Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

The edited UPF1 is correlated with elevated asparagine synthetase in pancreatic ductal adenocarcinomas.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) is a malignant disorder and is the most common pancreatic cancer type. The malignant cells depend on the uptake of asparagine (Asn) for growth. The synthesis of Asn occurs through the enzyme asparagine synthetase (ASNS). Interestingly, ASNS is known as is direct target of nonsense-mediated RNA decay (NMD). We have previously reported that NMD major factor UPF1 mutations in the pancreatic tumors. However, the relationship between NMD and the level of ASNS is unknown. METHOD: We constructed point mutations by site-specific mutagenesis. To evaluate NMD magnitude, we assessed the expression ratio of an exogenously expressed wild-type and mutated ß-globin mRNA with N39 allele, and five known NMD targets. Then, reverse transcription-polymerase chain reaction (RT-PCR), RT-qPCR and western bolt to determine RNA or protein levels, after knockdown of endogenous UPF1 by small RNA interference in the cells. RESULTS: An RNA editing event (c.3101 A > G) at UPF1 transcripts resulting in an Asparagine (p.1034) changed to a Serine is found in one primary PDAC patient. The edited UPF1 increases the ability of degrading of NMD provoking transcripts, such as ß-globin mRNA with N39 allele and 5 out of 5 known endogenous NMD substrate mRNAs, including ASNS. In addition, ASNS mRNA is subjected to NMD degradation by virtue of its possessing uORFs at the 5'UTR. A reduction of endogenous ASNS RNA and the increased protein expression level is found either in the PDAC patient or in the cells with edited UPF1 at c.3101 A > G relative to the controls. CONCLUSIONS: This edited UPF1 found in the PDAC results in hyperactivated NMD, which is tightly correlation to elevated expression level of ASNS. The targeting of knockdown of ASNS may improve the antitumor potency in PDACs.

Association of allele-specific methylation of the ASNS gene with asparaginase sensitivity and prognosis in T-ALL.

Asparaginase therapy is a key component of chemotherapy for patients with T-cell acute lymphoblastic leukemia (T-ALL). Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, whereas leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Because the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite polymerase chain reaction products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from pediatric patients with T-ALL in Japanese cohorts (N = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In pediatric T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status. These observations show that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

Associations between Genetic Variants in the Vitamin D Metabolism Pathway and Severity of COVID-19 among UAE Residents.

Vitamin D has many effects on cells in the immune system. Many studies have linked low vitamin D status with severity of COVID-19. Genetic variants involved in vitamin D metabolism have been implicated as potential risk factors for severe COVID-19 outcomes. This study investigated how genetic variations in humans affected the clinical presentation of COVID-19. In total, 646 patients with SARS-CoV-2 infection were divided into two groups: noncritical COVID-19 (n = 453; 70.12%) and a critical group (n = 193; 29.87%). Genotype data on the GC, NADSYN1, VDR, and CYP2R1 genes along with data on serum 25-hydroxyvitamin D levels were compiled in patients admitted to a major hospital in the United Arab Emirates between April 2020 and January 2021. We identified 12 single-nucleotide polymorphisms associated with the critical COVID-19 condition: rs59241277, rs113574864, rs182901986, rs60349934, and rs113876500; rs4944076, rs4944997, rs4944998, rs4944979, and rs10898210; and rs11574018 and rs11574024. We report significant associations between genetic determinants of vitamin D metabolism and COVID-19 severity in the UAE population. Further research needed to clarify the mechanism of action against viral infection in vitamin D deficiency. These variants could be used with vaccination to manage the spread of SARS-CoV-2 and could be particularly valuable in populations in which vitamin D deficiency is common.

Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells.

Asparaginase depletes extracellular asparagine in the blood and is an important treatment for acute lymphoblastic leukemia (ALL) due to asparagine auxotrophy of ALL blasts. Unfortunately, resistance occurs and has been linked to expression of the enzyme asparagine synthetase (ASNS), which generates asparagine from intracellular sources. Although TP53 is the most frequently mutated gene in cancer overall, TP53 mutations are rare in ALL. However, TP53 mutation is associated with poor therapy response and occurs at higher frequency in relapsed ALL. The mutant p53-reactivating compound APR-246 (Eprenetapopt/PRIMA-1Met) is currently being tested in phase II and III clinical trials in several hematological malignancies with mutant TP53. Here we present CEllular Thermal Shift Assay (CETSA) data indicating that ASNS is a direct or indirect target of APR-246 via the active product methylene quinuclidinone (MQ). Furthermore, combination treatment with asparaginase and APR-246 resulted in synergistic growth suppression in ALL cell lines. Our results thus suggest a potential novel treatment strategy for ALL.

Oncogenic KRAS creates an aspartate metabolism signature in colorectal cancer cells.

Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that the expression of KRASG13D upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS, and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1 supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.

Epistatic interactions of genetic loci associated with age-related macular degeneration.

The currently largest genome-wide association study (GWAS) for age-related macular degeneration (AMD) defines disease association with genome-wide significance for 52 independent common and rare genetic variants across 34 chromosomal loci. Overall, these loci contain over 7200 variants and are enriched for genes with functions indicating several shared cellular processes. Still, the precise mechanisms leading to AMD pathology are largely unknown. Here, we exploit the phenomenon of epistatic interaction to identify seemingly independent AMD-associated variants that reveal joint effects on gene expression. We focus on genetic variants associated with lipid metabolism, organization of extracellular structures, and innate immunity, specifically the complement cascade. Multiple combinations of independent variants were used to generate genetic risk scores allowing gene expression in liver to be compared between low and high-risk AMD. We identified genetic variant combinations correlating significantly with expression of 26 genes, of which 19 have not been associated with AMD before. This study defines novel targets and allows prioritizing further functional work into AMD pathobiology.

Nuclear targeted Saccharomyces cerevisiae asparagine synthetases associate with the mitotic spindle regardless of their enzymatic activity.

Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes.

ERK2 Phosphorylates PFAS to Mediate Posttranslational Control of De Novo Purine Synthesis.

The RAS-ERK/MAPK (RAS-extracellular signal-regulated kinase/mitogen-activated protein kinase) pathway integrates growth-promoting signals to stimulate cell growth and proliferation, at least in part, through alterations in metabolic gene expression. However, examples of direct and rapid regulation of the metabolic pathways by the RAS-ERK pathway remain elusive. We find that physiological and oncogenic ERK signaling activation leads to acute metabolic flux stimulation through the de novo purine synthesis pathway, thereby increasing building block availability for RNA and DNA synthesis, which is required for cell growth and proliferation. We demonstrate that ERK2, but not ERK1, phosphorylates the purine synthesis enzyme PFAS (phosphoribosylformylglycinamidine synthase) at T619 in cells to stimulate de novo purine synthesis. The expression of nonphosphorylatable PFAS (T619A) decreases purine synthesis, RAS-dependent cancer cell-colony formation, and tumor growth. Thus, ERK2-mediated PFAS phosphorylation facilitates the increase in nucleic acid synthesis required for anabolic cell growth and proliferation.

High Expression of Asparagine Synthetase Is Associated with Poor Prognosis of Breast Cancer in Chinese Population.

Aims: This study aimed to determine the expression of asparagine synthetase (ASNS) in breast cancer (BC) tissues and estimate its prognostic value for BC patients. Besides, the roles of ASNS in the proliferation of BC cells were also examined in the study. Methods: Quantitative real-time PCR was conducted to detect the expression of ASNS mRNA in BC tissues and normal controls. The relationship between ASNS expression and clinical characteristics of BC patients was analyzed using χ-square test. MTT assay was performed to explore the effect of ASNS expression on the proliferation of BC cells. Kaplan-Meier curves were plotted to describe the overall survival rate of BC patients. Cox regression analyses were implemented to investigate prognostic factors. Results: ASNS mRNA overexpression was observed in BC tissues (p < 0.05). High expression of ASNS was significantly related to histological grade (p = 0.017), vascular invasion (p = 0.009), and PR status (p = 0.014). The downregulation of ASNS affected the proliferation of BC cells (p < 0.05). Kaplan-Meier survival showed that patients with high ASNS expression lived shorter than those with low expressions (p < 0.001). Finally, Cox regression analyses revealed that ASNS could act as a prognostic marker for BC patients (p < 0.001, HR = 3.293, 95% CI = 1.790-6.058). Conclusion: Taken together, ASNS is a valuable prognostic biomarker for BC patients.

Different ways to transport ammonia in human and Mycobacterium tuberculosis NAD+ synthetases.

NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans. This bifunctional enzyme couples the NAD+ synthetase and glutaminase activities through an ammonia tunnel but free ammonia is also a substrate. Here we show that the Homo sapiens NAD+ synthetase (hsNadE) lacks substrate specificity for glutamine over ammonia and displays a modest activation of the glutaminase domain compared to tbNadE. We report the crystal structures of hsNadE and NAD+ synthetase from M. tuberculosis (tbNadE) with synthetase intermediate analogues. Based on the observed exclusive arrangements of the domains and of the intra- or inter-subunit tunnels we propose a model for the inter-domain communication mechanism for the regulation of glutamine-dependent activity and NH3 transport. The structural and mechanistic comparison herein reported between hsNadE and tbNadE provides also a starting point for future efforts in the development of anti-TB drugs.

Asparagine Synthetase Is Highly Expressed at Baseline in the Pancreas Through Heightened PERK Signaling.

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure.

Erythrocyte-encapsulated asparaginase (eryaspase) combined with chemotherapy in second-line treatment of advanced pancreatic cancer: An open-label, randomized Phase IIb trial.

PURPOSE: This Phase IIb (NCT02195180) open-label study evaluated erythrocyte-encapsulated asparaginase (eryaspase) in combination with chemotherapy in second-line advanced pancreatic adenocarcinoma. METHODS: Eligible patients were randomized 2:1 to either eryaspase in combination with gemcitabine or mFOLFOX6 (eryaspase arm), or to gemcitabine or mFOLFOX6 alone (control arm). Co-primary endpoints were overall survival (OS) and progression-free survival (PFS) in patients with low asparagine synthetase (ASNS) expression. Secondary endpoints included OS and PFS in the entire population. RESULTS: 141 patients were randomized (eryaspase arm, n = 95; control arm, n = 46). Median OS and PFS in patients with low ASNS expression were 6.2 months (95% CI, 5.1-8.8) in the eryaspase arm versus 4.9 months (3.1-7.1) in the control arm (HR, 0.63; 95% CI, 0.39-1.01; P = 0.056) and 2.0 months (95% CI, 1.8-3.4) in the eryaspase arm versus 1.8 months (1.4-3.8) in the control arm (HR, 0.67; 95% CI, 0.40-1.12; P = 0.127), respectively. In the entire population, median OS and PFS for the eryaspase arm versus control were 6.0 months versus 4.4 months (HR, 0.60; P = 0.008) and 2.0 months versus 1.6 months (HR, 0.56; 95% CI, 0.37-0.84; P = 0.005), respectively. The combination of eryaspase and chemotherapy was well tolerated. The most frequent Grade 3/4 adverse events in the eryaspase arm (n = 93) were gamma-glutamyltransferase increase (16 [17.2%]), neutropenia (12 [12.9%]), and physical health deterioration (12 [12.9%]). CONCLUSION: Eryaspase in combination with chemotherapy is associated with improvements in OS and PFS, irrespective of ASNS expression in second-line advanced pancreatic adenocarcinoma. A Phase III trial is underway.

Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import.

Protein nuclear translocation is highly regulated and crucial for diverse biological processes. However, our understanding concerning protein nuclear import is incomplete. Here we report that a cellular purine synthesis enzyme inhibits protein nuclear import via deamidation. Employing human Kaposi's sarcoma-associated herpesvirus (KSHV) to probe the role of protein deamidation, we identified a purine synthesis enzyme, phosphoribosylformylglycinamidine synthetase (PFAS) that inhibits KSHV transcriptional activation. PFAS deamidates the replication transactivator (RTA), a transcription factor crucial for KSHV lytic replication. Mechanistically, deamidation of two asparagines flanking a positively charged nuclear localization signal impaired the binding of RTA to an importin ß subunit, thus diminishing RTA nuclear localization and transcriptional activation. Finally, RTA proteins of all gamma herpesviruses appear to be regulated by PFAS-mediated deamidation. These findings uncover an unexpected function of a metabolic enzyme in restricting viral replication and a key role of deamidation in regulating protein nuclear import.

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan.

Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to ß-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.

The landscape of cancer cell line metabolism.

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography-mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.

Protein interactome of the deamidase phosphoribosylformylglycinamidine synthetase (PFAS) by LC-MS/MS.

Phosphoribosylformylglycinamidine synthase (PFAS) is an essential enzyme in de novo synthesis of purine. Previously, PFAS has been reported to modulate RIG-I activation during viral infection via deamidation. In this study, we sought to identify potential substrates that PFAS can deamidate. Flag-PFAS was transfected into HEK-293T cells and PFAS associated proteins were purified with anti-Flag M2 magnetic beads. PFAS associated proteins were identified using mass spectrometry and were analyzed using bioinformatics tools including KEGG pathway analysis, gene ontology annotation, and protein interaction network analysis. A total of 441 proteins is suggested to potentially interact with PFAS. Of this number, 12 were previously identified and 429 are newly identified. The interactions of PFAS with CAD, CCT2, PRDX1, and PHGDH were confirmed by co-immunoprecipitation and western blotting. This study is first to report the interaction of PFAS with several proteins which play physiological roles in tumor development including CAD, CCT2, PRDX1, and PHGDH. Furthermore, we show here that PFAS is able to deamidate PHGDH, and induce other posttranslational modification into CAD, CCT2 and PRDX1. The present data provide insight on the biological function of PFAS. Further study to explore the role of these protein interactions in tumorigenesis and other diseases is recommended.

NAD metabolic dependency in cancer is shaped by gene amplification and enhancer remodelling.

Precision oncology hinges on linking tumour genotype with molecularly targeted drugs1; however, targeting the frequently dysregulated metabolic landscape of cancer has proven to be a major challenge2. Here we show that tissue context is the major determinant of dependence on the nicotinamide adenine dinucleotide (NAD) metabolic pathway in cancer. By analysing more than 7,000 tumours and 2,600 matched normal samples of 19 tissue types, coupled with mathematical modelling and extensive in vitro and in vivo analyses, we identify a simple and actionable set of 'rules'. If the rate-limiting enzyme of de novo NAD synthesis, NAPRT, is highly expressed in a normal tissue type, cancers that arise from that tissue will have a high frequency of NAPRT amplification and be completely and irreversibly dependent on NAPRT for survival. By contrast, tumours that arise from normal tissues that do not express NAPRT highly are entirely dependent on the NAD salvage pathway for survival. We identify the previously unknown enhancer that underlies this dependence. Amplification of NAPRT is shown to generate a pharmacologically actionable tumour cell dependence for survival. Dependence on another rate-limiting enzyme of the NAD synthesis pathway, NAMPT, as a result of enhancer remodelling is subject to resistance by NMRK1-dependent synthesis of NAD. These results identify a central role for tissue context in determining the choice of NAD biosynthetic pathway, explain the failure of NAMPT inhibitors, and pave the way for more effective treatments.