Selected Ion Monitoring for Orbitrap-Based Metabolomics.

Orbitrap mass spectrometry in full scan mode enables the simultaneous detection of hundreds of metabolites and their isotope-labeled forms. Yet, sensitivity remains limiting for many metabolites, including low-concentration species, poor ionizers, and low-fractional-abundance isotope-labeled forms in isotope-tracing studies. Here, we explore selected ion monitoring (SIM) as a means of sensitivity enhancement. The analytes of interest are enriched in the orbitrap analyzer by using the quadrupole as a mass filter to select particular ions. In tissue extracts, SIM significantly enhances the detection of ions of low intensity, as indicated by improved signal-to-noise (S/N) ratios and measurement precision. In addition, SIM improves the accuracy of isotope-ratio measurements. SIM, however, must be deployed with care, as excessive accumulation in the orbitrap of similar m/z ions can lead, via space-charge effects, to decreased performance (signal loss, mass shift, and ion coalescence). Ion accumulation can be controlled by adjusting settings including injection time and target ion quantity. Overall, we suggest using a full scan to ensure broad metabolic coverage, in tandem with SIM, for the accurate quantitation of targeted low-intensity ions, and provide methods deploying this approach to enhance metabolome coverage.

Reprogramming neuroblastoma by diet-enhanced polyamine depletion.

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers.

Acidic Methanol Treatment Facilitates Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging of Energy Metabolism.

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.

Slow TCA flux and ATP production in primary solid tumours but not metastases.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Multifunctional Nanogenerator-Integrated Metamaterial Concrete Systems for Smart Civil Infrastructure.

Creating multifunctional concrete materials with advanced functionalities and mechanical tunability is a critical step toward reimagining the traditional civil infrastructure systems. Here, the concept of nanogenerator-integrated mechanical metamaterial concrete is presented to design lightweight and mechanically tunable concrete systems with energy harvesting and sensing functionalities. The proposed metamaterial concrete systems are created via integrating the mechanical metamaterial and nano-energy-harvesting paradigms. These advanced materials are composed of reinforcement auxetic polymer lattices with snap-through buckling behavior fully embedded inside a conductive cement matrix. We rationally design their composite structures to induce contact-electrification between the layers under mechanical excitations/triggering. The conductive cement enhanced with graphite powder serves as the electrode in the proposed systems, while providing the desired mechanical performance. Experimental studies are conducted to investigate the mechanical and electrical properties of the designed prototypes. The metamaterial concrete systems are tuned to achieve up to 15% compressibility under cycling loading. The power output of the nanogenerator-integrated metamaterial concrete prototypes reaches 330 µW. Furthermore, the self-powered sensing functionality of the nanogenerator concrete systems for distributed health monitoring of large-scale concrete structures is demonstrated. The metamaterial concrete paradigm can possibly enable the design of smart civil infrastructure systems with a broad range of advanced functionalities.

NAD precursors cycle between host tissues and the gut microbiome.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.

Photoproximity Labeling of Sialylated Glycoproteins (GlycoMap) Reveals Sialylation-Dependent Regulation of Ion Transport.

Sialylation, the addition of sialic acid to glycans, is a crucial post-translational modification of proteins, contributing to neurodevelopment, oncogenesis, and immune response. In cancer, sialylation is dramatically upregulated. Yet, the functional biochemical consequences of sialylation remain mysterious. Here, we establish a µMap proximity labeling platform that utilizes metabolically inserted azidosialic acid to introduce iridium-based photocatalysts on sialylated cell-surface glycoproteins as a means to profile local microenvironments across the sialylated proteome. In comparative experiments between primary cervical cells and a cancerous cell line (HeLa), we identify key differences in both the global sialome and proximal proteins, including solute carrier proteins that regulate metabolite and ion transport. In particular, we show that cell-surface interactions between receptors trafficking ethanolamine and zinc are sialylation-dependent and impact intracellular metabolite levels. These results establish a µMap method for interrogating proteoglycan function and support a role for sialylated glycoproteins in regulating cell-surface transporters.

G6PD-mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis.

Metastasizing cancer cells are able to withstand high levels of oxidative stress through mechanisms that are poorly understood. Here, we show that under various oxidative stress conditions, pancreatic cancer cells markedly expand NADPH and NADP+ pools. This expansion is due to up-regulation of glucose-6-phosphate dehydrogenase (G6PD), which stimulates the cytoplasmic nicotinamide adenine dinucleotide kinase (NADK1) to produce NADP+ while converting NADP+ to NADPH. G6PD is activated by the transcription factor TAp73, which is, in turn, regulated by two pathways. Nuclear factor-erythroid 2 p45-related factor-2 suppresses expression of the ubiquitin ligase PIRH2, stabilizing the TAp73 protein. Checkpoint kinases 1/2 and E2F1 induce expression of the TAp73 gene. Levels of G6PD and its upstream activators are elevated in metastatic pancreatic cancer. Knocking down G6PD impedes pancreatic cancer metastasis, whereas forced G6PD expression promotes it. These findings reveal an intracellular network that maintains redox homeostasis through G6PD-mediated increase in de novo NADP+ biosynthesis, which may be co-opted by tumor cells to enable metastasis.

Impaired Lymphocyte Responses in Pediatric Sepsis Vary by Pathogen Type and are Associated with Features of Immunometabolic Dysregulation.

BACKGROUND: Sepsis is the leading cause of death in hospitalized children worldwide. Despite its hypothesized immune-mediated mechanism, targeted immunotherapy for sepsis is not available for clinical use. OBJECTIVE: To determine the association between longitudinal cytometric, proteomic, bioenergetic, and metabolomic markers of immunometabolic dysregulation and pathogen type in pediatric sepsis. METHODS: Serial peripheral blood mononuclear cell (PBMC) samples were obtained from 14 sepsis patients (34 total samples) and 7 control patients for this observational study. Flow cytometry was used to define immunophenotype, including T cell subset frequency and activation state, and assess intracellular cytokine production. Global immune dysfunction was assessed by tumor necrosis factor-α (TNF-α) production capacity and monocyte human leukocyte antigen DR (HLA-DR) expression. Mitochondrial function was assessed by bulk respirometry. Plasma cytokine levels were determined via Luminex assay. Metabolites were measured by liquid chromatography-mass spectrometry. Results were compared by timepoint and pathogen type. RESULTS: Sepsis patients were older (15.9 years vs. 10.4 years, P = 0.02) and had higher illness severity by PRISM-III (12.0 vs. 2.0, P < 0.001) compared to controls; demographics were otherwise similar, though control patients were predominately male. Compared to controls, sepsis patients at timepoint 1 demonstrated lower monocyte HLA-DR expression (75% vs. 92%, P = 0.02), loss of peripheral of non-naïve CD4+ T cells (62.4% vs. 77.6%, P = 0.04), and reduced PBMC mitochondrial spare residual capacity (SRC; 4.0 pmol/s/106 cells vs. 8.4 pmol/s/106 cells, P = 0.01). At sepsis onset, immunoparalysis (defined as TNF-α production capacity < 200 pg/mL) was present in 39% of sepsis patients and not identified among controls. Metabolomic findings in sepsis patients were most pronounced at sepsis onset and included elevated uridine and 2-dehydrogluconate and depleted citrulline. Loss of peripheral non-naïve CD4+ T cells was associated with immune dysfunction and reduced cytokine production despite increased T cell activation. CD4+ T cell differentiation and corresponding pro- and anti-inflammatory cytokines varied by pathogen. CONCLUSION: Pediatric sepsis patients exhibit a complex, dynamic physiologic state characterized by impaired T cell function and immunometabolic dysregulation which varies by pathogen type.

Ketogenic diet and chemotherapy combine to disrupt pancreatic cancer metabolism and growth.

Background: Ketogenic diet is a potential means of augmenting cancer therapy. Here, we explore ketone body metabolism and its interplay with chemotherapy in pancreatic cancer. Methods: Metabolism and therapeutic responses of murine pancreatic cancer were studied using KPC primary tumors and tumor chunk allografts. Mice on standard high-carbohydrate diet or ketogenic diet were treated with cytotoxic chemotherapy (nab-paclitaxel, gemcitabine, cisplatin). Metabolic activity was monitored with metabolomics and isotope tracing, including 2H- and 13C-tracers, liquid chromatography-mass spectrometry, and imaging mass spectrometry. Findings: Ketone bodies are unidirectionally oxidized to make NADH. This stands in contrast to the carbohydrate-derived carboxylic acids lactate and pyruvate, which rapidly interconvert, buffering NADH/NAD. In murine pancreatic tumors, ketogenic diet decreases glucose's concentration and tricarboxylic acid cycle contribution, enhances 3-hydroxybutyrate's concentration and tricarboxylic acid contribution, and modestly elevates NADH, but does not impact tumor growth. In contrast, the combination of ketogenic diet and cytotoxic chemotherapy substantially raises tumor NADH and synergistically suppresses tumor growth, tripling the survival benefits of chemotherapy alone. Chemotherapy and ketogenic diet also synergize in immune-deficient mice, although long-term growth suppression was only observed in mice with an intact immune system. Conclusions: Ketogenic diet sensitizes murine pancreatic cancer tumors to cytotoxic chemotherapy. Based on these data, we have initiated a randomized clinical trial of chemotherapy with standard versus ketogenic diet for patients with metastatic pancreatic cancer (NCT04631445).

Metabolite discovery through global annotation of untargeted metabolomics data.

Liquid chromatography-high-resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantify all metabolites, but most LC-MS peaks remain unidentified. Here we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times and (when available) tandem mass spectrometry fragmentation patterns. Peaks are connected based on mass differences reflecting adduction, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically informative peak-peak relationships, including for peaks lacking tandem mass spectrometry spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.

NAD+ flux is maintained in aged mice despite lower tissue concentrations.

NAD+ is an essential coenzyme for all living cells. NAD+ concentrations decline with age, but whether this reflects impaired production or accelerated consumption remains unclear. We employed isotope tracing and mass spectrometry to probe age-related changes in NAD+ metabolism across tissues. In aged mice, we observed modest tissue NAD+ depletion (median decrease ∼30%). Circulating NAD+ precursors were not significantly changed, and isotope tracing showed the unimpaired synthesis of nicotinamide from tryptophan. In most tissues of aged mice, turnover of the smaller tissue NAD+ pool was modestly faster such that absolute NAD+ biosynthetic flux was maintained, consistent with more active NAD+-consuming enzymes. Calorie restriction partially mitigated age-associated NAD+ decline by decreasing consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, the decline in NAD+ with normal aging is relatively subtle and occurs despite maintained NAD+ production, likely due to increased consumption.

The Source of Glycolytic Intermediates in Mammalian Tissues.

Glycolysis plays a central role in organismal metabolism, but its quantitative inputs across mammalian tissues remain unclear. Here we use 13C-tracing in mice to quantify glycolytic intermediate sources: circulating glucose, intra-tissue glycogen, and circulating gluconeogenic precursors. Circulating glucose is the main source of circulating lactate, the primary end product of tissue glycolysis. Yet circulating glucose highly labels glycolytic intermediates in only a few tissues: blood, spleen, diaphragm, and soleus muscle. Most glycolytic intermediates in the bulk of body tissue, including liver and quadriceps muscle, come instead from glycogen. Gluconeogenesis contributes less but also broadly to glycolytic intermediates, and its flux persists with physiologic feeding (but not hyperinsulinemic clamp). Instead of suppressing gluconeogenesis, feeding activates oxidation of circulating glucose and lactate to maintain glucose homeostasis. Thus, the bulk of the body slowly breaks down internally stored glycogen while select tissues rapidly catabolize circulating glucose to lactate for oxidation throughout the body.

Upregulation of Antioxidant Capacity and Nucleotide Precursor Availability Suffices for Oncogenic Transformation.

The emergence of cancer from diverse normal tissues has long been rationalized to represent a common set of fundamental processes. However, these processes are not fully defined. Here, we show that forced expression of glucose-6-phosphate dehydrogenase (G6PD) affords immortalized mouse and human cells anchorage-independent growth in vitro and tumorigenicity in animals. Mechanistically, G6PD augments the NADPH pool by stimulating NAD+ kinase-mediated NADP+ biosynthesis in addition to converting NADP+ to NADPH, bolstering antioxidant defense. G6PD also increases nucleotide precursor levels through the production of ribose and NADPH, promoting cell proliferation. Supplementation of antioxidants or nucleosides suffices to convert immortalized mouse and human cells into a tumorigenic state, and supplementation of both is required when their overlapping metabolic consequences are minimized. These results suggest that normal cells have a limited capacity for redox balance and nucleotide synthesis, and overcoming this limit might represent a key aspect of oncogenic transformation.

Bisphosphoglycerate Mutase Deficiency Protects against Cerebral Malaria and Severe Malaria-Induced Anemia.

The replication cycle and pathogenesis of the Plasmodium malarial parasite involves rapid expansion in red blood cells (RBCs), and variants of certain RBC-specific proteins protect against malaria in humans. In RBCs, bisphosphoglycerate mutase (BPGM) acts as a key allosteric regulator of hemoglobin/oxyhemoglobin. We demonstrate here that a loss-of-function mutation in the murine Bpgm (BpgmL166P) gene confers protection against both Plasmodium-induced cerebral malaria and blood-stage malaria. The malaria protection seen in BpgmL166P mutant mice is associated with reduced blood parasitemia levels, milder clinical symptoms, and increased survival. The protective effect of BpgmL166P involves a dual mechanism that enhances the host's stress erythroid response to Plasmodium-driven RBC loss and simultaneously alters the intracellular milieu of the RBCs, including increased oxyhemoglobin and reduced energy metabolism, reducing Plasmodium maturation, and replication. Overall, our study highlights the importance of BPGM as a regulator of hemoglobin/oxyhemoglobin in malaria pathogenesis and suggests a new potential malaria therapeutic target.

Quantitative Fluxomics of Circulating Metabolites.

Mammalian organs are nourished by nutrients carried by the blood circulation. These nutrients originate from diet and internal stores, and can undergo various interconversions before their eventual use as tissue fuel. Here we develop isotope tracing, mass spectrometry, and mathematical analysis methods to determine the direct sources of circulating nutrients, their interconversion rates, and eventual tissue-specific contributions to TCA cycle metabolism. Experiments with fifteen nutrient tracers enabled extensive accounting for both circulatory metabolic cycles and tissue TCA inputs, across fed and fasted mice on either high-carbohydrate or ketogenic diet. We find that a majority of circulating carbon flux is carried by two major cycles: glucose-lactate and triglyceride-glycerol-fatty acid. Futile cycling through these pathways is prominent when dietary content of the associated nutrients is low, rendering internal metabolic activity robust to food choice. The presented in vivo flux quantification methods are broadly applicable to different physiological and disease states.

Metabolic excretion associated with nutrient-growth dysregulation promotes the rapid evolution of an overt metabolic defect.

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine-auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A subpopulation repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly reduced glutathione (GSH) and GSH conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program, including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.

Improved Annotation of Untargeted Metabolomics Data through Buffer Modifications That Shift Adduct Mass and Intensity.

Annotation of untargeted high-resolution full-scan LC-MS metabolomics data remains challenging due to individual metabolites generating multiple LC-MS peaks arising from isotopes, adducts, and fragments. Adduct annotation is a particular challenge, as the same mass difference between peaks can arise from adduct formation, fragmentation, or different biological species. To address this, here we describe a buffer modification workflow (BMW) in which the same sample is run by LC-MS in both liquid chromatography solvent with 14NH3-acetate buffer and in solvent with the buffer modified with 15NH3-formate. Buffer switching results in characteristic mass and signal intensity changes for adduct peaks, facilitating their annotation. This relatively simple and convenient chromatography modification annotated yeast metabolomics data with similar effectiveness to growing the yeast in isotope-labeled media. Application to mouse liver data annotated both known metabolite and known adduct peaks with 95% accuracy. Overall, it identified 26% of ∼27 000 liver LC-MS features as putative metabolites, of which ∼2600 showed HMDB or KEGG database formula match. This workflow is well suited to biological samples that cannot be readily isotope labeled, including plants, mammalian tissues, and tumors.

mTORC1 restrains adipocyte lipolysis to prevent systemic hyperlipidemia.

OBJECTIVE: Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents and can extend the lifespan of model organisms. An undesirable side effect of these drugs is hyperlipidemia. Although multiple roles have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo. METHODS: We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit Raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in their adipocytes. RESULTS: Mice lacking mTORC1 activity in their adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in their adipose tissue restored normal levels of triglycerides and cholesterol in the fed state as well as the ability to clear triglycerides in an oral fat tolerance test. CONCLUSIONS: Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis.

Elevated Choline Kinase α-Mediated Choline Metabolism Supports the Prolonged Survival of TRAF3-Deficient B Lymphocytes.

Specific deletion of the tumor suppressor TRAF3 from B lymphocytes in mice leads to the prolonged survival of mature B cells and expanded B cell compartments in secondary lymphoid organs. In the current study, we investigated the metabolic basis of TRAF3-mediated regulation of B cell survival by employing metabolomic, lipidomic, and transcriptomic analyses. We compared the polar metabolites, lipids, and metabolic enzymes of resting splenic B cells purified from young adult B cell-specific Traf3 -/- and littermate control mice. We found that multiple metabolites, lipids, and enzymes regulated by TRAF3 in B cells are clustered in the choline metabolic pathway. Using stable isotope labeling, we demonstrated that phosphocholine and phosphatidylcholine biosynthesis was markedly elevated in Traf3 -/- mouse B cells and decreased in TRAF3-reconstituted human multiple myeloma cells. Furthermore, pharmacological inhibition of choline kinase α, an enzyme that catalyzes phosphocholine synthesis and was strikingly increased in Traf3 -/- B cells, substantially reversed the survival phenotype of Traf3 -/- B cells both in vitro and in vivo. Taken together, our results indicate that enhanced phosphocholine and phosphatidylcholine synthesis supports the prolonged survival of Traf3 -/- B lymphocytes. Our findings suggest that TRAF3-regulated choline metabolism has diagnostic and therapeutic value for B cell malignancies with TRAF3 deletions or relevant mutations.