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Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of developing carcinomas arising in squamous mucosal epithelia lining the mouth, proximal esophagus, vulva and anus. The origin of these cancers is not understood, and no effective way has been identified to prevent or delay their appearance. FA-associated carcinomas are also therapeutically challenging, as they may be multi-focal and stage-advanced at diagnosis making surgical control challenging. Moreover, individuals with FA have systemic DNA damage hypersensitivity and thus an elevated risk of toxicity when treated with standard-of-care therapies such as DNA cross-linking drugs and ionizing radiation.We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) in order to foster new research on the origins, treatment, and prevention of FA-associated cancers. The FA-CCLR consists of FANC-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs from cancers arising in individuals with FA, or newly engineered from sporadic HNSCC cell lines. Molecular, cellular, and biochemical analyses were used to demonstrate the causal dependence of key FA-associated phenotypes on FANC genotype, expression and pathway activity. These FANC-isogenic cell line pairs are available to academic and non-profit investigators, with ordering information available at the ‘Fanconi Anemia Research Materials’ Resource and Repository at Oregon Health & Sciences University, Portland OR.SignificanceWe have generated new isogenic cancer cell line models to investigate the origins, treatment and prevention of Fanconi anemia-associated squamous carcinomas that target the oral mucosa, proximal esophagus, and anogenital region.

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression ofFoxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymusin vivofollowing acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration.SUMMARYThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration.Damage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes.Elevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage.Extracellular ATP release promotesFoxn1expression in cTECs via activation of P2Y2Therapeutic targeting of the P2Y2 receptor promotes thymic regeneration.

Metabolic disruption is a mainstay of cancer therapy, prompting research aimed at identifying novel metabolic targets. Despite strong effects observed in culture, three recent studies found pancreatic tumors are refractory to disruption of the metabolic enzyme GOT2, revealing complex interactions within the tumor microenvironment that bypass its conventional metabolic roles.

Metabolic pathway regulates cell fate Lineage-specific regulators direct cell fate decisions, but the precise mechanisms are not well known. Using an in vivo chemical suppressor screen of a bloodless zebrafish mutant, Rossmann et al. show that the lineage-specific chromatin factor tif1γ directly regulates mitochondrial genes to drive red blood cell differentiation. Loss of tif1γ reduces coenzyme Q synthesis and function, impeding mitochondrial respiration and leading to epigenetic alterations and repression of erythropoiesis. The loss of blood in the mutant fish can be rescued by the addition of coenzyme Q. This work establishes a mechanism by which a chromatin factor tunes a metabolic pathway in a tissue-specific manner. Science , this issue p. 716

Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can be a proliferation bottleneck that is influenced by environmental conditions. However, a comprehensive quantitative understanding of metabolic processes that may be affected by NAD+ deficiency is currently missing. Here, we use computational and experimental approaches to demonstrate that in addition to requiring oxygen for lipid desaturation reactions, de novo lipid biosynthesis can impose a substantial NAD+ consumption cost in proliferating cancer cells. Our results reveal that when electron acceptors are limited environmental lipids become crucial for proliferation because NAD+ is required to generate precursors for fatty acid biosynthesis. We find that both oxidative and even net reductive pathways for lipogenic citrate synthesis are gated by reactions that depend on NAD+ availability. We also show that access to acetate can relieve lipid auxotrophy by bypassing the NAD+ consuming reactions involved in de novo lipid synthesis. Consistent with biochemical results, analysis of gene expression across diverse tumor types demonstrates that lipid biosynthesis strongly anticorrelates with expression of hypoxia markers, whereas lipid uptake genes are consistently upregulated in hypoxia. Overall, our results define a requirement for oxidative metabolism to support de novo lipid biosynthesis and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptors limited conditions such as hypoxia.

The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC). Mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports cell proliferation through aspartate synthesis but, unlike other ETC impairments, is not restored by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context are dependent on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. Collectively, these data identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal that compartmentalized redox changes can impact cellular fitness.

Lactate dehydrogenase (LDH) accounts for the fermentative component of aerobic glycolysis, a near ubiquitous metabolic alteration in cancer. Recently, Oshima et al. developed a bioavailable LDH inhibitor that decreases tumor growth in mice and functions synergistically with mitochondrial respiration inhibitors. These findings suggest a cooperative mechanism of action that targets redox homeostasis.

Many metabolic phenotypes in cancer cells are also characteristic of proliferating non-transformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate, and provide a model to distinguish the metabolic influences of oncogenic mutations and non-oncogenic growth.

Skeletal muscle has long been recognized as an inhospitable site for disseminated tumour cells (DTCs). Yet its antimetastatic nature has eluded a thorough mechanistic examination. Here, we show that DTCs traffic to and persist within skeletal muscle in mice and in humans, which raises the question of how this tissue suppresses colonization. Results from mouse and organotypic culture models along with metabolomic profiling suggested that skeletal muscle imposes a sustained oxidative stress on DTCs that impairs their proliferation. Functional studies demonstrated that disrupting reduction-oxidation homeostasis via chemogenetic induction of reactive oxygen species slowed proliferation in a more fertile organ: the lung. Conversely, enhancement of the antioxidant potential of tumour cells through ectopic expression of catalase in the tumour or host mitochondria allowed robust colonization of skeletal muscle. These findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by skeletal muscle. A thorough understanding of this biology could reveal previously undocumented DTC vulnerabilities that can be exploited to prevent metastasis in other more susceptible tissues.

The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of TCA cycle products for cell viability and proliferation, mammalian cells display diversity in TCA cycle activity(1,2). How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially-derived citrate exported to the cytoplasm is metabolized via ATP citrate lyase (ACL), ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating expression of ACL or the canonical TCA cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells (ESCs) revealed that changes in TCA cycle configuration accompany cell fate transitions. During exit from pluripotency, ESCs switch from canonical to non-canonical TCA cycle metabolism; accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA cycle engagement is required for changes in cell state.

Colorectal cancer (CRC) is driven by mutations that activate canonical WNT/β-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a β-catenin independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to CRC because these can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or β-catenin mutations) of GSK3, thus allowing WNT/β-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or β-catenin mutant CRC, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or β-catenin mutant CRC, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for CRC therapy.

Production of oxidized biomass, which requires regeneration of the cofactor NAD

It is not well understood how physiological environmental conditions and nutrient availability influence cancer cell proliferation. Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can limit cancer cell proliferation1-5. However, it is currently unclear which specific metabolic processes are constrained by electron acceptor availability, and how they affect cell proliferation. Here, we use computational and experimental approaches to demonstrate that de novo lipid biosynthesis can impose an increased demand for NAD+ in proliferating cancer cells. While some cancer cells and tumors synthesize a substantial fraction of their lipids de novo6, we find that environmental lipids are crucial for proliferation in hypoxia or when the mitochondrial electron transport chain is inhibited. Surprisingly, we also find that even the reductive glutamine carboxylation pathway to produce fatty acids is impaired when cancer cells are limited for NAD+. Furthermore, gene expression analysis of 34 heterogeneous tumor types shows that lipid biosynthesis is strongly and consistently negatively correlated with hypoxia, whereas expression of genes involved in lipid uptake is positively correlated with hypoxia. These results demonstrate that electron acceptor availability and access to environmental lipids can play an important role in determining whether cancer cells engage in de novo lipogenesis to support proliferation.

The development of effective antifungal therapeutics remains a formidable challenge because of the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability because of its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin that kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. Using genetic, biochemical, and metabolomic approaches, we established ML316 as the first Mir1 inhibitor. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption, resulting in an unusual metabolic catastrophe marked by citrate accumulation and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much-needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.

Altered cell metabolism is ubiquitous in cancer cells; however, it remains challenging to exploit these alterations for cancer therapy. A new study reveals that metabolic alterations to the urea cycle promote tumor growth but unexpectedly also trigger mutations that mark cancer cells for recognition by immunotherapy.

Small cell lung cancer (SCLC) is a highly aggressive and lethal neoplasm. To identify candidate tumor suppressors we applied CRISPR-Cas9 gene inactivation screens to a cellular model of early-stage SCLC. Among the top hits was MAX, the obligate heterodimerization partner for MYC family proteins that is mutated in human SCLC. Max deletion increases growth and transformation in cells and dramatically accelerates SCLC progression in an Rb1/Trp53-deleted mouse model. In contrast, deletion of Max abrogates tumorigenesis in MYCL-overexpressing SCLC. Max deletion in SCLC resulted in de-repression of metabolic genes involved in serine and one carbon metabolism. By increasing serine biosynthesis, Max deleted cells exhibit resistance to serine depletion. Thus, Max loss results in metabolic rewiring and context-specific tumor suppression.

Cultured cells convert glucose to lactate, and glutamine is the major source of tricarboxylic acid (TCA)-cycle carbon, but whether the same metabolic phenotype is found in tumors is less studied. We infused mice with lung cancers with isotope-labeled glucose or glutamine and compared the fate of these nutrients in tumor and normal tissue. As expected, lung tumors exhibit increased lactate production from glucose. However, glutamine utilization by both lung tumors and normal lung was minimal, with lung tumors showing increased glucose contribution to the TCA cycle relative to normal lung tissue. Deletion of enzymes involved in glucose oxidation demonstrates that glucose carbon contribution to the TCA cycle is required for tumor formation. These data suggest that understanding nutrient utilization by tumors can predict metabolic dependencies of cancers in vivo. Furthermore, these data argue that the in vivo environment is an important determinant of the metabolic phenotype of cancer cells., National Science Foundation (U.S.) (Grant T32GM007287)

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is a hallmark of proliferative metabolism that is observed across many organisms and conditions. To better understand why aerobic glycolysis is associated with cell proliferation, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase mitochondrial pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the nicotinamide adenine dinucleotide cofactor ratio (NAD+/NADH). This change in NAD+/NADH ratio is caused by an increase in mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling mitochondrial 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 the demand for NAD+ to support oxidation reactions exceeds the demand for ATP consumption in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation despite available oxygen. This argues that cells engage in aerobic glycolysis when the cellular demand for NAD+ is in excess of the cellular demand for ATP.

Cells utilize diverse metabolic strategies to support cell type-specific bioenergetic requirements. The enzymes that mediate cellular metabolic processes have been well studied. However, how cells coordinate these factors into metabolic networks remains poorly understood. Here we use measurements of gene dependency across hundreds of cancer cell lines to cluster metabolic genes into co-dependent modules. These analyses identified two distinct tricarboxylic acid (TCA) cycle modules with specific genetic dependencies. Isotope tracing combined with genetic manipulation of TCA cycle modules revealed that both normal stem cells and cancer cells can engage two different modes of TCA cycle metabolism. Enforcing self-renewal or differentiation induced preferential use of one TCA cycle module over another, indicating that TCA cycle dynamics are determined by cell state. In turn, the mode of TCA cycle metabolism employed by cells dictated nutrient dependencies, enabling cell-type specific responses to nutrient deprivation. Collectively, these studies reveal that TCA cycle behavior is dynamic and can be rerouted to confer a selective advantage to cells under different nutrient conditions. Citation Format: Paige K. Arnold, Benjamin T. Jackson, Sydney Alibeckoff, Esther Drill, Lucas B. Sullivan, Lydia W.S. Finley. Cell identity determines TCA cycle dynamics [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-021.

Understanding in-vivo mechanisms of erythropoiesis is critical for directed differentiation approaches to treat blood disorders such as leukemias. Zebrafish moonshine (mon) mutant embryos defective for the conserved transcriptional intermediary factor 1 gamma (tif1γ) do not specify enough erythroid progenitors due to a transcription elongation block characterized by aberrantly paused RNA polymerase II. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we performed a chemical suppressor screen using 3,100 compounds and identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH). Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar rescue the formation of erythroid progenitors in 61% (38/62) and 68% (50/74) of mon embryos, respectively. Beyond changes in nucleotide metabolism, in-vivo metabolic analyses revealed low levels of TCA cycle metabolites which were functionally linked to a reduced oxygen consumption rate. In addition, an increased 2HG/αKG ratio was associated with higher histone methylation states at H3K27, H3K36 and H4K20 as assessed by quantitative targeted mass spectrometry, which may contribute to the erythroid differentiation block upon tif1γ loss. DHODH is the only pyrimidine de novo synthesis enzyme located in mitochondria and its activity is coupled to that of the electron transport chain (ETC) via coenzyme Q (CoQ). Rotenone, a potent ETC complex I inhibitor reversed the rescue by DHODH inhibition in mon embryos. Through parallel genome-wide transcriptome and chromatin immunoprecipitation analyses, we found that genes encoding CoQ metabolic enzymes are direct TIF1γ targets. Treatment with the CoQ analog decylubiquinone rescued erythroid progenitors in 26% (33/126) of mon embryos. These results demonstrate a tight coordination of nucleotide and mitochondrial metabolism as a key function of tif1γ-dependent transcription and reveal that TIF1γ activity regulates a metabolic program that drives cell fate decisions in the early blood lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases. Citation Format: Marlies P. Rossmann, Karen Hoi, Victoria Chan, Brian J. Abraham, Song Yang, James Mullahoo, Malvina Papanastasiou, Ilaria Elia, Sejal Vyas, Partha P. Nag, Lucas B. Sullivan, Julie R. Perlin, Elliott J. Hagedorn, Sara Hetzel, Raha Weigert, Curtis R. Warren, Bilguujin Dorjsuren, Eugenia Custo Greig, Chad A. Cowan, Stuart L. Schreiber, Richard A. Young, Alexander Meissner, Marcia Haigis, Steven A. Carr, Leonard I. Zon. Transcriptional regulation of mitochondrial metabolism by TIF1γ drives erythroid progenitor differentiation [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR04.

Understanding in-vivo mechanisms of erythropoiesis is critical for directed differentiation approaches to treat blood disorders. Zebrafish moonshine (mon) mutant embryos defective for the conserved transcriptional intermediary factor 1 gamma (tif1γ) do not specify enough erythroid progenitors. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we performed a chemical suppressor screen using 3,100 compounds and identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH). Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar rescue the formation of erythroid progenitors in 61% (38/62) and 68% (50/74) of mon embryos, respectively. Beyond changes in nucleotide metabolism, in-vivo metabolic analyses revealed low levels of TCA cycle metabolites which were functionally linked to a reduced oxygen consumption rate. In addition, an increased 2HG/αKG ratio was associated with higher histone methylation states at H3K27, H3K36 and H4K20 as assessed by quantitative targeted mass spectrometry, which may contribute to the erythroid differentiation block upon tif1γ loss. DHODH is the only pyrimidine de novo synthesis enzyme located in mitochondria and its activity is coupled to that of the electron transport chain (ETC) via coenzyme Q (CoQ). Rotenone, a potent ETC complex I inhibitor reversed the rescue by DHODH inhibition in mon embryos. Through parallel genome-wide transcriptome and chromatin immunoprecipitation analyses, we found that genes encoding CoQ metabolic enzymes are direct TIF1γ targets. Treatment with the CoQ analog decylubiquinone rescued erythroid progenitors in 26% (33/126) of mon embryos. Our work highlights the importance of transcription regulatory processes for tuning metabolism during lineage differentiation and could have therapeutic implications.

SummaryMitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

Defining the metabolic limitations of tumour growth will help to develop cancer therapies1. Cancer cells proliferate slower in tumours than in standard culture conditions, indicating that a metabolic limitation may restrict cell proliferation in vivo. Aspartate synthesis can limit cancer cell proliferation when respiration is impaired2–4; however, whether acquiring aspartate is endogenously limiting for tumour growth is unknown. We confirm that aspartate has poor cell permeability, which prevents environmental acquisition, whereas the related amino acid asparagine is available to cells in tumours, but cancer cells lack asparaginase activity to convert asparagine to aspartate. Heterologous expression of guinea pig asparaginase 1 (gpASNase1), an enzyme that produces aspartate from asparagine5, confers the ability to use asparagine to supply intracellular aspartate to cancer cells in vivo. Tumours expressing gpASNase1 grow at a faster rate, indicating that aspartate acquisition is an endogenous metabolic limitation for the growth of some tumours. Tumours expressing gpASNase1 are also refractory to the growth suppressive effects of metformin, suggesting that metformin inhibits tumour growth by depleting aspartate. These findings suggest that therapeutic aspartate suppression could be effective to treat cancer.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. While it has been shown that cells can traffic metabolic enzymes via EVs much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Both our metabolomics and functional analyses revealed that EVs harbour L-asparaginase activity catalysed by the enzyme Asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC-derived EVs traffic ASRGL1. Our results demonstrate for the first time that NSC EVs function as independent, extracellular metabolic units able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Understanding the cell-autonomous as well as niche contributions governing erythropoiesis is critical for directed differentiation approaches of hematopoietic stem cells into differentiated red blood cells (RBCs) to treat blood disorders such as anemias and leukemias. Transcriptional intermediary factor 1 gamma (TIF1γ) is essential for erythropoiesis from zebrafish to mammals. Zebrafish moonshine mutant embryos defective for tif1γ do not make red blood cells (RBCs) due to a transcription elongation block characterized by aberrantly paused RNA polymerase II. Loss of factors involved in transcription elongation control, PAF1 and spt5, rescues the moonshine RBC defect. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we have performed a high-content chemical suppressor screen in the bloodless moonshine mutant using 3,500 compounds. Among the suppressors, we identified leflunomide, an inhibitor of dihydroorotate dehydrogenase (DHODH), an essential enzyme for de novo pyrimidine synthesis. Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar both rescue the formation of primitive erythroid cells in 61% (38/62) and 68% (50/74) of moonshine embryos, respectively. Blastula transplant experiments revealed that tif1γ, in addition to its cell-autonomous role, plays a role in the hematopoietic niche for RBC development. Through in-vivo metabolomics analyses we have identified nucleotide metabolism as the most significantly altered process in moonshine mutants, including elevated levels of uridine monophosphate and low levels of nicotinamide adenine dinucleotide (NAD+). Low NAD+ levels are accompanied by a reduced oxygen consumption rate in tif1γ-depleted embryos by Seahorse analysis. In support, genome-wide transcriptome analysis coupled with chromatin immunoprecipitation studies revealed genes encoding coenzyme Q (CoQ) metabolic enzymes as direct TIF1γ targets. DHODH is the only enzyme of the pyrimidine de novo synthesis pathway located on the inner mitochondrial membrane and its activity is coupled to that of the electron transport chain (ETC). Rotenone, a potent ETC complex I inhibitor reverses the rescue of the blood defect by DHODH inhibition in moonshine embryos. Since DHODH function is linked to mitochondrial oxidative phosphorylation via CoQ activity, we asked whether alterations in mitochondrial metabolism might be causal for the RBC defect in moonshine mutants. Indeed, treatment with the CoQ analog decylubiquinone results in rescue of βe3 globin expression in 26% (33/126) of moonshine embryos. These results demonstrate a tight coordination of nucleotide and mitochondrial metabolism as a key function of tif1γ-dependent transcription and reveal that TIF1γ activity regulates a metabolic program that drives cell fate decisions in the early blood lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases and consequences for erythroid differentiation protocols. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

Altered cell metabolism is a characteristic feature of many cancers. Aside from well-described changes in nutrient consumption and waste excretion, altered cancer cell metabolism also results in changes to intracellular metabolite concentrations. Increased levels of metabolites that result directly from genetic mutations and cancer-associated modifications in protein expression can promote cancer initiation and progression. Changes in the levels of specific metabolites, such as 2-hydroxyglutarate, fumarate, succinate, aspartate and reactive oxygen species, can result in altered cell signalling, enzyme activity and/or metabolic flux. In this Review, we discuss the mechanisms that lead to changes in metabolite concentrations in cancer cells, the consequences of these changes for the cells and how they might be exploited to improve cancer therapy.

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use 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 suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Extracellular vesicles (EVs) are a class of secreted membrane particles capable of transferring biological molecules between cells. Metabolomics measurements indicate that isolated EVs also have autonomous metabolic enzyme activities, including the unexpected identification of endogenous human asparaginase activity.

Changes to the biophysical properties of lymphocytes are identified as an adaptive response to acute nutrient stress that occurs before the induction of autophagy., Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-xL expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-xL cells depleted of IL-3 and primary CD8+ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.

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.

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting., Broad Institute of MIT and Harvard. SPARC Program, Burroughs Wellcome Fund

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Keywords: cancer metabolism; metformin; biguanide; NAD+/NADH ratio; drug sensitivity; complex I; mitochondria; aspartate, National Institutes of Health (U.S.) (Grant P30CA1405141), National Institutes of Health (U.S.) (Grant GG006413), National Institutes of Health (U.S.) (Grant R01 CA168653), National Institutes of Health (U.S.) (Grant R01 CA201276)

Mitochondria produce reactive oxygen species (mROS) as a natural by-product of electron transport chain activity. While initial studies focused on the damaging effects of reactive oxygen species, a recent paradigm shift has shown that mROS can act as signaling molecules to activate pro-growth responses. Cancer cells have long been observed to have increased production of ROS relative to normal cells, although the implications of this increase were not always clear. This is especially interesting considering cancer cells often also induce expression of antioxidant proteins. Here, we discuss how cancer-associated mutations and microenvironments can increase production of mROS, which can lead to activation of tumorigenic signaling and metabolic reprogramming. This tumorigenic signaling also increases expression of antioxidant proteins to balance the high production of ROS to maintain redox homeostasis. We also discuss how cancer-specific modifications to ROS and antioxidants may be targeted for therapy.

Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I. DOI: http://dx.doi.org/10.7554/eLife.02242.001, eLife digest Metformin is widely used to reduce the high blood sugar levels caused by diabetes. Recently, several studies have suggested that patients taking metformin who also develop cancer have tumors that grow more slowly than average. As clinical trials have already started to investigate if metformin is an effective anti-cancer treatment, it is important to understand how it might restrict tumor growth. Researchers have proposed two ways that metformin could affect tumors. First, insulin is known to prompt cancer cells to divide, so the slower rate of tumor growth could just be a side-effect of the metformin reducing the amount of insulin in the blood. Alternatively, metformin could target cancer cells more directly by cutting the energy supply produced by their mitochondria. Metformin has been shown to disrupt complex I of the electron transport chain that is used by cells to generate energy. However, it is not known if disrupting complex I would actually stop cells dividing because they can generate energy in other ways. Wheaton, Weinberg et al. have now demonstrated that metformin does target complex I in cancer cells, and that its effects depend on the amount of glucose available for cells to convert, without involving mitochondria, into energy. When there is plenty of glucose, metformin slows down the rate at which cancer cells divide, which slows down tumor growth. When the cells are deprived of glucose, metformin kills the cells instead. Metformin also inhibits the pathways that regulate hypoxia inducible factors (HIFs), which are part of a system that helps cells to survive low-oxygen conditions, a prominent feature of many tumors. This means that metformin may combat cancer more effectively if used alongside other treatments that reduce the availability of both oxygen and glucose inside cells. Metformin could also potentially treat conditions that are linked to overactive HIFs, such as pulmonary hypertension. DOI: http://dx.doi.org/10.7554/eLife.02242.002

The tricarboxylic acid cycle enzyme fumarate hydratase (FH) has been identified as a tumor suppressor in a subset of human renal cell carcinomas. Human FH-deficient cancer cells display high fumarate concentration and ROS levels along with activation of HIF-1. The underlying mechanisms by which FH loss increases ROS and HIF-1 are not fully understood. Here, we report that glutamine-dependent oxidative citric acid cycle metabolism is required to generate fumarate and increase ROS and HIF-1 levels. Accumulated fumarate directly bonds the antioxidant glutathione in vitro and in vivo to produce the metabolite succinated glutathione (GSF). GSF acts as an alternative substrate to glutathione reductase to decrease NADPH levels and enhance mitochondrial ROS and HIF-1 activation. Increased ROS also correlates with hypermethylation of histones in these cells. Thus, fumarate serves as a proto-oncometabolite by binding to glutathione which results in the accumulation of ROS.

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.

Normal cells and cancer cells rely on mitochondria for a range of essential functions including production of ATP, biosynthetic intermediates, heme and iron-sulfur clusters, and reactive oxygen species (ROS) for signaling. These functions are essential for cell survival and proliferation. Most cancer cells have functional mitochondria to fulfill these essential requirements; however, there is a subset of cancers which derive from a loss of function in tricarboxylic acid (TCA) cycle enzymes, including fumarate hydratase (FH).1 Cancer cells with loss of FH function are respiratory-deficient and accumulate high levels of fumarate. This raises an intriguing question: how do cancer cells with dysfunctional mitochondria fulfill their requirements for cell proliferation? The isolation of FH-deficient renal cancer cells (UOK262) has allowed examination of how loss of FH impacts mitochondrial metabolism and cell proliferation.2 Loss of FH causes mitochondrial dysfunction; however, the essential cellular processes that typically occur in the mitochondria are maintained. ATP is maintained by increasing glycolysis. The mitochondrial TCA cycle generates intermediates that are required for biosynthesis reactions, such as citrate for fatty acid synthesis. Normal cells produce citrate by oxidative condensation of oxaloacetate and glycolytic acetyl-CoA; however, UOK262 cells do not produce acetyl-CoA from glycolysis. In UOK262 cells, glutamine-derived mitochondrial 2-oxoglutarate is metabolized in the reverse direction of the canonical TCA cycle to produce citrate by reductive carboxylation.3 Thus, cells are capable of generating the TCA cycle intermediates required for proliferation, whether it is by oxidative mitochondrial metabolism in normal cells or reductive mitochondrial metabolism in FH mutant cells. A byproduct of oxidative metabolism is the generation of ROS by the electron transport chain. Increased ROS at low nM levels in cancer cells promotes signaling pathways to activate HIFs for metabolic adaptation, NFκB for cell survival, and MAP kinases for cell proliferation.4 However, ROS at higher levels can oxidize lipids, proteins, or DNA to promote damage or mutagenesis. To exploit ROS signaling, cancer cells both increase ROS to promote tumorigenic signaling and also increase antioxidant proteins and metabolites to keep ROS at levels compatible with growth. Antioxidants such as glutathione are oxidized by ROS and restored by consumption of NADPH by antioxidant enzymes. ROS levels can be increased by either increased generation of ROS or by decreased supply of antioxidants. How FH-null cells that engage in reductive metabolism increase ROS to promote pro-tumorigenic pathways is not fully understood. Recently we reported the production and accumulation of fumarate in FH-deficient cells is required to increase ROS levels.5 Accumulated fumarate undergoes a covalent reaction with intracellular glutathione (GSH) to produce a novel metabolite, succinated glutathione (GSF). Surprisingly, GSF serves as a substrate for glutathione reductase. Normally, glutathione reductase reduces oxidized glutathione (GSSG) to GSH by coupling it to NADPH oxidation to NADP+. An in vitro mixture of glutathione reductase, NADPH, and GSF showed rapid depletion of NADPH and production of glutathione. Thus, glutathione reductase uses NADPH to split GSF into glutathione and, likely, succinate. Decreased levels of NADPH will result in less antioxidant activity and increased ROS levels. Indeed, FH-null cells show decreased NADPH and increased ROS compared with FH reconstituted cells. Also, treatment with GSF caused depletion of NADPH and enhanced endogenous ROS. At first glance, it would seem the accumulation of fumarate binding to glutathione would deplete glutathione levels, raising ROS to excessively high levels, resulting in cell death or suppression of proliferation. However, previous reports indicated that fumarate can cause the post-translational modification succination on KEAP1, the negative regulator of the master antioxidant transcription factor NRF2.6,7 Succination of KEAP1 allows NRF2 to be active and stimulate antioxidant genes. Thus, NRF2 is activated to mitigate increased ROS of FH-null cells. Indeed, we observed that RNAi of NRF2 in FH-deficient tumor cells caused excessively high levels of ROS, resulting in suppression of proliferation. A consequence of this increased ROS production in FH-deficient cancer cells was the activation of HIF-1 and hyper-methylation of histones, regulated by inhibition of the 2-oxoglutarate dioxygenases prolyl hydroxylases and Jumonji-domain-containing histone demethylases, respectively.8 Fumarate is known to be a competitive inhibitor with 2-oxoglutrate for these dioxygenases. However, our data suggest, in addition to fumarate, these dioxygenases might be susceptible to inhibition through an unknown ROS-dependent mechanism. Based on our observations and previous research on ROS, we propose that loss of FH is advantageous for tumor cells, because it allows the accumulation of fumarate, which ultimately triggers an increase in ROS levels. The accumulation of fumarate and ROS can impinge on variety of pathways that control tumorigenesis (Fig. 1). Figure 1. FH mutant cancer cells maintain proliferation requirements supplied by the mitochondria. Normal cells utilize oxidative metabolism to supply metabolites for biosynthesis, ATP for energy, and ROS for signaling. FH-null cells supply these ... The role of the mitochondria in cancer cells has been contentious for decades. Otto Warburg’s initial hypothesis was that mitochondrial impairment was a prerequisite for transformation. Thus, many investigators have assumed that most cancer cells have impaired or dampened mitochondrial function. However, cancer cells can exhibit increased, decreased, or unchanged mitochondrial metabolism compared with normal cells. Our data indicate that mitochondria provide essential functions for cell proliferation even if they harbor mutations that impair their oxidative metabolism. It will be of interest in future studies to determine whether mitochondrial metabolism is an attractive target for cancer therapy.