[Clinical and genetic spectrum of 6 cases with asparagine synthetase deficiency].

Objective: To explore the clinical and genetic characteristics of asparagine synthase deficiency. Methods: Case series studies. Retrospective analysis and summary of the clinical data of 6 cases with asparagine synthase deficiency who were diagnosed by genetic testing and admitted to the Third Affiliated Hospital of Zhengzhou University from May 2017 to April 2023 were analyzed retrospectively. The main clinical features, laboratory and imaging examination characteristics of the 6 cases were summarized, and the gene variation sites of them were analyzed. Results: All of the 6 cases were male, with onset ages ranging from 1 month to 1 year and 4 months. All of the 6 cases had cognitive and motor developmental delay, with 3 cases starting with developmental delay, 3 cases starting with convulsions and later experiencing developmental arrest or even regression. All of 6 cases had epilepsy, in whom 2 cases with severe microcephaly developed epileptic encephalopathy in the early stages of infancy with spasms as the main form of convulsions, 4 cases with mild or no microcephaly gradually evolved into convulsions with no fever after multiple febrile convulsions with focal seizures, tonic clonic seizures and tonic seizure as the main forms of convulsions. Three cases of 4 gradually developed into stagnation or even regression of development and ataxia after multiple convulsions with no fever. There were normal cranial imaging in 2 cases, dysplasia of the brains in 1 cases, frontal lobe apex accompanied by abnormal white matter signal in the frontal lobe and thin corpus callosum in 1 case, thin corpus callosum and abnormal lateral ventricular morphology in 1 case, and normal in early stage, but gradually developing into cerebellar atrophy at the age of 5 years and 9 months in 1 case. Two cases underwent visual evoked potential tests, the results of which were both abnormal. Three cases underwent auditory evoked potential examination, with 1 being normal and 2 being abnormal. All of 6 cases had variations in the asparagine synthase gene, with 2 deletion variations and 7 missense variations. The variations of 2 cases had not been reported so far, including c.1341_1343del and c.1283A>G, c.1165_1167del and c.1075G>A. The follow-up time ranged from 3 months to 53 months. Two cases who had severe microcephaly died in infancy, while the other 4 cases with mild or no microcephaly were in survival states until the follow-up days but the control of epilepsy was poor. Conclusions: Asparagine synthase deficiency has a certain degree of heterogeneity in clinical phenotype. Children with obvious microcephaly often present as severe cases, while children with mild or no microcephaly have relatively mild clinical manifestations. The variation of asparagine synthetase gene is mainly missense variation.

Bisabosqual A: A novel asparagine synthetase inhibitor suppressing the proliferation and migration of human non-small cell lung cancer A549 cells.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

Utility of ASNS gene methylation evaluated with the HPLC method as a pharmacogenomic biomarker to predict asparaginase sensitivity in BCP-ALL.

Asparaginase is an important agent for the treatment of acute lymphoblastic leukaemia (ALL), but it is occasionally associated with severe adverse events. Thus, for safer and more efficacious therapy, a clinical biomarker predicting asparaginase sensitivity is highly anticipated. Asparaginase depletes serum asparagine by deaminating asparagine into aspartic acid, and ALL cells are thought to be sensitive to asparaginase due to reduced asparagine synthetase (ASNS) activity. We have recently shown that allele-specific methylation of the ASNS gene is highly involved in asparaginase sensitivity in B-precursor ALL (BCP-ALL) by using next-generation sequence (NGS) analysis of bisulphite PCR products of the genomic DNA. Here, we sought to confirm the utility of methylation status of the ASNS gene evaluated with high-performance liquid chromatography (HPLC) analysis of bisulphite PCR products for future clinical applications. In the global methylation status of 23 CpG sites at the boundary region of promoter and exon 1 of the ASNS gene, a strong positive correlation was confirmed between the mean percent methylation evaluated with the HPLC method and that with the NGS method in 79 BCP-ALL cell lines (R2 = 0.85, p = 1.3 × 10-33) and in 63 BCP-ALL clinical samples (R2 = 0.84, p = 5.0 × 10-26). Moreover, methylation status of the ASNS gene evaluated with the HPLC method was significantly associated with in vitro asparaginase sensitivities as well as gene and protein expression levels of ASNS. These observations indicated that the ASNS gene methylation status evaluated with the HPLC method is a reliable biomarker for predicting the asparaginase sensitivity of BCP-ALL.

L-Asparaginase delivery systems targeted to minimize its side-effects.

L-asparaginase (L-ASP) is one of the key enzymes used in therapeutic applications, particularly to treat Acute Lymphocytic Leukemia (ALL). L-asparagine is a non-essential amino acid, which means that it can be synthesized by the body and is not required to be obtained through the diet. The synthesis of L-asparagine occurs primarily in the liver, but it also takes place in other tissues throughout the body. In contrast, leukemic cells cannot synthesize L-asparagine due the absence of L-asparagine synthetase and should obtain it from circulating sources for protein synthesis and cell division processes to ensure their vital functions. L-ASP catalyzes the deamination process of L-asparagine amino-acid into aspartic acid and ammonia, depriving leukemic cells of asparagine. This leads to decreased protein synthesis and cell division in tumor cells. However, using L-ASP has side effects, such as hypersensitivity or allergic reaction, antigenicity, short half-life, temporary blood clearance, and toxicity. L-ASP immobilization can minimize the side effects of L-ASP by stopping the immune system from attacking non-human enzymes and improving the enzyme's performance. The first strategy includes modification of enzyme structure, such as covalent binding (conjugation), adsorption to the support material and cross-linking of the enzyme. The chemical modification of residues, often nonspecific, changes the enzyme's hydrophobicity and surface charge, lowering the enzyme's activity. Also, the first strategy exposes the enzyme's surface to the environment. This eliminates its performance and does not allow targeted delivery of the enzyme. The second strategy is based on the entrapment of the enzyme inside the protecting structure or encapsulation. This strategy offers the same benefits as the first. Still, it also enables reducing toxicity, prolonging in vivo half-life, enhancing stability and activity, enables a targeted delivery and controlled release of the enzyme. Compared to the first strategy, encapsulation does not modify the chemical structure of the enzyme since L-ASP is only effective against leukemia in its native tetrameric form. This review aims to present state of the art in L-ASP formulations developed for reducing the side effects of L-ASP, focusing on describing improvements in their safety. The primary focus in the field remains to be improving the overall performance of the L-ASP formulations. Almost all encapsulation systems allow reducing immune response due to screening the enzyme from antibodies and prolonging its half-life. However, the enzyme's activity and stability depend on the encapsulation system type. Therefore, the selection of the right encapsulation system is crucial in therapy due to its effect on the performance parameters of the L-ASP. Biodegradable and biocompatible materials, such as chitosan, alginate and liposomes, mainly attract the researcher's interest in enzyme encapsulation. The research trends are also moving towards developing formulations with targeted delivery and increased selectivity.

Metabolomic Profiling of Asparagine Deprivation in Asparagine Synthetase Deficiency Patient-Derived Cells.

The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.

In vivo stabilization of a less toxic asparaginase variant leads to a durable antitumor response in acute leukemia.

Asparagine is a non-essential amino acid since it can either be taken up via the diet or synthesized by asparagine synthetase. Acute lymphoblastic leukemia (ALL) cells do not express asparagine synthetase or express it only minimally, which makes them completely dependent on extracellular asparagine for their growth and survival. This dependency makes ALL cells vulnerable to treatment with L-asparaginase, an enzyme that hydrolyzes asparagine. To date, all clinically approved L-asparaginases have significant L-glutaminase co-activity, associated with non-immune related toxic side effects observed during therapy. Therefore, reduction of L-glutaminase co-activity with concomitant maintenance of its anticancer L-asparaginase effect may effectively improve the tolerability of this unique drug. Previously, we designed a new alternative variant of Erwinia chrysanthemi (ErA; Erwinaze) with decreased L-glutaminase co-activity, while maintaining its L-asparaginase activity, by the introduction of three key mutations around the active site (ErA-TM). However, Erwinaze and our ErA-TM variant have very short half-lives in vivo. Here, we show that the fusion of ErA-TM with an albumin binding domain (ABD)-tag significantly increases its in vivo persistence. In addition, we evaluated the in vivo therapeutic efficacy of ABD-ErA-TM in a B-ALL xenograft model of SUP-B15. Our results show a comparable long-lasting durable antileukemic effect between the standard-of-care pegylated-asparaginase and ABD-ErA-TM L-asparaginase, but with fewer co-glutaminase-related acute side effects. Since the toxic side effects of current L-asparaginases often result in treatment discontinuation in ALL patients, this novel ErA-TM variant with ultra-low L-glutaminase co-activity and long in vivo persistence may have great clinical potential.

Targeting Asparagine Synthetase in Tumorgenicity Using Patient-Derived Tumor-Initiating Cells.

Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency.

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.

Anticancer Properties of N,N-dibenzylasparagine as an Asparagine (Asp) analog, Using Colon Cancer Caco-2 Cell Line.

OBJECTIVES: This study was conducted to investigate the potential anticancer properties of N, N-dibenzyl asparagine (NNDAsp), as an Asparagine (Asp) analog, using colon cancer Caco-2 cell and the normal NCM-460 cell line. METHODS: Cell viability rate and levels of produced lactate dehydrogenase (LDH) were achieved upon treatment with NNDAsp compared to Asp treatment using MTT assay and LDH production kit. The protein expression profile of asparagine synthetase (ASNS) was achieved by using ELISA and flow cytometry assay. The levels of released inflammatory cytokines, including interleukin-1 alpha (IL-1α) and IL-1 beta (IL-ß), were monitored using an ELISA assay. RESULTS: Our findings showed significant inhibition of colon cancer cell proliferation accompanied by a high level of produced LDH in a dose-dependent of an NNDAsp treatment without detectable toxic effect in normal cells. Interestingly, NNDAsp showed competitive inhibition of ASNS protein expression, in almost 3% of stained cancer cells, compared to 18% and 35% of untreated cells and cells pre-treated with Asp, respectively. Likewise, the concentration of ASNS protein was dramatically depleted in a dose and time-dependent of NNDAsp treatment in comparison with Asp treatment indicated by ELISA assay. Furthermore, as an apoptotic indicator, the expression of P53 and Caspase 3 (Caps3) was significantly increased in Caco-2 cells treated with NNDAsp at both RNA and protein levels. In contrast, their expression was markedly depleted in Asp-treated cells. In addition, the expression of both IL-1α and IL-1 ß was markedly increased in Caco-2 cells in a dose and time-dependent of NNDAsp exogenous treatment. Moreover, targeting of ASNS by the Asp analog, NNDAsp, was further confirmed by the docking analysis of inhibitors ligands and crystal structure of ASNS protein. CONCLUSION: These data provide evidence for the effectiveness of NNDAsp in cancer treatment via selective degradation of ASNS protein expression in colon cancer cells.

Tumoral microenvironment prevents de novo asparagine biosynthesis in B cell lymphoma, regardless of ASNS expression.

Depletion of circulating asparagine with l-asparaginase (ASNase) is a mainstay of leukemia treatment and is under investigation in many cancers. Expression levels of asparagine synthetase (ASNS), which catalyzes asparagine synthesis, were considered predictive of cancer cell sensitivity to ASNase treatment, a notion recently challenged. Using [U-13C5]-l-glutamine in vitro and in vivo in a mouse model of B cell lymphomas (BCLs), we demonstrated that supraphysiological or physiological concentrations of asparagine prevent de novo asparagine biosynthesis, regardless of ASNS expression levels. Overexpressing ASNS in ASNase-sensitive BCL was insufficient to confer resistance to ASNase treatment in vivo. Moreover, we showed that ASNase's glutaminase activity enables its maximal anticancer effect. Together, our results indicate that baseline ASNS expression (low or high) cannot dictate BCL dependence on de novo asparagine biosynthesis and predict BCL sensitivity to dual ASNase activity. Thus, except for ASNS-deficient cancer cells, ASNase's glutaminase activity should be considered in the clinic.

Amino acid stress response genes promote L-asparaginase resistance in pediatric acute lymphoblastic leukemia.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.

Asparagine synthetase regulates lung-cancer metastasis by stabilizing the ß-catenin complex and modulating mitochondrial response.

The availability of asparagine is the limitation of cell growth and metastasis. Asparagine synthetase (ASNS) was an essential enzyme for endogenous asparagine products. In our study, ASNS-induced asparagine products were essential to maintain tumor growth and colony formations in vitro. But mutated ASNS which defected endogenous asparagine products still upregulated cell invasiveness, which indicated that ASNS promoted invasiveness by alternative pathways. Mechanically, ASNS modulated Wnt signal transduction by promoting GSK3ß phosphorylation on ser9 and stabilizing the ß-catenin complex, as result, ASNS could promote more ß-catenin translocation into nucleus independent of endogenous asparagine. At the same time, ASNS modulated mitochondrial response to Wnt stimuli with increased mitochondrial potential and membrane fusion. In summary, ASNS promoted metastasis depending on Wnt pathway and mitochondrial functions even without endogenous asparagine products.

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.

Analysis of Enzyme Activity and Cellular Function for the N80S and S480F Asparagine Synthetase Variants Expressed in a Child with Asparagine Synthetase Deficiency.

Asparagine Synthetase Deficiency (ASNSD) is a disease caused by mutations in asparagine synthetase (ASNS). Newborns exhibit microcephaly, intractable epileptic-like seizures, progressive brain atrophy, and axial hypotonia. ASNSD results in global developmental delays and premature death. The present report describes a 9-year-old child who is a compound heterozygote with ASNS mutations c.1439C > T and c.239A > G leading to variants p.S480F and p.N80S, respectively. When grown in a complete culture medium, primary fibroblasts from the child contained ASNS mRNA and protein levels similar to an unrelated wild-type fibroblast cell line. When the child's fibroblasts were cultured for up to 72 h in a medium lacking asparagine, proliferation was reduced by about 50%. Purification of ASNS proteins harboring either the S480F or the N80S substitution had reduced enzymatic activity by 80% and 50%, respectively. Ectopic expression of either variant in ASNS-null Jensen rat sarcoma (JRS) cells did not support proliferation in the absence of medium-supplied asparagine, whereas expression of wild-type enzyme completely restored growth. These studies add to the list of pathogenic ASNS variants and use enzyme activity and protein expression in ASNS-null cells to expand our knowledge of the biological impact of mutations in the ASNS gene.

Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway.

Asparagine synthetase is a key enzyme that catalyses the conversion of amide groups from glutamine or ammonium to aspartate, which leads to the generation of asparagine. However, the role of asparagine synthetase in plant immunity remains largely unknown. Here, we identified a Nicotiana benthamiana asparagine synthetase B (NbAS-B) that associates with tomato mosaic virus coat protein-interacting protein L (IP-L) using the yeast two-hybrid assay and examined its role in tobacco mosaic virus (TMV) resistance. The association of IP-L with NbAS-B was further confirmed by in vivo co-immunoprecipitation, luciferase complementation imaging, and bimolecular fluorescence complementation assays. IP-L and NbAS-B interact in the nucleus and cytosol and IP-L apparently stabilizes NbAS-B, thus enhancing its accumulation. The expressions of IP-L and NbAS-B are continuously induced on TMV-green fluorescent protein (GFP) infection. Co-silencing of IP-L and NbAS-B facilitates TMV-GFP infection. Overexpression of NbAS-B in tobacco reduces TMV-GFP infection by significantly improving the synthesis of asparagine. Furthermore, the external application of asparagine significantly inhibits the infection of TMV-GFP by activating the salicylic acid signalling pathway. These findings hold the potential for the future application of asparagine in the control of TMV.

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.

Consumption of meat containing ractopamine might enhance tumor growth through induction of asparagine synthetase.

There is currently no evidence of the carcinogenic effect of the ß-adrenergic agonist ractopamine added in finishing swine and cattle feed for promoting leanness. Nonetheless, it has the capability of stimulating expression of asparagine synthetase (ASNS) through activating transcription factor 5, and many other genes involved in the stress reaction in the skeletal muscle of pigs according to published scientific articles. Because overexpression of ASNS has been detected as a key player in amino acid response and unfolded protein response during the development of not a few malignant diseases, especially those with KRAS mutations, and found to be closely related to tumor proliferation, invasion and metastasis, it seems reasonable to hypothesize that intake of ractopamine residue in meat might bring negative effects to cancer patients.

Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer.

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.

Canagliflozin Improves Liver Function in Rats by Upregulating Asparagine Synthetase.

INTRODUCTION: Canagliflozin (CANA) is a sodium-glucose cotransporter 2 inhibitor that was recently approved for treating diabetes. However, its effects on liver function are not well understood. The function of asparagine synthetase (ASNS) has been studied in several cancers but not in liver injury. Therefore, we investigated the connection between CANA and ASNS in alleviating damage (i.e., their hepatoprotective effect) in a rat liver injury model. METHODS: The rat model of liver injury was established using carbon tetrachloride treatment. Rats with liver injury were administered CANA orally for 8 weeks daily. After week 8, peripheral blood was collected to measure serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels. Liver histopathology was examined using hematoxylin and eosin staining to determine the degree of liver injury. Protein expression in the rat livers was examined using Western blotting. RESULTS: CANA treatment decreased serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels compared with those of the untreated group, demonstrating diminished liver injury. Mechanistically, CANA treatment activated AMP-activated protein kinase (AMPK), leading to increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and activating transcription factor 4 (ATF4), which upregulated ASNS expression in liver-injured rats. CONCLUSION: CANA significantly alleviated liver injury by activating the AMPK/Nrf2/ATF4 axis and upregulating ASNS expression, indicating its potential for treating patients with type 2 diabetes mellitus with impaired liver function.

Compound NSC84167 selectively targets NRF2-activated pancreatic cancer by inhibiting asparagine synthesis pathway.

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.