A metabolic switch orchestrated by IL-18 and the cyclic dinucleotide cGAMP programs intestinal tolerance.

Tissues are exposed to diverse inflammatory challenges that shape future inflammatory responses. While cellular metabolism regulates immune function, how metabolism programs and stabilizes immune states within tissues and tunes susceptibility to inflammation is poorly understood. Here, we describe an innate immune metabolic switch that programs long-term intestinal tolerance. Intestinal interleukin-18 (IL-18) stimulation elicited tolerogenic macrophages by preventing their proinflammatory glycolytic polarization via metabolic reprogramming to fatty acid oxidation (FAO). FAO reprogramming was triggered by IL-18 activation of SLC12A3 (NCC), leading to sodium influx, release of mitochondrial DNA, and activation of stimulator of interferon genes (STING). FAO was maintained in macrophages by a bistable switch that encoded memory of IL-18 stimulation and by intercellular positive feedback that sustained the production of macrophage-derived 2'3'-cyclic GMP-AMP (cGAMP) and epithelial-derived IL-18. Thus, a tissue-reinforced metabolic switch encodes durable immune tolerance in the gut and may enable reconstructing compromised immune tolerance in chronic inflammation.

Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase.

A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.

Dihydropyrimidine accumulation is required for the epithelial-mesenchymal transition.

It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.

Dietary modifications for enhanced cancer therapy.

Tumours depend on nutrients supplied by the host for their growth and survival. Modifications to the host's diet can change nutrient availability in the tumour microenvironment, which might represent a promising strategy for inhibiting tumour growth. Dietary modifications can limit tumour-specific nutritional requirements, alter certain nutrients that target the metabolic vulnerabilities of the tumour, or enhance the cytotoxicity of anti-cancer drugs. Recent reports have suggested that modification of several nutrients in the diet can alter the efficacy of cancer therapies, and some of the newest developments in this quickly expanding field are reviewed here. The results discussed indicate that the dietary habits and nutritional state of a patient must be taken into account during cancer research and therapy.

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.

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.

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.

Histidine catabolism is a major determinant of methotrexate sensitivity.

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.

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.

Potential Benefits and Pitfalls of Histidine Supplementation for Cancer Therapy Enhancement.

Dietary supplementation of the amino acid histidine has demonstrable benefits in various clinical conditions. Recent work in a pediatric leukemia mouse model exposed a surprising potential application of histidine supplementation for cancer therapy enhancement. These findings demand a deeper reassessment of the physiological effects and potential drawbacks of histidine supplementation. As pertinent to this question, we discuss the safety of high doses of histidine and its relevant metabolic fates in the human body. We refrain from recommendations or final conclusions because comprehensive preclinical evidence for safety and efficacy of histidine supplementation is still lacking. However, we emphasize the incentive to study the safety of histidine supplementation and its potential to improve the clinical outcome of pediatric blood cancers through a simple dietary supplementation. The need for comprehensive preclinical testing of histidine supplementation in healthy and tumor-bearing mice is fundamental, and we hope that this review will facilitate such studies.

mTOR generates an auto-amplification loop by triggering the ßTrCP- and CK1α-dependent degradation of DEPTOR.

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein ßTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the ßTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the ßTrCP-dependent degradation of DEPTOR via ßTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind ßTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and ßTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways.

Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCF{beta}-TrCP by single substrate depletion.

beta-TrCP, the substrate recognition subunit of a Skp1-Cul1-F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible beta-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two beta-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon beta-TrCP restoration. Remarkably, testicular depletion of a single beta-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation.

Critical role for IL-1ß in DNA damage-induced mucositis.

ß-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of ß-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1ß as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1ß is induced by DNA damage via an NF-κB-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1ß receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1ß prevents epithelial tight junction dysfunction and alleviates mucositis in ß-TrCP-deficient mice. IL-1ß antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Regulation of NF-κB by ubiquitination and degradation of the IκBs.

The nuclear factor-κB (NF-κB) signaling pathway is a busy ground for the action of the ubiquitin-proteasome system; many of the signaling steps are coordinated by protein ubiquitination. The end point of this pathway is to induce transcription, and to this end, there is a need to overcome a major obstacle, a set of inhibitors (IκBs) that bind NF-κB and prohibit either the nuclear entry or the DNA binding of the transcription factor. Two major signaling steps are required for the elimination of the inhibitors: activation of the IκB kinase (IKK) and degradation of the phosphorylated inhibitors. IKK activation and IκB degradation involve different ubiquitination modes; the latter is mediated by a specific E3 ubiquitin ligase SCF(ß-TrCP) . The F-box component of this E3, ß-TrCP, recognizes the IκB degron formed following phosphorylation by IKK and thus couples IκB phosphorylation to ubiquitination. SCF(ß-TrCP) -mediated IκB ubiquitination and degradation is a very efficient process, often resulting in complete degradation of the key inhibitor IκBα within a few minutes of cell stimulation. In vivo ablation of ß-TrCP results in accumulation of all the IκBs and complete NF-κB inhibition. As many details of IκB-ß-TrCP interaction have been worked out, the development of ß-TrCP inhibitors might be a feasible therapeutic approach for NF-κB-associated human disease. However, we may still need to advance our understanding of the mechanism of IκB degradation as well as of the diverse functions of ß-TrCP in vivo.

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.

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.

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.

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 [...].