Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy.

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites in Rodent Cerebrospinal Fluid.

Thyroid hormones (TH) are required for brain development and function. Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH as free hormones or as bound to transthyretin (TTR). Tight TH level regulation in the central nervous system is essential for developmental gene expression, which governs neurogenesis, myelination, and synaptogenesis. This integrated function of TH highlights the importance of developing precise and reliable methods for assessing TH levels in CSF. We report an optimized liquid chromatography-mass spectrometry (LC-MS)-based method to measure TH in rodent CSF and serum, applicable to both fresh and frozen samples. Using this new method, we find distinct differences in CSF TH in pregnant dams vs. non-pregnant adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic profiling uncovers distinct central carbon metabolism in the CSF of these populations. TH detection and metabolite profiling of related metabolic pathways open new avenues of rigorous research into CSF TH and will inform future studies on metabolic alterations in CSF during normal development.

Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis.

Folate, an essential vitamin, is a one-carbon acceptor and donor in key metabolic reactions. Erythroid cells harbor a unique sensitivity to folate deprivation, as revealed by the primary pathological manifestation of nutritional folate deprivation: megaloblastic anemia. To study this metabolic sensitivity, we applied mild folate depletion to human and mouse erythroid cell lines and primary murine erythroid progenitors. We show that folate depletion induces early blockade of purine synthesis and accumulation of the purine synthesis intermediate and signaling molecule, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), followed by enhanced heme metabolism, hemoglobin synthesis, and erythroid differentiation. This is phenocopied by inhibition of folate metabolism using the inhibitor SHIN1, and by AICAR supplementation. Mechanistically, the metabolically driven differentiation is independent of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine 5'-monophosphate-activated protein kinase (AMPK) and is instead mediated by protein kinase C. Our findings suggest that folate deprivation-induced premature differentiation of erythroid progenitor cells is a molecular etiology to folate deficiency-induced anemia.

Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites in Rodent Cerebrospinal Fluid.

BACKGROUND: Thyroid hormones (TH) are required for brain development and function. Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH as free or transthyretin (TTR)-bound. Tight thyroid hormone level regulation in the central nervous system is essential for developmental gene expression that governs neurogenesis, myelination, and synaptogenesis. This integrated function of TH highlights the importance of developing precise and reliable methods for assessing TH levels in CSF. METHODS: we report an optimized LC-MS based method to measure thyroid hormones in rodent CSF and serum, applicable to both fresh and frozen samples. RESULTS: We find distinct differences in CSF thyroid hormone in pregnant dams vs. non-pregnant adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic profiling uncovers distinct central carbon metabolism in the CSF of these populations. CONCLUSIONS: TH detection and metabolite profiling of related metabolic pathways open new avenues of rigorous research into CSF thyroid hormone and will inform future studies on metabolic alterations in CSF during normal development.

Metabolomics of Mouse Embryonic CSF Following Maternal Immune Activation.

The cerebrospinal fluid (CSF) serves various roles in the developing central nervous system (CNS), from neurogenesis to lifelong cognitive functions. Changes in CSF composition due to inflammation can impact brain function. We recently identified an abnormal cytokine signature in embryonic CSF (eCSF) following maternal immune activation (MIA), a mouse model of autism spectrum disorder (ASD). We hypothesized that MIA leads to other alterations in eCSF composition and employed untargeted metabolomics to profile changes in the eCSF metabolome in mice after inducing MIA with polyI:C. We report these data here as a resource, include a comprehensive MS1 and MS2 reference dataset, and present additional datasets comparing two mouse strains (CD-1 and C57Bl/6) and two developmental time points (E12.5 and E14.5). Targeted metabolomics further validated changes upon MIA. We show a significant elevation of glucocorticoids and kynurenine pathway related metabolites. Both pathways are relevant for suppressing inflammation or could be informative as disease biomarkers. Our resource should inform future mechanistic studies regarding the etiology of MIA neuropathology and roles and contributions of eCSF metabolites to brain development.

Regulatory mechanisms of one-carbon metabolism enzymes.

One-carbon metabolism is a central metabolic pathway critical for the biosynthesis of several amino acids, methyl group donors, and nucleotides. The pathway mostly relies on the transfer of a carbon unit from the amino acid serine, through the cofactor folate (in its several forms), and to the ultimate carbon acceptors that include nucleotides and methyl groups used for methylation of proteins, RNA, and DNA. Nucleotides are required for DNA replication, DNA repair, gene expression, and protein translation, through ribosomal RNA. Therefore, the one-carbon metabolism pathway is essential for cell growth and function in all cells, but is specifically important for rapidly proliferating cells. The regulation of one-carbon metabolism is a critical aspect of the normal and pathological function of the pathway, such as in cancer, where hijacking these regulatory mechanisms feeds an increased need for nucleotides. One-carbon metabolism is regulated at several levels: via gene expression, posttranslational modification, subcellular compartmentalization, allosteric inhibition, and feedback regulation. In this review, we aim to inform the readers of relevant one-carbon metabolism regulation mechanisms and to bring forward the need to further study this aspect of one-carbon metabolism. The review aims to integrate two major aspects of cancer metabolism-signaling downstream of nutrient sensing and one-carbon metabolism, because while each of these is critical for the proliferation of cancerous cells, their integration is critical for comprehensive understating of cellular metabolism in transformed cells and can lead to clinically relevant insights.

Regulators of mitonuclear balance link mitochondrial metabolism to mtDNA expression.

Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells requiring coordinated gene expression across organelles. To identify genes involved in dual-origin protein complex synthesis, we performed fluorescence-activated cell-sorting-based genome-wide screens analysing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of Complex IV. We identified genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically impacts Complex IV biogenesis by acting at the intersection of mitochondrial lipid metabolism and protein synthesis, whereas NME6, an uncharacterized nucleoside diphosphate kinase, controls OXPHOS biogenesis through multiple mechanisms reliant on its NDPK domain. Firstly, NME6 forms a complex with RCC1L, which together perform nucleoside diphosphate kinase activity to maintain local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Secondly, NME6 modulates the activity of mitoribosome regulatory complexes, altering mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression.

Simultaneous isolation of hormone receptor-positive breast cancer organoids and fibroblasts reveals stroma-mediated resistance mechanisms.

Recurrent hormone receptor-positive (HR+) breast cancer kills more than 600,000 women annually. Although HR+ breast cancers typically respond well to therapies, approximately 30% of patients relapse. At this stage, the tumors are usually metastatic and incurable. Resistance to therapy, particularly endocrine therapy is typically thought to be tumor intrinsic (e.g., estrogen receptor mutations). However, tumor-extrinsic factors also contribute to resistance. For example, stromal cells, such as cancer-associated fibroblasts (CAFs), residing in the tumor microenvironment, are known to stimulate resistance and disease recurrence. Recurrence in HR+ disease has been difficult to study due to the prolonged clinical course, complex nature of resistance, and lack of appropriate model systems. Existing HR+ models are limited to HR+ cell lines, a few HR+ organoid models, and xenograft models that all lack components of the human stroma. Therefore, there is an urgent need for more clinically relevant models to study the complex nature of recurrent HR+ breast cancer, and the factors contributing to treatment relapse. Here, we present an optimized protocol that allows a high take-rate, and simultaneous propagation of patient-derived organoids (PDOs) and matching CAFs, from primary and metastatic HR+ breast cancers. Our protocol allows for long-term culturing of HR+ PDOs that retain estrogen receptor expression and show responsiveness to hormone therapy. We further show the functional utility of this system by identifying CAF-secreted cytokines, such as growth-regulated oncogene α , as stroma-derived resistance drivers to endocrine therapy in HR+ PDOs.

FDX1 regulates cellular protein lipoylation through direct binding to LIAS.

Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D, and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore-induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post-translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 directly regulates protein lipoylation by binding the lipoyl synthase (LIAS) enzyme promoting its functional binding to the lipoyl carrier protein GCSH and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss of function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling established that FDX1 loss-of-function results in the induction of both compensatory metabolism-related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-function is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting its role in cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.

Correction: Maynard et al. Notes from the 2022 Folate, Vitamin B12, and One-Carbon Metabolism Conference. Metabolites 2023, 13, 486.

Thanks to feedback from several speakers, text was amended, and citations updated, in the original article [...].

Defining diurnal fluctuations in mouse choroid plexus and CSF at high molecular, spatial, and temporal resolution.

Transmission and secretion of signals via the choroid plexus (ChP) brain barrier can modulate brain states via regulation of cerebrospinal fluid (CSF) composition. Here, we developed a platform to analyze diurnal variations in male mouse ChP and CSF. Ribosome profiling of ChP epithelial cells revealed diurnal translatome differences in metabolic machinery, secreted proteins, and barrier components. Using ChP and CSF metabolomics and blood-CSF barrier analyses, we observed diurnal changes in metabolites and cellular junctions. We then focused on transthyretin (TTR), a diurnally regulated thyroid hormone chaperone secreted by the ChP. Diurnal variation in ChP TTR depended on Bmal1 clock gene expression. We achieved real-time tracking of CSF-TTR in awake TtrmNeonGreen mice via multi-day intracerebroventricular fiber photometry. Diurnal changes in ChP and CSF TTR levels correlated with CSF thyroid hormone levels. These datasets highlight an integrated platform for investigating diurnal control of brain states by the ChP and CSF.

Notes from the 2022 Folate, Vitamin B12, and One-Carbon Metabolism Conference.

Here, we present notes from the Folate, Vitamin B12, and One-Carbon Metabolism Conference organized by The Federation of American Societies for Experimental Biology (FASEB), held in Asheville, North Carolina, USA, 14-19 August 2022. We aim to share the most recent findings in the field with members of our scientific community who did not attend the meeting and who are interested in the research that was presented. The research described includes discussions of one-carbon metabolism at the biochemical and physiological levels and studies of the role of folate and B12 in development and in the adult, and from bacteria to mammals. Furthermore, the summarized studies address the role of one-carbon metabolism in disease, including COVID-19, neurodegeneration, and cancer.

Cycloguanil and Analogues Potently Target DHFR in Cancer Cells to Elicit Anti-Cancer Activity.

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.

FDX1 regulates cellular protein lipoylation through direct binding to LIAS.

Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 regulates protein lipoylation by directly binding to the lipoyl synthase (LIAS) enzyme and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss-of-function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling of cells growing in either normal or low glucose conditions established that FDX1 loss-of-function results in the induction of both compensatory metabolism related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-functions is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.

Genome-wide screens for mitonuclear co-regulators uncover links between compartmentalized metabolism and mitochondrial gene expression.

Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells, in which gene expression must be coordinated across organelles using distinct pools of ribosomes. How cells produce and maintain the accurate subunit stoichiometries for these OXPHOS complexes remains largely unknown. To identify genes involved in dual-origin protein complex synthesis, we performed FACS-based genome-wide screens analyzing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of cytochrome c oxidase (Complex IV). We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6 . We found that PREPL specifically regulates Complex IV biogenesis by interacting with mitochondrial protein synthesis machinery, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS complex biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 maintains local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, through stabilizing interactions with RCC1L, NME6 modulates the activity of mitoribosome regulatory complexes, leading to disruptions in mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression. Finally, we present these screens as a resource, providing a catalog of genes involved in mitonuclear gene regulation and OXPHOS biogenesis.

Choroid plexus-CSF-targeted antioxidant therapy protects the brain from toxicity of cancer chemotherapy.

For many cancer patients, chemotherapy produces untreatable life-long neurologic effects termed chemotherapy-related cognitive impairment (CRCI). We discovered that the chemotherapy methotrexate (MTX) adversely affects oxidative metabolism of non-cancerous choroid plexus (ChP) cells and the cerebrospinal fluid (CSF). We used a ChP-targeted adeno-associated viral (AAV) vector approach in mice to augment CSF levels of the secreted antioxidant SOD3. AAV-SOD3 gene therapy increased oxidative defense capacity of the CSF and prevented MTX-induced lipid peroxidation in the hippocampus. Furthermore, this gene therapy prevented anxiety and deficits in short-term learning and memory caused by MTX. MTX-induced oxidative damage to cultured human cortical neurons and analyses of CSF samples from MTX-treated lymphoma patients demonstrated that MTX diminishes antioxidant capacity of patient CSF. Collectively, our findings motivate the advancement of ChP- and CSF-targeted anti-oxidative prophylactic measures to relieve CRCI.

Copper induces cell death by targeting lipoylated TCA cycle proteins.

Copper is an essential cofactor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copper-dependent, regulated cell death is distinct from known death mechanisms and is dependent on mitochondrial respiration. We show that copper-dependent death occurs by means of direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss, which leads to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms.

The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase.

Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS.

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell's metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.