administered, highly specific, ATP-competitive inhibitor of CK2 and is under clinical investigation for treating malignancies. Methods: We investigated the anticancer activity of CX-4945 in 3 aggressive SCCHN cell lines. Autophagy induction and interference with cellular metabolism were characterized. Results: CX-4945 induced autophagic cell death in SCCHN cells. CX-4945 treatment caused time- and dose-dependent lipid droplet accumulation, inhibition of ACC phosphorylation, depressed cellular levels of acetyl Co-A, and severely suppressed mitochondrial respiration, without altering the expression of stress-related proteins in the endoplasmic reticulum. Conclusions: The findings demonstrated that inhibition of CK2 by CX-4945 contributed to deranged cellular energy metabolism in SCCHN cells, leading to autophagy-mediated programmed cell death. CX-4945 targeting to CK2 may be a promising therapeutic strategy against SCCHN cancer.

Pyruvate Oxidase as a Critical Link between Metabolism and Capsule Biosynthesis in Streptococcus pneumoniae.

The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation.

Polyamine regulating protein antizyme binds to ATP citrate lyase to accelerate acetyl-CoA production in cancer cells.

Antizyme (AZ) regulates cellular polyamines (i.e., putrescine, spermidine, and spermine) through binding to ornithine decarboxylase and subsequent ubiquitin-independent degradation of the enzyme protein by the 26S proteasome. Screening for AZ-binding proteins using a yeast two-hybrid system identified ATP citrate lyase (ACLY), a cytosolic enzyme which catalyzes the production of acetyl-CoA that is used for lipid anabolism or acetylation of cellular components. We confirmed that both AZ1 and AZ2 bind to ACLY and AZ colocalizes with ACLY to the cytoplasm. Unexpectedly, neither AZ1 nor AZ2 accelerated ACLY degradation. Additionally, purified AZ, particularly AZ1, increased the activity of purified ACLY in a dose-dependent manner invitro, suggesting that AZ activates ACLY through protein-protein interaction. Polyamines themselves had no effect on the ACLY activity invitro. Knockdown of AZ1 and/or AZ2 in human cancer cells significantly decreased the ACLY activity as well as cellular levels of acetyl-CoA and cholesterol. Our results are the first to show the crosstalk between polyamine and acetyl-CoA metabolism. We hypothesize that AZ may promote acetyl-CoA synthesis to downregulate spermidine and spermine through acetylation.