Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.

Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate.

A central problem in biology is to identify gene function. One approach is to infer function in large supergenomic networks of interactions and ancestral relationships among genes; however, their analysis can be computationally prohibitive. We show here that these biological networks are compressible. They can be shrunk dramatically by eliminating redundant evolutionary relationships, and this process is efficient because in these networks the number of compressible elements rises linearly rather than exponentially as in other complex networks. Compression enables global network analysis to computationally harness hundreds of interconnected genomes and to produce functional predictions. As a demonstration, we show that the essential, but functionally uncharacterized Plasmodium falciparum antigen EXP1 is a membrane glutathione S-transferase. EXP1 efficiently degrades cytotoxic hematin, is potently inhibited by artesunate, and is associated with artesunate metabolism and susceptibility in drug-pressured malaria parasites. These data implicate EXP1 in the mode of action of a frontline antimalarial drug.

MIF/NR3C2 Axis Regulates Glucose Metabolism Reprogramming in Pancreatic Cancer through MAPK-ERK and AP-1 Pathways.

Inflammation and aberrant cellular metabolism are widely recognized as hallmarks of cancer. In pancreatic ductal adenocarcinoma (PDAC), inflammatory signaling and metabolic reprogramming are tightly interwoven, playing pivotal roles in the pathogenesis and progression of the disease. However, the regulatory functions of inflammatory mediators in metabolic reprogramming in pancreatic cancer have not been fully explored. Earlier, we demonstrated that pro-inflammatory mediator macrophage migration inhibitory factor (MIF) enhances disease progression by inhibiting its downstream transcriptional factor nuclear receptor subfamily 3 group C member 2 (NR3C2). Here, we provide evidence that MIF and NR3C2 interactively regulate metabolic reprogramming, resulting in MIF-induced cancer growth and progression in PDAC. MIF positively correlates with the HK1 (hexokinase 1), HK2 (hexokinase 2), and LDHA (lactate dehydrogenase) expression and increased pyruvate and lactate production in PDAC patients. Additionally, MIF augments glucose uptake and lactate efflux by upregulating HK1, HK2 and LDHA expression in pancreatic cancer cells in vitro and in mouse models of PDAC. Conversely, a reduction in HK1, HK2, LDHA expression is observed in tumors with high NR3C2 expression in PDAC patients. NR3C2 suppresses HK1, HK2, and LDHA expression, thereby inhibiting glucose uptake and lactate efflux in pancreatic cancer. Mechanistically, MIF-mediated regulation of glycolytic metabolism involves the activation of MAPK-ERK signaling pathway, whereas NR3C2 interacts with the activator protein 1 (AP-1) to regulate glycolysis. Our findings reveal an interactive role of the MIF/NR3C2 axis in regulating glucose metabolism supporting tumor growth and progression and may be a potential target for designing novel approaches for improving disease outcome.

Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase (TERT) enzyme and telomerase RNA component (TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer.

Alterations in both cell metabolism and transcriptional programs are hallmarks of cancer that sustain rapid proliferation and metastasis 1 . However, the mechanisms that control the interaction between metabolic reprogramming and transcriptional regulation remain unclear. Here we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). We used a kinome-wide RNA interference-based screening method to identify potential kinases that modulate the intrinsic SRC-3 transcriptional response. PFKFB4, a regulatory enzyme that synthesizes a potent stimulator of glycolysis 2 , is found to be a robust stimulator of SRC-3 that coregulates oestrogen receptor. PFKFB4 phosphorylates SRC-3 at serine 857 and enhances its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient Ser857Ala mutant SRC-3 abolishes the SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway and enables purine synthesis by transcriptionally upregulating the expression of the enzyme transketolase. In addition, the two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH), which are involved in purine metabolism, were identified as SRC-3 targets that may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation of SRC-3 at Ser857 increases its interaction with the transcription factor ATF4 by stabilizing the recruitment of SRC-3 and ATF4 to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses breast tumour growth in mice and prevents metastasis to the lung from an orthotopic setting, as does Ser857Ala-mutant SRC-3. PFKFB4 and phosphorylated SRC-3 levels are increased and correlate in oestrogen receptor-positive tumours, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer. These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumours.

Myocardial Rev-erb-Mediated Diurnal Metabolic Rhythm and Obesity Paradox.

BACKGROUND: The nuclear receptor Rev-erbα/ß, a key component of the circadian clock, emerges as a drug target for heart diseases, but the function of cardiac Rev-erb has not been studied in vivo. Circadian disruption is implicated in heart diseases, but it is unknown whether cardiac molecular clock dysfunction is associated with the progression of any naturally occurring human heart diseases. Obesity paradox refers to the seemingly protective role of obesity for heart failure, but the mechanism is unclear. METHODS: We generated mouse lines with cardiac-specific Rev-erbα/ß knockout (KO), characterized cardiac phenotype, conducted multi-omics (RNA-sequencing, chromatin immunoprecipitation sequencing, proteomics, and metabolomics) analyses, and performed dietary and pharmacological rescue experiments to assess the time-of-the-day effects. We compared the temporal pattern of cardiac clock gene expression with the cardiac dilation severity in failing human hearts. RESULTS: KO mice display progressive dilated cardiomyopathy and lethal heart failure. Inducible ablation of Rev-erbα/ß in adult hearts causes similar phenotypes. Impaired fatty acid oxidation in the KO myocardium, in particular, in the light cycle, precedes contractile dysfunctions with a reciprocal overreliance on carbohydrate utilization, in particular, in the dark cycle. Increasing dietary lipid or sugar supply in the dark cycle does not affect cardiac dysfunctions in KO mice. However, obesity coupled with systemic insulin resistance paradoxically ameliorates cardiac dysfunctions in KO mice, associated with rescued expression of lipid oxidation genes only in the light cycle in phase with increased fatty acid availability from adipose lipolysis. Inhibition of glycolysis in the light cycle and lipid oxidation in the dark cycle, but not vice versa, ameliorate cardiac dysfunctions in KO mice. Altered temporal patterns of cardiac Rev-erb gene expression correlate with the cardiac dilation severity in human hearts with dilated cardiomyopathy. CONCLUSIONS: The study delineates temporal coordination between clock-mediated anticipation and nutrient-induced response in myocardial metabolism at multi-omics levels. The obesity paradox is attributable to increased cardiac lipid supply from adipose lipolysis in the fasting cycle due to systemic insulin resistance and adiposity. Cardiac molecular chronotypes may be involved in human dilated cardiomyopathy. Myocardial bioenergetics downstream of Rev-erb may be a chronotherapy target in treating heart failure and dilated cardiomyopathy.

Evolution of cisplatin resistance through coordinated metabolic reprogramming of the cellular reductive state.

BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.

P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality.

The liver plays a significant role in regulating a wide range of metabolic, homeostatic, and host-defense functions. However, the impact of liver injury on the host's ability to control bacteremia and morbidity in sepsis is not well understood. Leukocyte recruitment and activation lead to cytokine and chemokine release, which, in turn, trigger hepatocellular injury and elevate nucleotide levels in the extracellular milieu. P2Y2 purinergic receptors, G protein-coupled and activated by extracellular ATP/UTP, are expressed at the cell surface of hepatocytes and nonparenchymal cells. We sought to determine whether P2Y2 purinergic receptor function is necessary for the maladaptive host response to bacterial infection and endotoxin-mediated inflammatory liver injury and mortality in mice. We report that P2Y2 purinergic receptor knockout mice (P2Y2-/-) had attenuated inflammation and liver injury, with improved survival in response to LPS/galactosamine (LPS/GalN; inflammatory liver injury) and cecal ligation and puncture (CLP; polymicrobial sepsis). P2Y2-/- livers had attenuated c-Jun NH2-terminal kinase activation, matrix metallopeptidase-9 expression, and hepatocyte apoptosis in response to LPS/GalN and attenuated inducible nitric oxide synthase and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 protein expression in response to CLP. Implicating liver injury in the disruption of amino acid homeostasis, CLP led to lower serum arginine and higher bacterial load and morbidity in the WT mice, whereas serum arginine levels were comparable to sham-operated controls in P2Y2-/- mice, which had attenuated bacteremia and improved survival. Collectively, our studies highlight the pathophysiological relevance of P2Y2 purinergic receptor function in inflammatory liver injury and dysregulation of systemic amino acid homeostasis with implications for sepsis-associated immune dysfunction and morbidity in mice.NEW & NOTEWORTHY Our studies provide experimental evidence for P2Y2 purinergic receptor-mediated potentiation of inflammatory liver injury, morbidity, and mortality, in two well-established animal models of inflammatory liver injury. Our findings highlight the potential to target P2Y2 purinergic signaling to attenuate the induction of "cytokine storm" and prevent its deleterious consequences on liver function, systemic amino acid homeostasis, host response to bacterial infection, and sepsis-associated morbidity and mortality.

Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming.

Blockade of angiogenesis can retard tumour growth, but may also paradoxically increase metastasis. This paradox may be resolved by vessel normalization, which involves increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability, and consequently mitigated hypoxia. Although these processes alter tumour progression, their regulation is poorly understood. Here we show that type 1 T helper (TH1) cells play a crucial role in vessel normalization. Bioinformatic analyses revealed that gene expression features related to vessel normalization correlate with immunostimulatory pathways, especially T lymphocyte infiltration or activity. To delineate the causal relationship, we used various mouse models with vessel normalization or T lymphocyte deficiencies. Although disruption of vessel normalization reduced T lymphocyte infiltration as expected, reciprocal depletion or inactivation of CD4+ T lymphocytes decreased vessel normalization, indicating a mutually regulatory loop. In addition, activation of CD4+ T lymphocytes by immune checkpoint blockade increased vessel normalization. TH1 cells that secrete interferon-γ are a major population of cells associated with vessel normalization. Patient-derived xenograft tumours growing in immunodeficient mice exhibited enhanced hypoxia compared to the original tumours in immunocompetent humans, and hypoxia was reduced by adoptive TH1 transfer. Our findings elucidate an unexpected role of TH1 cells in vasculature and immune reprogramming. TH1 cells may be a marker and a determinant of both immune checkpoint blockade and anti-angiogenesis efficacy.

Metabolic dysregulation in the Atp7b-/- Wilson's disease mouse model.

Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b-/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b-/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b-/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b-/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b-/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b-/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b-/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b-/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b-/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b-/- mice.

Differential Effects of Pancreatic Cancer-Derived Extracellular Vesicles Driving a Suppressive Environment.

Pancreatic ductal adenocarcinoma (PDAC) cells display extensive crosstalk with their surrounding environment to regulate tumor growth, immune evasion, and metastasis. Recent advances have attributed many of these interactions to intercellular communication mediated by small extracellular vesicles (sEVs), involving cancer-associated fibroblasts (CAF). To explore the impact of sEVs on monocyte lineage transition as well as the expression of checkpoint receptors and activation markers, peripheral blood monocytes from healthy subjects were exposed to PDAC-derived sEVs. Additionally, to analyze the role of sEV-associated HA in immune regulation and tissue-resident fibroblasts, monocytes and pancreatic stellate cells were cultured in the presence of PDAC sEVs with or depleted of HA. Exposure of monocytes to sEVs resulted in unique phenotypic changes in HLA-DR, PD-L1, CD86 and CD64 expression, and cytokine secretion that was HA-independent except for IL-1ß and MIP1ß. In contrast, monocyte suppression of autologous T cell proliferation was reduced following exposure to HA-low sEVs. In addition, exposure of stellate cells to sEVs upregulated the secretion of various cytokines, including MMP-9, while removal of HA from PDAC-derived sEVs attenuated the secretion of MMP-9, demonstrating the role of sEV-associated HA in regulating expression of this pro-tumorigenic cytokine from stellate cells. This observation lends credence to the findings from the TCGA database that PDAC patients with high levels of enzymes in the HA synthesis pathway had worse survival rates compared with patients having low expression of these enzymes. PDAC-derived sEVs have an immune modulatory role affecting the activation state of monocyte subtypes. However, sEV-associated HA does not affect monocyte phenotype but alters cytokine secretion and suppression of autologous T cell proliferation and induces secretion of pro-tumorigenic factors by pancreatic stellate cells (PSC), as has been seen following the conversion of PSCs to cancer-associated fibroblasts (CAFs). Interruption of the hexosamine biosynthetic pathway, activated in PDAC producing the key substrate (UDP-GlcNAc) for HA synthesis, thus, represents a potential clinical interception strategy for PDAC patients. Findings warrant further investigations of underlying mechanisms involving larger sample cohorts.

Metabolic stress induces GD2+ cancer stem cell-like phenotype in triple-negative breast cancer.

BACKGROUND: Metabolic stress resulting from nutrient deficiency is one of the hallmarks of a growing tumour. Here, we tested the hypothesis that metabolic stress induces breast cancer stem-like cell (BCSC) phenotype in triple-negative breast cancer (TNBC). METHODS: Flow cytometry for GD2 expression, mass spectrometry and Ingenuity Pathway Analysis for metabolomics, bioinformatics, in vitro tumorigenesis and in vivo models were used. RESULTS: Serum/glucose deprivation not only increased stress markers but also enhanced GD2+ BCSC phenotype and function in TNBC cells. Global metabolomics profiling identified upregulation of glutathione biosynthesis in GD2high cells, suggesting a role of glutamine in the BCSC phenotype. Cueing from the upregulation of the glutamine transporters in primary breast tumours, inhibition of glutamine uptake using small-molecule inhibitor V9302 reduced GD2+ cells by 70-80% and BCSC characteristics in TNBC cells. Mechanistic studies revealed inhibition of the mTOR pathway and induction of ferroptosis by V9302 in TNBC cells. Finally, inhibition of glutamine uptake significantly reduced in vivo tumour growth in a TNBC patient-derived xenograft model using NSG (non-obese diabetic/severe combined immunodeficiency with a complete null allele of the IL-2 receptor common gamma chain) mice. CONCLUSION: Here, we show metabolic stress results in GD2+ BCSC phenotype in TNBC and glutamine contributes to GD2+ phenotype, and targeting the glutamine transporters could complement conventional chemotherapy in TNBC.

Race reporting and disparities regarding clinical trials in bladder cancer: a systematic review.

PURPOSE: To systematically review the literature to investigate racial disparities among bladder cancer clinical trial enrollees. METHODS: A systematic review was conducted using Ovid, MEDLINE® to identify clinical trials between 1970 and 2020. Articles were reviewed and were included if they assessed race in their outcomes reporting among bladder cancer patients enrolled in clinical trials. The review was conducted in accordance with the PRISMA statement. RESULTS: We identified 544 clinical trials meeting our initial search criteria, with only 24 (4.4%) studies reporting racial demographic data. Enrollees were largely Caucasian (81-98%), with a strikingly small proportion of enrolled patients consisting of African-Americans (2-8%) and Hispanics (2-5%). Only one of the studies reported results on the efficacy and safety/tolerability of the tested treatment separately for racial groups and performed analyses stratified by race. CONCLUSION: Race is poorly studied in bladder cancer clinical trials. Trial cohorts may not reflect multicultural populations. The potential association between race and efficacy, safety or tolerability of the tested interventions is unknown. Given the up to twofold increase in bladder cancer-specific death among African-Americans, further research is needed to address the impact of race in clinical trials, while encompassing socioeconomic factors and disease risk factor exposures.

Dnmt3a loss and Idh2 neomorphic mutations mutually potentiate malignant hematopoiesis.

Mutations in the epigenetic regulators DNMT3A and IDH1/2 co-occur in patients with acute myeloid leukemia and lymphoma. In this study, these 2 epigenetic mutations cooperated to induce leukemia. Leukemia-initiating cells from Dnmt3a-/- mice that express an IDH2 neomorphic mutant have a megakaryocyte-erythroid progenitor-like immunophenotype, activate a stem-cell-like gene signature, and repress differentiated progenitor genes. We observed an epigenomic dysregulation with the gain of repressive H3K9 trimethylation and loss of H3K9 acetylation in diseased mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). HDAC inhibitors rapidly reversed the H3K9 methylation/acetylation imbalance in diseased mouse HSPCs while reducing the leukemia burden. In addition, using targeted metabolomic profiling for the first time in mouse leukemia models, we also showed that prostaglandin E2 is overproduced in double-mutant HSPCs, rendering them sensitive to prostaglandin synthesis inhibition. These data revealed that Dnmt3a and Idh2 mutations are synergistic events in leukemogenesis and that HSPCs carrying both mutations are sensitive to induced differentiation by the inhibition of both prostaglandin synthesis and HDAC, which may reveal new therapeutic opportunities for patients carrying IDH1/2 mutations.

Metabolome and microbiome multi-omics integration from a murine lung inflammation model of bronchopulmonary dysplasia.

BACKGROUND: Respiratory tract microbial dysbiosis can exacerbate inflammation and conversely inflammation may cause dysbiosis. Dysbiotic microbiome metabolites may lead to bronchopulmonary dysplasia (BPD). Hyperoxia and lipopolysaccharide (LPS) interaction alters lung microbiome and metabolome, mediating BPD lung injury sequence. METHODS: C57BL6/J mice were exposed to 21% (normoxia) or 70% (hyperoxia) oxygen during postnatal days (PND) 1-14. Pups were injected with LPS (6 mg/kg) or equal PBS volume, intraperitoneally on PND 3, 5, and 7. At PND14, the lungs were collected for microbiome and metabolomic analyses (n = 5/group). RESULTS: Microbiome alpha and beta diversity were similar between groups. Metabolic changes included hyperoxia 31 up/18 down, LPS 7 up/4 down, exposure interaction 8. Hyperoxia increased Intestinimonas abundance, whereas LPS decreased Clostridiales, Dorea, and Intestinimonas; exposure interaction affected Blautia. Differential co-expression analysis on multi-omics data identified exposure-altered modules. Hyperoxia metabolomics response was integrated with a published matching transcriptome, identifying four induced genes (ALDOA, GAA, NEU1, RENBP), which positively correlated with BPD severity in a published human newborn cohort. CONCLUSIONS: We report hyperoxia and LPS lung microbiome and metabolome signatures in a clinically relevant BPD model. We identified four genes correlating with BPD status in preterm infants that are promising targets for therapy and prevention. IMPACT: Using multi-omics, we identified and correlated key biomarkers of hyperoxia and LPS on murine lung micro-landscape and examined their potential clinical implication, which shows strong clinical relevance for future research. Using a double-hit model of clinical relevance to bronchopulmonary dysplasia, we are the first to report integrated metabolomic/microbiome landscape changes and identify novel disease biomarker candidates.

Gut Microbiota-Derived Short-Chain Fatty Acids Promote Poststroke Recovery in Aged Mice.

RATIONALE: The elderly experience profound systemic responses after stroke, which contribute to higher mortality and more severe long-term disability. Recent studies have revealed that stroke outcomes can be influenced by the composition of gut microbiome. However, the potential benefits of manipulating the gut microbiome after injury is unknown. OBJECTIVE: To determine if restoring youthful gut microbiota after stroke aids in recovery in aged subjects, we altered the gut microbiome through young fecal transplant gavage in aged mice after experimental stroke. Further, the effect of direct enrichment of selective bacteria producing short-chain fatty acids (SCFAs) was tested as a more targeted and refined microbiome therapy. METHODS AND RESULTS: Aged male mice (18-20 months) were subjected to ischemic stroke by middle cerebral artery occlusion. We performed fecal transplant gavage 3 days after middle cerebral artery occlusion using young donor biome (2-3 months) or aged biome (18-20 months). At day 14 after stroke, aged stroke mice receiving young fecal transplant gavage had less behavioral impairment, and reduced brain and gut inflammation. Based on data from microbial sequencing and metabolomics analysis demonstrating that young fecal transplants contained much higher SCFA levels and related bacterial strains, we selected 4 SCFA-producers (Bifidobacterium longum, Clostridium symbiosum, Faecalibacterium prausnitzii, and Lactobacillus fermentum) for transplantation. These SCFA-producers alleviated poststroke neurological deficits and inflammation, and elevated gut, brain and plasma SCFA concentrations in aged stroke mice. CONCLUSIONS: This is the first study suggesting that the poor stroke recovery in aged mice can be reversed via poststroke bacteriotherapy following the replenishment of youthful gut microbiome via modulation of immunologic, microbial, and metabolomic profiles in the host.

IDH1 p.R132H ctDNA and D-2-hydroxyglutarate as CSF biomarkers in patients with IDH-mutant gliomas.

INTRODUCTION: We aimed to evaluate IDH1 p.R132H mutation and 2-hydroxyglutarate (2HG) in cerebrospinal fluid (CSF) as biomarkers for patients with IDH-mutant gliomas. METHODS: CSF was collected from patients with infiltrating glioma, and 2HG levels were measured by liquid chromatography-mass spectrometry. IDH1 p.R132H mutant allele frequency (MAF) in CSF-ctDNA was measured by digital droplet PCR (ddPCR). Tumor volume was measured from standard-of-care magnetic resonance images. RESULTS: The study included 48 patients, 6 with IDH-mutant and 42 with IDH-wildtype gliomas, and 57 samples, 9 from the patients with IDH-mutant and 48 from the patients with IDH-wildtype gliomas. ctDNA was detected in 7 of the 9 samples from patients with IDH-mutant glioma, and IDH1 p.R132H mutation was detected in 5 of the 7 samples. The MAF ranged from 0.3 to 39.95%. Total 2HG level, D-2HG level, and D/L-2HG ratio in CSF were significantly higher in patients with IDH-mutant gliomas than in patients with IDH-wildtype gliomas. D-2HG level and D/L-2HG ratio correlated with total tumor volume in patients with IDH-mutant gliomas but not in patients with IDH-wildtype gliomas. CONCLUSION: Our results suggest that detection of IDH1 p.R132H mutation by ddPCR and increased D-2HG level in CSF may help identify IDH-mutant gliomas. Our results also suggest that D-2HG level and D/L-2HG ratio correlate with tumor volume in patients with IDH-mutant gliomas. Further prospective studies with larger cohorts are needed to validate these findings.

Early Systemic Glycolytic Shift After Aneurysmal Subarachnoid Hemorrhage is Associated with Functional Outcomes.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (aSAH) leads to a robust systemic inflammatory response. We hypothesized that an early systemic glycolytic shift occurs after aSAH, resulting in a unique metabolic signature and affecting systemic inflammation. METHODS: Control patients and patients with aSAH were analyzed. Samples from patients with aSAH were collected within 24 h of aneurysmal rupture. Mass spectrometry-based metabolomics was performed to assess relative abundance of 16 metabolites involved in the tricarboxylic acid cycle, glycolysis, and pentose phosphate pathway. Principal component analysis was used to segregate control patients from patients with aSAH. Dendrograms were developed to depict correlations between metabolites and cytokines. Analytic models predicting functional outcomes were developed, and receiver operating curves were compared. RESULTS: A total of 122 patients with aSAH and 38 control patients were included. Patients with aSAH had higher levels of glycolytic metabolites (3-phosphoglycerate/2-phosphoglycerate, lactate) but lower levels of oxidative metabolites (succinate, malate, fumarate, and oxalate). Patients with higher clinical severity (Hunt-Hess Scale score ≥ 4) had higher levels of glyceraldehyde 3-phosphate and citrate but lower levels of α-ketoglutarate and glutamine. Principal component analysis readily segregated control patients from patients with aSAH. Correlation analysis revealed distinct clusters in control patients that were not observed in patients with aSAH. Higher levels of fumarate were associated with good functional outcomes at discharge (odds ratio [OR] 1.76, 95% confidence interval [CI] 1.15-2.82) in multivariable models, whereas higher levels of citrate were associated with poor functional outcomes at discharge (OR 0.36, 95% CI 0.16-0.73) and at 3 months (OR 0.35, 95% CI 0.14-0.81). No associations were found with delayed cerebral ischemia. Levels of α-ketoglutarate and glutamine correlated with lower levels of interleukin-8, whereas fumarate was associated with lower levels of tumor necrosis factor alpha. CONCLUSIONS: Aneurysmal subarachnoid hemorrhage results in a unique pattern of plasma metabolites, indicating a shift toward glycolysis. Higher levels of fumarate and lower levels of citrate were associated with better functional outcomes. These metabolites may represent targets to improve metabolism after aSAH.

Development of a rational strategy for integration of lactate dehydrogenase A suppression into therapeutic algorithms for head and neck cancer.

BACKGROUND: Lactate dehydrogenase (LDH) is a critical metabolic enzyme. LDH A (LDHA) overexpression is a hallmark of aggressive malignancies and has been linked to tumour initiation, reprogramming and progression in multiple tumour types. However, successful LDHA inhibition strategies have not materialised in the translational and clinical space. We sought to develop a rational strategy for LDHA suppression in the context of solid tumour treatment. METHODS: We utilised a doxycycline-inducible short hairpin RNA (shRNA) system to generate LDHA suppression. Lactate and LDH activity levels were measured biochemically and kinetically using hyperpolarised 13C-pyruvate nuclear magnetic resonance spectroscopy. We evaluated effects of LDHA suppression on cellular proliferation and clonogenic survival, as well as on tumour growth, in orthotopic models of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), alone or in combination with radiation. RESULTS: shRNA suppression of LDHA generated a time-dependent decrease in LDH activity with transient shifts in intracellular lactate levels, a decrease in carbon flux from pyruvate into lactate and compensatory shifts in metabolic flux in glycolysis and the Krebs cycle. LDHA suppression decreased cellular proliferation and temporarily stunted tumour growth in ATC and HNSCC xenografts but did not by itself result in tumour cure, owing to the maintenance of residual viable cells. Only when chronic LDHA suppression was combined with radiation was a functional cure achieved. CONCLUSIONS: Successful targeting of LDHA requires exquisite dose and temporal control without significant concomitant off-target toxicity. Combinatorial strategies with conventional radiation are feasible as long as the suppression is targeted, prolonged and non-toxic.

Rapid affinity purification of intracellular organelles using a twin strep tag.

Cells are internally organized into compartmentalized organelles that execute specialized functions. To understand the functions of individual organelles and their regulations, it is critical to resolve the compositions of individual organelles, which relies on a rapid and efficient isolation method for specific organellar populations. Here, we introduce a robust affinity purification method for rapid isolation of intracellular organelles (e.g. lysosomes, mitochondria and peroxisomes) by taking advantage of the extraordinarily high affinity between the twin strep tag and streptavidin variants. With this method, we can isolate desired organelles with high purity and yield in 3 min from the post-nuclear supernatant of mammalian cells or less than 8 min for the whole purification process. Using lysosomes as an example, we show that the rapid procedure is especially useful for studying transient and fast cellular activities, such as organelle-initiated signaling and organellar contents of small-molecular metabolites. Therefore, our method offers a powerful tool to dissect spatiotemporal regulation and functions of intracellular organelles.