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Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.

The PI3K/AKT/mTOR pathway is commonly dysregulated in cancer. Rapalogs exhibit modest clinical benefit, likely owing to their lack of effects on 4EBP1. We hypothesized that bi-steric mTORC1-selective inhibitors would have greater potential for clinical benefit than rapalogs in tumors with mTORC1 dysfunction. We assessed this hypothesis in tumor models with high mTORC1 activity both in vitro and in vivo. Bi-steric inhibitors had strong growth inhibition, eliminated phosphorylated 4EBP1, and induced more apoptosis than rapamycin or MLN0128. Multiomics analysis showed extensive effects of the bi-steric inhibitors in comparison with rapamycin. De novo purine synthesis was selectively inhibited by bi-sterics through reduction in JUN and its downstream target PRPS1 and appeared to be the cause of apoptosis. Hence, bi-steric mTORC1-selective inhibitors are a therapeutic strategy to treat tumors driven by mTORC1 hyperactivation., Introduction The PI3K/AKT/mTOR network is a critical intracellular signaling pathway directing cell growth and metabolism in physiological and pathological conditions (1). The evolutionarily conserved mammalian target of rapamycin (mTOR) is [...]

Activation of anaplerosis takes away glutamine from the biosynthetic pathways to the energy-producing TCA cycle. Especially, induction of hyperoxia driven anaplerosis in neurovascular tissues such as the retina during early stages of development could deplete biosynthetic precursors from newly proliferating endothelial cells impeding physiological angiogenesis and leading to vasoobliteration. Using an oxygen-induced retinopathy (OIR) mouse model, we investigated the metabolic differences between OIR-resistant BALB/cByJ and OIR susceptible C57BL/6J strains at system levels to understand the molecular underpinnings that potentially contribute to hyperoxia-induced vascular abnormalities in the neural retina. Our systems level in vivo RNA-seq, proteomics, and lipidomic profiling and ex-vivo retinal explant studies show that the medium-chain fatty acids serves as an alternative source to feed the TCA cycle. Our findings strongly implicate that medium-chain fatty acids could suppress glutamine-fueled anaplerosis and ameliorate hyperoxia-induced vascular abnormalities in conditions such as retinopathy of prematurity. Medium chain fatty acid metabolism differs between OIR resistant and susceptible strains. Hyperoxia induces medium chain fatty acid utilization in retinal explant preventing glutamine-fueled anaplerosis in hyperoxic conditions. [ABSTRACT FROM AUTHOR]

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance. [ABSTRACT FROM AUTHOR]

Neuronal cells are highly specialized cells and have a specific metabolic profile to support their function. It has been demonstrated that the metabolic profiles of different cells/tissues undergo significant reprogramming with advancing age, which has often been considered a contributing factor towards aging‐related diseases including Alzheimer's (AD) and Parkinson's (PD) diseases. However, it is unclear if the metabolic changes associated with normal aging predispose neurons to disease conditions or a distinct set of metabolic alterations happen in neurons in AD or PD which might contribute to disease pathologies. To decipher the changes in neuronal metabolism with age, in AD, or in PD, we performed high‐throughput steady‐state metabolite profiling on heads in wildtype Drosophila and in Drosophila models relevant to AD and PD. Intriguingly, we found that the spectrum of affected metabolic pathways is dramatically different between normal aging, Tau, or Synuclein overexpressing neurons. Genetic targeting of the purine and glutamate metabolism pathways, which were dysregulated in both old age and disease conditions partially rescued the neurodegenerative phenotype associated with the overexpression of wildtype and mutant tau. Our findings support a "two‐hit model" to explain the pathological manifestations associated with AD where both aging‐ and Tau/Synuclein‐ driven metabolic reprogramming events cooperate with each other, and targeting both could be a potent therapeutic strategy. [ABSTRACT FROM AUTHOR]

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Inactivation of the RB1 tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies against RBI-deficient cancers remain elusive. Here, we showed that RB1 loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression of ACSL4 and enriching ACSL4-dependent arachidonic acid-containing phospholipids, which are key components of ferroptosis execution. ACSL4 appeared to be a direct E2F target gene and was critical to RB1 loss-induced sensitization to ferroptosis. Importantly, using cell line-derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blocked RBI-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis and also suggest a promising approach for the treatment of RBI-deficient malignancies., Introduction Mutations in the retinoblastoma tumor suppressor gene, RB1, are common in a wide spectrum of pediatric and adult cancers, including retinoblastoma, sarcoma, small cell lung and prostate cancers, and [...]

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Preoperative risk biomarkers for delirium may aid in identifying high-risk patients and developing intervention therapies, which would minimize the health and economic burden of postoperative delirium. Previous studies have typically used single omics approaches to identify such biomarkers. Preoperative cerebrospinal fluid (CSF) from the Healthier Postoperative Recovery study of adults ≥ 63 years old undergoing elective major orthopedic surgery was used in a matched pair delirium case–no delirium control design. We performed metabolomics and lipidomics, which were combined with our previously reported proteomics results on the same samples. Differential expression, clustering, classification, and systems biology analyses were applied to individual and combined omics datasets. Probabilistic graph models were used to identify an integrated multi-omics interaction network, which included clusters of heterogeneous omics interactions among lipids, metabolites, and proteins. The combined multi-omics signature of 25 molecules attained an AUC of 0.96 [95% CI: 0.85–1.00], showing improvement over individual omics-based classification. We conclude that multi-omics integration of preoperative CSF identifies potential risk markers for delirium and generates new insights into the complex pathways associated with delirium. With future validation, this hypotheses-generating study may serve to build robust biomarkers for delirium and improve our understanding of its pathophysiology. [ABSTRACT FROM AUTHOR]

Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.