Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Sex, life, and death in MYC-driven lymphomagenesis.

Gong et al. (2021) demonstrate that MYC-induced proteotoxic stress could be relieved by inactivating RNA helicase DDX3X for tumor initiation, and in male MYC-driven lymphomas, the homologous helicase DDX3Y, encoded on the Y chromosome, is subsequently induced for disease progression.

MYC on the path to cancer.

The MYC oncogene contributes to the genesis of many human cancers. Recent insights into its expression and function have led to therapeutic opportunities. MYC's activation by bromodomain proteins could be inhibited by drug-like molecules, resulting in tumor inhibition in vivo. Tumor growth can also be curbed by pharmacologically uncoupling bioenergetic pathways involving glucose or glutamine metabolism from Myc-induced cellular biomass accumulation. Other approaches to halt Myc on the path to cancer involve targeting Myc-Max dimerization or Myc-induced microRNA expression. Here the richness of our understanding of MYC is reviewed, highlighting new biological insights and opportunities for cancer therapies.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1.

T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis.

The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites in healthy tissues, is disrupted across many human cancers. Deregulated expression of the MYC oncoprotein has been shown to alter expression of molecular clock genes, leading to a disruption of molecular clock oscillation across cancer types. It remains unclear what benefit cancer cells gain from suppressing clock oscillation, and how this loss of molecular clock oscillation impacts global gene expression and metabolism in cancer. We hypothesized that MYC or its paralog N-MYC (collectively termed MYC herein) suppress oscillation of gene expression and metabolism to upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, cells from distinct cancer types with inducible MYC were examined, using time-series RNA-sequencing and metabolomics, to determine the extent to which MYC activation disrupts global oscillation of genes, gene expression pathways, and metabolites. We focused our analyses on genes, pathways, and metabolites that changed in common across multiple cancer cell line models. We report here that MYC disrupted over 85% of oscillating genes, while instead promoting enhanced ribosomal and mitochondrial biogenesis and suppressed cell attachment pathways. Notably, when MYC is activated, biosynthetic programs that were formerly circadian flipped to being upregulated in an oscillation-free manner. Further, activation of MYC ablates the oscillation of nutrient transporter proteins while greatly upregulating transporter expression, cell surface localization, and intracellular amino acid pools. Finally, we report that MYC disrupts metabolite oscillations and the temporal segregation of amino acid metabolism from nucleotide metabolism. Our results demonstrate that MYC disruption of the molecular circadian clock releases metabolic and biosynthetic processes from circadian control, which may provide a distinct advantage to cancer cells.

ACLY alternative splicing correlates with cancer phenotypes.

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

Epigenetic state determines inflammatory sensing in neuroblastoma.

Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically "cold" pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling, and all but one lack functional cytosolic DNA sensing via cGAS-STING. However, double-stranded RNA (dsRNA) sensing via Toll-like receptor 3 (TLR3) was heterogeneous, as was signaling through other dsRNA sensors and TLRs more broadly. Seven cell lines showed robust response to dsRNA, six of which are in the mesenchymal epigenetic state, while all unresponsive cell lines are in the adrenergic state. Genetically switching adrenergic cell lines toward the mesenchymal state fully restored responsiveness. In responsive cells, dsRNA sensing results in the secretion of proinflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T cells in vitro. Using single-cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intratumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.

Partial Regret After Gender Affirmation Surgery of a 35-Year-Old Taiwanese Transgender Woman.

Gender-affirming surgery (GAS) is often sought after to alleviate the distress of those who suffer from gender dysphoria (GD). While many studies have shown that a significant percentage of people benefit from this procedure, a number of individuals later regret their decision of undergoing surgery. Studies have illustrated what regret depicts, categorizing regret based on intensity, persistency, and sources, in the hopes to prevent an unwanted irreversible intervention. Here, an in-depth interview with a 35-year-old transwoman from Taiwan who underwent feminizing GAS at the age of 31 illustrates her unique cultural upbringing and the course of her regret. Her experience best matches the characteristics of true regret and major regret based on the classifications of Pfäfflin and Wiepjes, respectively, indicating that she expected GAS to be the solution to her personal acceptance issue, but, in retrospect, regretted the diagnosis and treatment as her problems were not solved and worsened to the extent of secondary dysphoria. This case report hopes to shed light on the complexity of GD and regret after GAS, while encouraging the pre-surgical evaluation of psychological comorbidities and post-surgical psychotherapy, and ensuring that patients are informed and give full consent. In addition, more elaborate, long-term, large-scale qualitative research, especially within more conservative cultural settings, is needed.

Redefining the Etiologic Landscape of Cerebellar Malformations.

Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.

Acute ischemic stroke induces magnetic resonance susceptibility signs dominated by endothelial nitric oxide synthase activation.

PURPOSE: Acute ischemic stroke induces deoxyhemoglobin accumulation around the ischemic region while activating endothelial nitric oxide synthase (eNOS) coupling and the subsequent release of nitric oxide (NO). Because deoxyhemoglobin is a natural NO spin trap, its interplay with NO could be prominent during acute stroke. Its interaction with NO has been shown to induce overt paramagnetic signals in vitro; our goal was to investigate whether this interplay can be detected using MRI. METHODS: To verify the in vivo image effects using the deoxyhemoglobin-NO interaction during acute stroke, eNOS states were manipulated in an animal model of acute ischemia, and the susceptibility signals, cerebral perfusion, and infarction were assessed noninvasively via MR susceptibility weighted imaging (SWI). RESULTS: Occlusion of the right middle cerebral artery increased eNOS coupling and susceptibility signals in the ischemic cortex while abolishing regional cerebral blood flow. Pharmacological eNOS blockage led to weakened susceptibility signals in the ischemic cortex as well as worsened tissue survival. Consistently, abolishment of eNOS coupling through genetic editing reduced the regional susceptibility signals in the ischemic cortex, causing large infarcts. CONCLUSION: Upregulation of eNOS during acute ischemia sustains tissue viability through the interaction between NO and deoxyhemoglobin. This interplay can be traced in vivo using SWI and can be considered a sensitive marker revealing the delicate oxygenation status of the ischemic tissue, therefore, guiding the management of acute stroke in clinical settings.

Survival after out-of-hospital cardiac arrest, Viet Nam: multicentre prospective cohort study.

OBJECTIVE: To investigate factors associated with survival after out-of-hospital cardiac arrest in Viet Nam. METHODS: We did a multicentre prospective observational study of people (> 18 years) presenting with out-of-hospital cardiac arrest (not caused by trauma) to three tertiary hospitals in Viet Nam from February 2014 to December 2018. We collected data on characteristics, management and outcomes of patients with out-of-hospital cardiac arrest and compared these data by type of transportation to hospital and survival to hospital admission. We assessed factors associated with survival to admission to and discharge from hospital using logistic regression analysis. FINDINGS: Of 590 eligible people with out-of-hospital cardiac arrest, 440 (74.6%) were male and the mean age was 56.1 years (standard deviation: 17.2). Only 24.2% (143/590) of these people survived to hospital admission and 14.1% (83/590) survived to hospital discharge. Most cardiac arrests (67.8%; 400/590) occurred at home, 79.4% (444/559) were witnessed by bystanders and 22.3% (124/555) were given cardiopulmonary resuscitation by a bystander. Only 8.6% (51/590) of the people were taken to hospital by the emergency medical services and 32.2% (49/152) received pre-hospital defibrillation. Pre-hospital defibrillation (odds ratio, OR: 3.90; 95% confidence interval, CI: 1.54-9.90) and return of spontaneous circulation in the emergency department (OR: 2.89; 95% CI: 1.03-8.12) were associated with survival to hospital admission. Hypothermia therapy during post-resuscitation care was associated with survival to discharge (OR: 5.44; 95% CI: 2.33-12.74). CONCLUSION: Improvements are needed in the emergency medical services in Viet Nam such as increasing bystander cardiopulmonary resuscitation and public access defibrillation, and improving ambulance and post-resuscitation care.

mTOR Senses Intracellular pH through Lysosome Dispersion from RHEB.

Acidity, generated in hypoxia or hypermetabolic states, perturbs homeostasis and is a feature of solid tumors. That acid peripherally disperses lysosomes is a three-decade-old observation, yet one little understood or appreciated. However, recent work has recognized the inhibitory impact this spatial redistribution has on mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of metabolism. This finding argues for a paradigm shift in localization of mTORC1 activator Ras homolog enriched in brain (RHEB), a conclusion several others have now independently reached. Thus, mTORC1, known to sense amino acids, mitogens, and energy to restrict biosynthesis to times of adequate resources, also senses pH and, via dampened mTOR-governed synthesis of clock proteins, regulates the circadian clock to achieve concerted responses to metabolic stress. While this may allow cancer to endure metabolic deprivation, immune cell mTOR signaling likewise exhibits pH sensitivity, suggesting that suppression of antitumor immune function by solid tumor acidity may additionally fuel cancers, an obstacle potentially reversible through therapeutic pH manipulation.

Myc-mediated transcriptional regulation of the mitochondrial chaperone TRAP1 controls primary and metastatic tumor growth.

The role of mitochondria in cancer continues to be debated, and whether exploitation of mitochondrial functions is a general hallmark of malignancy or a tumor- or context-specific response is still unknown. Using a variety of cancer cell lines and several technical approaches, including siRNA-mediated gene silencing, ChIP assays, global metabolomics and focused metabolite analyses, bioenergetics, and cell viability assays, we show that two oncogenic Myc proteins, c-Myc and N-Myc, transcriptionally control the expression of the mitochondrial chaperone TNFR-associated protein-1 (TRAP1) in cancer. In turn, this Myc-mediated regulation preserved the folding and function of mitochondrial oxidative phosphorylation (OXPHOS) complex II and IV subunits, dampened reactive oxygen species production, and enabled oxidative bioenergetics in tumor cells. Of note, we found that genetic or pharmacological targeting of this pathway shuts off tumor cell motility and invasion, kills Myc-expressing cells in a TRAP1-dependent manner, and suppresses primary and metastatic tumor growth in vivo We conclude that exploitation of mitochondrial functions is a general trait of tumorigenesis and that this reliance of cancer cells on mitochondrial OXPHOS pathways could offer an actionable therapeutic target in the clinic.

De novo synthesis of serine and glycine fuels purine nucleotide biosynthesis in human lung cancer tissues.

Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [13C6]glucose, D2-glycine, [13C2]glycine, and D3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro Surprisingly, [13C6]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D3-Ser was preferentially incorporated into purine rings over D2-glycine in both tissues and cell lines. MYC suppression attenuated [13C6]glucose, D3-serine, and [13C2]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy.

Links between metabolism and cancer.

Metabolism generates oxygen radicals, which contribute to oncogenic mutations. Activated oncogenes and loss of tumor suppressors in turn alter metabolism and induce aerobic glycolysis. Aerobic glycolysis or the Warburg effect links the high rate of glucose fermentation to cancer. Together with glutamine, glucose via glycolysis provides the carbon skeletons, NADPH, and ATP to build new cancer cells, which persist in hypoxia that in turn rewires metabolic pathways for cell growth and survival. Excessive caloric intake is associated with an increased risk for cancers, while caloric restriction is protective, perhaps through clearance of mitochondria or mitophagy, thereby reducing oxidative stress. Hence, the links between metabolism and cancer are multifaceted, spanning from the low incidence of cancer in large mammals with low specific metabolic rates to altered cancer cell metabolism resulting from mutated enzymes or cancer genes.

Turning publicly available gene expression data into discoveries using gene set context analysis.

Gene Set Context Analysis (GSCA) is an open source software package to help researchers use massive amounts of publicly available gene expression data (PED) to make discoveries. Users can interactively visualize and explore gene and gene set activities in 25,000+ consistently normalized human and mouse gene expression samples representing diverse biological contexts (e.g. different cells, tissues and disease types, etc.). By providing one or multiple genes or gene sets as input and specifying a gene set activity pattern of interest, users can query the expression compendium to systematically identify biological contexts associated with the specified gene set activity pattern. In this way, researchers with new gene sets from their own experiments may discover previously unknown contexts of gene set functions and hence increase the value of their experiments. GSCA has a graphical user interface (GUI). The GUI makes the analysis convenient and customizable. Analysis results can be conveniently exported as publication quality figures and tables. GSCA is available at https://github.com/zji90/GSCA. This software significantly lowers the bar for biomedical investigators to use PED in their daily research for generating and screening hypotheses, which was previously difficult because of the complexity, heterogeneity and size of the data.

MYC oncogene overexpression drives renal cell carcinoma in a mouse model through glutamine metabolism.

The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization-mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.

MYC and metabolism on the path to cancer.

The MYC proto-oncogene is frequently deregulated in human cancers, activating genetic programs that orchestrate biological processes to promote growth and proliferation. Altered metabolism characterized by heightened nutrients uptake, enhanced glycolysis and glutaminolysis and elevated fatty acid and nucleotide synthesis is the hallmark of MYC-driven cancer. Recent evidence strongly suggests that Myc-dependent metabolic reprogramming is critical for tumorigenesis, which could be attenuated by targeting specific metabolic pathways using small drug-like molecules. Understanding the complexity of MYC-mediated metabolic re-wiring in cancers as well as how MYC cooperates with other metabolic drivers such as mammalian target of rapamycin (mTOR) will provide translational opportunities for cancer therapy.