Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.

Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.

The gut protist Tritrichomonas arnold restrains virus-mediated loss of oral tolerance by modulating dietary antigen-presenting dendritic cells.

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Drp1 splice variants regulate ovarian cancer mitochondrial dynamics and tumor progression.

Aberrant mitochondrial fission/fusion dynamics are frequently associated with pathologies, including cancer. We show that alternative splice variants of the fission protein Drp1 (DNM1L) contribute to the complexity of mitochondrial fission/fusion regulation in tumor cells. High tumor expression of the Drp1 alternative splice variant lacking exon 16 relative to other transcripts is associated with poor outcome in ovarian cancer patients. Lack of exon 16 results in Drp1 localization to microtubules and decreased association with mitochondrial fission sites, culminating in fused mitochondrial networks, enhanced respiration, changes in metabolism, and enhanced pro-tumorigenic phenotypes in vitro and in vivo. These effects are inhibited by siRNAs designed to specifically target the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the pathophysiological importance of Drp1 alternative splicing, highlight the divergent functions and consequences of changing the relative expression of Drp1 splice variants in tumor cells, and strongly warrant consideration of alternative splicing in future studies focused on Drp1.

Sex-based differences in persistent lung inflammation following influenza infection of juvenile outbred mice.

Children are susceptible to influenza infections and can experience severe disease presentation due to a lack of or limited pre-existing immunity. Despite the disproportionate impact influenza has on this population, there is a lack of focus on pediatric influenza research, particularly when it comes to identifying the pathogenesis of long-term outcomes that persist beyond the point of viral clearance. In this study, juvenile outbred male and female mice were infected with influenza and analyzed following viral clearance to determine how sex impacts the persistent inflammatory responses to influenza. It was found that females maintained a broader cytokine response in the lung following clearance of influenza, with innate, type I and type II cytokine signatures in almost all mice. Males, on the other hand, had higher levels of IL-6 and other macrophage-related cytokines, but no evidence of a type I or type II response. The immune landscape was similar in the lungs between males and females postinfection, but males had a higher regulatory T cell to TH1 ratio compared with female mice. Cytokine production positively correlated with the frequency of TH1 cells and exudate macrophages, as well as the number of cells in the bronchoalveolar lavage fluid. Furthermore, female lungs were enriched for metabolites involved in the glycolytic pathway, suggesting glycolysis is higher in female lungs compared with males after viral clearance. These data suggest juvenile female mice have persistent and excessive lung inflammation beyond the point of viral clearance, whereas juvenile males had a more immunosuppressive phenotype.NEW & NOTEWORTHY This study identifies sex-based differences in persistent lung inflammation following influenza infection in an outbred, juvenile animal model of pediatric infection. These findings indicate the importance of considering sex and age as variable in infectious disease research.

Serum metabolomic signatures of fatty acid oxidation defects differentiate host-response subphenotypes of acute respiratory distress syndrome.

BACKGROUND: Fatty acid oxidation (FAO) defects have been implicated in experimental models of acute lung injury and associated with poor outcomes in critical illness. In this study, we examined acylcarnitine profiles and 3-methylhistidine as markers of FAO defects and skeletal muscle catabolism, respectively, in patients with acute respiratory failure. We determined whether these metabolites were associated with host-response ARDS subphenotypes, inflammatory biomarkers, and clinical outcomes in acute respiratory failure. METHODS: In a nested case-control cohort study, we performed targeted analysis of serum metabolites of patients intubated for airway protection (airway controls), Class 1 (hypoinflammatory), and Class 2 (hyperinflammatory) ARDS patients (N = 50 per group) during early initiation of mechanical ventilation. Relative amounts were quantified by liquid chromatography high resolution mass spectrometry using isotope-labeled standards and analyzed with plasma biomarkers and clinical data. RESULTS: Of the acylcarnitines analyzed, octanoylcarnitine levels were twofold increased in Class 2 ARDS relative to Class 1 ARDS or airway controls (P = 0.0004 and < 0.0001, respectively) and was positively associated with Class 2 by quantile g-computation analysis (P = 0.004). In addition, acetylcarnitine and 3-methylhistidine were increased in Class 2 relative to Class 1 and positively correlated with inflammatory biomarkers. In all patients within the study with acute respiratory failure, increased 3-methylhistidine was observed in non-survivors at 30 days (P = 0.0018), while octanoylcarnitine was increased in patients requiring vasopressor support but not in non-survivors (P = 0.0001 and P = 0.28, respectively). CONCLUSIONS: This study demonstrates that increased levels of acetylcarnitine, octanoylcarnitine, and 3-methylhistidine distinguish Class 2 from Class 1 ARDS patients and airway controls. Octanoylcarnitine and 3-methylhistidine were associated with poor outcomes in patients with acute respiratory failure across the cohort independent of etiology or host-response subphenotype. These findings suggest a role for serum metabolites as biomarkers in ARDS and poor outcomes in critically ill patients early in the clinical course.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Metabolic Adaptation of Macrophages as Mechanism of Defense against Crystalline Silica.

Silicosis is a lethal pneumoconiosis for which no therapy is available. Silicosis is a global threat, and more than 2.2 million people per year are exposed to silica in the United States. The initial response to silica is mediated by innate immunity. Phagocytosis of silica particles by macrophages is followed by recruitment of mitochondria to phagosomes, generation of mitochondrial reactive oxygen species, and cytokine (IL-1ß, TNF-α, IFN-ß) release. In contrast with LPS, the metabolic remodeling of silica-exposed macrophages is unclear. This study contrasts mitochondrial and metabolic alterations induced by LPS and silica on macrophages and correlates them with macrophage viability and cytokine production, which are central to the pathogenesis of silicosis. Using high-resolution respirometer and liquid chromatography-high-resolution mass spectrometry, we determined the effects of silica and LPS on mitochondrial respiration and determined changes in central carbon metabolism of murine macrophage cell lines RAW 264.7 and IC-21. We show that silica induces metabolic reprogramming of macrophages. Silica, as well as LPS, enhances glucose uptake and increases aerobic glycolysis in macrophages. In contrast with LPS, silica affects mitochondria respiration, reducing complex I and enhancing complex II activity, to sustain cell viability. These mitochondrial alterations are associated in silica, but not in LPS-exposed macrophages, with reductions of tricarboxylic acid cycle intermediates, including succinate, itaconate, glutamate, and glutamine. Furthermore, in contrast with LPS, these silica-induced metabolic adaptations do not correlate with IL-1ß or TNF-α production, but with the suppressed release of IFN-ß. Our data highlight the importance of complex II activity and tricarboxylic acid cycle remodeling to macrophage survival and cytokine-mediated inflammation in silicosis.

Liraglutide Protects Against Diastolic Dysfunction and Improves Ventricular Protein Translation.

PURPOSE: Diastolic dysfunction is an increasingly common cardiac pathology linked to heart failure with preserved ejection fraction. Previous studies have implicated glucagon-like peptide 1 (GLP-1) receptor agonists as potential therapies for improving diastolic dysfunction. In this study, we investigate the physiologic and metabolic changes in a mouse model of angiotensin II (AngII)-mediated diastolic dysfunction with and without the GLP-1 receptor agonist liraglutide (Lira). METHODS: Mice were divided into sham, AngII, or AngII+Lira therapy for 4 weeks. Mice were monitored for cardiac function, weight change, and blood pressure at baseline and after 4 weeks of treatment. After 4 weeks of treatment, tissue was collected for histology, protein analysis, targeted metabolomics, and protein synthesis assays. RESULTS: AngII treatment causes diastolic dysfunction when compared to sham mice. Lira partially prevents this dysfunction. The improvement in function in Lira mice is associated with dramatic changes in amino acid accumulation in the heart. Lira mice also have improved markers of protein translation by Western blot and increased protein synthesis by puromycin assay, suggesting that increased protein turnover protects against fibrotic remodeling and diastolic dysfunction seen in the AngII cohort. Lira mice also lost lean muscle mass compared to the AngII cohort, raising concerns about peripheral muscle scavenging as a source of the increased amino acids in the heart. CONCLUSIONS: Lira therapy protects against AngII-mediated diastolic dysfunction, at least in part by promoting amino acid uptake and protein turnover in the heart. Liraglutide therapy is associated with loss of mean muscle mass, and long-term studies are warranted to investigate sarcopenia and frailty with liraglutide therapy in the setting of diastolic disease.

Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions.

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae-contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under the control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is expressed in vitro only under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse-phase chromatography column (RPCC) and showed that the virulence-activating molecules were retained on the RPCC column and recovered in the eluate. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to noninducing medium and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that the ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regionally specific bile acid species in the intestine.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

Adropin treatment restores cardiac glucose oxidation in pre-diabetic obese mice.

Exposure to a high fat (HF) diet promotes increased fatty acid uptake, fatty acid oxidation and lipid accumulation in the heart. These maladaptive changes impact cellular energy metabolism and may promote the development of cardiac dysfunction. Attempts to increase cardiac glucose utilization have been proposed as a way to reverse cardiomyopathy in obese and diabetic individuals. Adropin is a nutrient-regulated metabolic hormone shown to promote glucose oxidation over fatty acid oxidation in skeletal muscle homogenates in vitro. The focus of the current study was to investigate whether adropin can regulate substrate metabolism in the heart following prolonged exposure to a HF diet in vivo. Mice on a long-term HF diet received serial intraperitoneal injections of vehicle or adropin over three days. Cardiac glucose oxidation was significantly reduced in HF animals, which was rescued by acute adropin treatment. Significant decreases in cardiac pyruvate dehydrogenase activity were observed in HF animals, which were also reversed by adropin treatment. In contrast to previous studies, this change was unrelated to Pdk4 expression, which remained elevated in both vehicle- and adropin-treated HF mice. Instead, we show that adropin modulated the expression of the mitochondrial acetyltransferase enzyme GCN5L1, which altered the acetylation status and activity of fuel metabolism enzymes to favor glucose utilization. Our findings indicate that adropin exposure leads to increased cardiac glucose oxidation under HF conditions, and may provide a future therapeutic avenue in the treatment of diabetic cardiomyopathy.

Lipoxin A4 yields an electrophilic 15-oxo metabolite that mediates FPR2 receptor-independent anti-inflammatory signaling.

The enzymatic oxidation of arachidonic acid is proposed to yield trihydroxytetraene species (termed lipoxins) that resolve inflammation via ligand activation of the formyl peptide receptor, FPR2. While cell and murine models activate signaling responses to synthetic lipoxins, primarily 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (lipoxin A4, LXA4), there are expanding concerns about the biological formation, detection and signaling mechanisms ascribed to LXA4 and related di- and tri-hydroxy ω-6 and ω-3 fatty acids. Herein, the generation and actions of LXA4 and its primary 15-oxo metabolite were assessed in control, LPS-activated and arachidonic acid supplemented RAW 264.7 macrophages. Despite protein expression of all enzymes required for LXA4 synthesis, both LXA4 and its 15-oxo-LXA4 metabolite were undetectable. Moreover, synthetic LXA4 and the membrane permeable 15-oxo-LXA4 methyl ester that is rapidly de-esterified to 15-oxo-LXA4, displayed no ligand activity for the putative LXA4 receptor FPR2, as opposed to the FPR2 ligand WKYMVm. Alternatively, 15-oxo-LXA4, an electrophilic α,ß-unsaturated ketone, alkylates nucleophilic amino acids such as cysteine to modulate redox-sensitive transcriptional regulatory protein and enzyme function. 15-oxo-LXA4 activated nuclear factor (erythroid related factor 2)-like 2 (Nrf2)-regulated gene expression of anti-inflammatory and repair genes and inhibited nuclear factor (NF)-κB-regulated pro-inflammatory mediator expression. LXA4 did not impact these macrophage anti-inflammatory and repair responses. In summary, these data show an absence of macrophage LXA4 formation and receptor-mediated signaling actions. Rather, if LXA4 were present in sufficient concentrations, this, and other more abundant mono- and poly-hydroxylated unsaturated fatty acids can be readily oxidized to electrophilic α,ß-unsaturated ketone products that modulate the redox-sensitive cysteine proteome via G-protein coupled receptor-independent mechanisms.

Alternative splice variants of the mitochondrial fission protein DNM1L/Drp1 regulate mitochondrial dynamics and tumor progression in ovarian cancer.

Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

Dietary dicarboxylic acids provide a non-storable alternative fat source that protects mice against obesity.

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Intestinal microbial circadian rhythms drive sex differences in host immunity and metabolism.

Circadian rhythms dynamically regulate sex differences in metabolism and immunity, and circadian disruption increases the risk of metabolic disorders. We investigated the role of sex-specific intestinal microbial circadian rhythms in host metabolism using germ-free and conventionalized mice and manipulation of dietary-derived fat, fiber, and microbiota-accessible carbohydrates. Our findings demonstrate that sex differences in circadian rhythms of genes involved in immunity and metabolism depend on oscillations in microbiota, microbial metabolic functions, and microbial metabolites. Further, we show that consuming an obesogenic, high-fat, low-fiber diet produced sex-specific changes in circadian rhythms in microbiota, metabolites, and host gene expression, which were linked to sex differences in the severity of metabolic dysfunction. Our results reveal that microbial circadian rhythms contribute to sex differences in immunity and metabolism and that dietary factors can entrain new circadian rhythms and modify the magnitude of sex differences in host-microbe circadian dynamics.

Targeting mitochondrial energetics reverses panobinostat- and marizomib-induced resistance in pediatric and adult high-grade gliomas.

In previous studies, we demonstrated that panobinostat, a histone deacetylase inhibitor, and bortezomib, a proteasomal inhibitor, displayed synergistic therapeutic activity against pediatric and adult high-grade gliomas. Despite the remarkable initial response to this combination, resistance emerged. Here, in this study, we aimed to investigate the molecular mechanisms underlying the anticancer effects of panobinostat and marizomib, a brain-penetrant proteasomal inhibitor, and the potential for exploitable vulnerabilities associated with acquired resistance. RNA sequencing followed by gene set enrichment analysis (GSEA) was employed to compare the molecular signatures enriched in resistant compared with drug-naïve cells. The levels of adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD)+ content, hexokinase activity, and tricarboxylic acid (TCA) cycle metabolites required for oxidative phosphorylation to meet their bioenergetic needs were analyzed. Here, we report that panobinostat and marizomib significantly depleted ATP and NAD+ content, increased mitochondrial permeability and reactive oxygen species generation, and promoted apoptosis in pediatric and adult glioma cell lines at initial treatment. However, resistant cells exhibited increased levels of TCA cycle metabolites, which required for oxidative phosphorylation to meet their bioenergetic needs. Therefore, we targeted glycolysis and the electron transport chain (ETC) with small molecule inhibitors, which displayed substantial efficacy, suggesting that resistant cell survival is dependent on glycolytic and ETC complexes. To verify these observations in vivo, lonidamine, an inhibitor of glycolysis and mitochondrial function, was chosen. We produced two diffuse intrinsic pontine glioma (DIPG) models, and lonidamine treatment significantly increased median survival in both models, with particularly dramatic effects in panobinostat- and marizomib-resistant cells. These data provide new insights into mechanisms of treatment resistance in gliomas.