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.

Increased demand for NAD+ relative to ATP drives aerobic glycolysis.

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD+/NADH ratio. This change in NAD+/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD+ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD+ is in excess of the demand for ATP.

Metabolomics in cancer research and emerging applications in clinical oncology.

Cancer has myriad effects on metabolism that include both rewiring of intracellular metabolism to enable cancer cells to proliferate inappropriately and adapt to the tumor microenvironment, and changes in normal tissue metabolism. With the recognition that fluorodeoxyglucose-positron emission tomography imaging is an important tool for the management of many cancers, other metabolites in biological samples have been in the spotlight for cancer diagnosis, monitoring, and therapy. Metabolomics is the global analysis of small molecule metabolites that like other -omics technologies can provide critical information about the cancer state that are otherwise not apparent. Here, the authors review how cancer and cancer therapies interact with metabolism at the cellular and systemic levels. An overview of metabolomics is provided with a focus on currently available technologies and how they have been applied in the clinical and translational research setting. The authors also discuss how metabolomics could be further leveraged in the future to improve the management of patients with cancer.

CLN3 is required for the clearance of glycerophosphodiesters from lysosomes.

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

MFSD12 mediates the import of cysteine into melanosomes and lysosomes.

Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome-the organelle, related to the lysosome, that synthesizes pigment-but have unclear functions1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3,4. We find that MFSD12 is required to maintain normal levels of cystine-the oxidized dimer of cysteine-in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7-9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes.

InterFeriNg with Acetγlation: Stress-Levels of Acetate Improve Memory T Cell Function.

Catabolic stress can lead to changes in circulating acetate levels. In this issue of Immunity, Balmer et al. (2016) report that serum acetate increases in response to acute infection and describe a mechanism by which this results in acetylation of the glycolytic enzyme GAPDH and improves the recall function of memory CD8(+) T cells.

Squalene accumulation in cholesterol auxotrophic lymphomas prevents oxidative cell death.

Cholesterol is essential for cells to grow and proliferate. Normal mammalian cells meet their need for cholesterol through its uptake or de novo synthesis1, but the extent to which cancer cells rely on each of these pathways remains poorly understood. Here, using a competitive proliferation assay on a pooled collection of DNA-barcoded cell lines, we identify a subset of cancer cells that is auxotrophic for cholesterol and thus highly dependent on its uptake. Through metabolic gene expression analysis, we pinpoint the loss of squalene monooxygenase expression as a cause of cholesterol auxotrophy, particularly in ALK+ anaplastic large cell lymphoma (ALCL) cell lines and primary tumours. Squalene monooxygenase catalyses the oxidation of squalene to 2,3-oxidosqualene in the cholesterol synthesis pathway and its loss results in accumulation of the upstream metabolite squalene, which is normally undetectable. In ALK+ ALCLs, squalene alters the cellular lipid profile and protects cancer cells from ferroptotic cell death, providing a growth advantage under conditions of oxidative stress and in tumour xenografts. Finally, a CRISPR-based genetic screen identified cholesterol uptake by the low-density lipoprotein receptor as essential for the growth of ALCL cells in culture and as patient-derived xenografts. This work reveals that the cholesterol auxotrophy of ALCLs is a targetable liability and, more broadly, that systematic approaches can be used to identify nutrient dependencies unique to individual cancer types.

SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

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.

Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.

Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.

Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells.

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo.

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

Enhanced phosphocholine metabolism is essential for terminal erythropoiesis.

Red cells contain a unique constellation of membrane lipids. Although much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here, we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine (PC) and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and, in adult mice, depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; adenosine triphosphate (ATP) production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1-knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of PC and phosphocholine metabolism in energy balance and amino acid supply.

Femoral nerve block at time of ACL reconstruction causes lasting quadriceps strength deficits and may increase short-term risk of re-injury.

PURPOSE: To determine whether femoral nerve blockade (FNB) at the time of primary ACL reconstruction is associated with meeting isokinetic extension strength return to sport criteria near completion of physical therapy and whether FNB affects 1-year or 2-year risk of ipsilateral ACL graft rupture or contralateral native ACL injury. METHODS: Three-hundred and sixty patients (n = 244 with FNB, n = 116 no FNB) underwent primary ACL reconstruction. All patients completed rehabilitation and underwent functional strength testing towards the end of knee rehabilitation (mean 5.6 months post-surgery). Association between FNB and isokinetic extension strength limb symmetry index (LSI) (goal LSI ≥ 90% for return to sport) as well as risk of recurrent ACL injury within first or second year after surgery was evaluated. RESULTS: Ipsilateral or contralateral ACL injury within 2 years occurred in 11.2% of patients with FNB and 5.7% without FNB (p = 0.01). Patients with FNB had higher incidence of ipsilateral graft rupture within the first year after surgery but no difference in graft rupture during the second. Two-year risk of contralateral ACL injury was similar in both groups. At the time of initial testing, patients who received FNB had lower fast isokinetic extension LSI versus patients without FNB and were less likely achieve a goal ≥ 90% LSI; slow extension LSI was unaffected. CONCLUSION: Use of FNB at the time of primary ACL reconstruction can negatively affect achievement of isokinetic extension strength return to sport criteria. FNB increases risk of graft rupture within the first year after surgery but does not affect re-injury risk during the second. FNB may not be appropriate for use in patients already at high risk of ACL re-injury. LEVEL OF EVIDENCE: III.

Cartilage damage at the time of anterior cruciate ligament reconstruction is associated with weaker quadriceps function and lower risk of future ACL injury.

PURPOSE: To determine whether articular cartilage damage noted at the time of primary anterior cruciate ligament reconstruction (ACLR) affects the likelihood of achieving ≥ 90% symmetry for isokinetic extension strength at 6 months after surgery or risk of recurrent ACL injury. METHODS: Five hundred and eight patients underwent primary ACLR and diagnostic arthroscopy. All identified cartilage lesions were graded using the Outerbridge system. All patients underwent isokinetic strength testing. The association between cartilage Outerbridge grade and a ≥ 90% Limb Symmetry Index (LSI) and recurrent ACL injury risk at mean 38.7 month follow-up (SD 31.8) was evaluated via multivariate regression analysis. RESULTS: Grade 2 or higher damage was present in 394 (77.5%) of patients, grade 3 or higher in 143 (28.1%) and grade 4 in 83 (16.4%) at time of ACLR. Ipsilateral ACLR graft rupture occurred in 31 (6.1%) of patients. Contralateral ACL injury occurred in 19 (3.7%). Patients with grade 2 or higher damage were significantly less likely to meet an LSI goal of ≥ 90% for fast (300°/s) isokinetic extension. There was no association with slow isokinetic extension. Cartilage lesion severity at or beyond grade 2 had a similar effect on isokinetic testing results regardless of compartment involvement or performance of microfracture. Patients with grade 2-4 cartilage damage were less likely to sustain a second ipsilateral ACL injury or a contralateral native ACL injury. CONCLUSIONS: Cartilage damage seen at time of ACL reconstruction is common and associated with lower likelihood of achieving ≥ 90% symmetry for isokinetic extension strength at 6 months after surgery. However, lower recurrent ACL injury rates are seen in patients with concurrent cartilage damage. These data may inform future clinical decisions regarding operative managment of recurrent ACL injuries. LEVEL OF EVIDENCE: III.

A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate.

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Atypical Scleredema Involving the Hands in an Adolescent.

Scleredema is a rare and incompletely understood disorder characterized by progressive skin thickening and induration typically affecting the trunk, neck, and proximal upper extremities. Hand and finger involvement is exceptionally rare, having been reported in only two infection-related cases. We present an atypical case of postinfectious scleredema involving the hands and discuss scleredema as an important potential cause of hand tightness and induration in adolescents.

Faecalibacterium prausnitzii A2-165 metabolizes host- and media-derived chemicals and induces transcriptional changes in colonic epithelium in GuMI human gut microphysiological system.

Aim: Recently, a GuMI gut microphysiological system has been established to coculture oxygen-intolerant Faecalibacterium prausnitzii (F. prausnitzii) A2-165 with organoids-derived primary human colonic epithelium. This study aims to test if this GuMI system applies to different donors with different healthy states and uses metabolomics to reveal the role of gut microbes in modulating host- and diet-derived molecules in the gut lumen. Methods: Organoids-derived colonic monolayers were generated from an uninflamed region of diverticulitis, ulcerative colitis, and Crohn's disease patients and then integrated into the GuMI system to coculture with F. prausnitzii A2-165 for 2 to 4 days. Apical media was collected for metabolomic analysis. Targeted metabolomics was performed to profile 169 polar chemicals under three conditions: conventional static culture without bacteria, GuMI without bacteria, and GuMI with F. prausnitzii. The barrier function of monolayers was measured using transepithelial resistance. Results: GuMI successfully cocultured patient-derived monolayers and F. prausnitzii for up to 4 days, with active bacterial growth. Introducing flow and oxygen gradient significantly increases the barrier function, while exposure to F. prausnitzii slightly increases the barrier function. Targeted metabolomics screened 169 compounds and detected 76 metabolites, of which 70 significantly differed between at least two conditions. F. prausnitzii significantly modulates the levels of nucleosides, nucleobases, and amino acids on the apical side. Further analysis suggests that F. prausnitzii changes the mRNA level of 260 transcription factor genes in colonic epithelial cells. Conclusion: The GuMI physiomimetic system can maintain the coculture of F. prausnitzii and colonic epithelium from different donors. Together with metabolomics, we identified the modulation of F. prausnitzii in extracellular chemicals and colonic epithelial cell transcription in coculture with human colonic epithelium, which may reflect its function in gut lumen in vivo.