NAGS, CPS1, and SLC25A13 (Citrin) at the Crossroads of Arginine and Pyrimidines Metabolism in Tumor Cells.

Urea cycle enzymes and transporters collectively convert ammonia into urea in the liver. Aberrant overexpression of carbamylphosphate synthetase 1 (CPS1) and SLC25A13 (citrin) genes has been associated with faster proliferation of tumor cells due to metabolic reprogramming that increases the activity of the CAD complex and pyrimidine biosynthesis. N-acetylglutamate (NAG), produced by NAG synthase (NAGS), is an essential activator of CPS1. Although NAGS is expressed in lung cancer derived cell lines, expression of the NAGS gene and its product was not evaluated in tumors with aberrant expression of CPS1 and citrin. We used data mining approaches to identify tumor types that exhibit aberrant overexpression of NAGS, CPS1, and citrin genes, and evaluated factors that may contribute to increased expression of the three genes and their products in tumors. Median expression of NAGS, CPS1, and citrin mRNA was higher in glioblastoma multiforme (GBM), glioma, and stomach adenocarcinoma (STAD) samples compared to the matched normal tissue. Median expression of CPS1 and citrin mRNA was higher in the lung adenocarcinoma (LUAD) sample while expression of NAGS mRNA did not differ. High NAGS expression was associated with an unfavorable outcome in patients with glioblastoma and GBM. Low NAGS expression was associated with an unfavorable outcome in patients with LUAD. Patterns of DNase hypersensitive sites and histone modifications in the upstream regulatory regions of NAGS, CPS1, and citrin genes were similar in liver tissue, lung tissue, and A549 lung adenocarcinoma cells despite different expression levels of the three genes in the liver and lung. Citrin gene copy numbers correlated with its mRNA expression in glioblastoma, GBM, LUAD, and STAD samples. There was little overlap between NAGS, CPS1, and citrin sequence variants found in patients with respective deficiencies, tumor samples, and individuals without known rare genetic diseases. The correlation between NAGS, CPS1, and citrin mRNA expression in the individual glioblastoma, GBM, LUAD, and STAD samples was very weak. These results suggest that the increased cytoplasmic supply of either carbamylphosphate, produced by CPS1, or aspartate may be sufficient to promote tumorigenesis, as well as the need for an alternative explanation of CPS1 activity in the absence of NAGS expression and NAG.

Metabolic Modulation of Intracellular Ammonia via Intravesical Instillation of Nanoporter-Encased Hydrogel Eradicates Bladder Carcinoma.

Tumor protein 53 (TP53) mutation in bladder carcinoma (BC), upregulates the transcription of carbamoyl phosphate synthetase 1 (CPS1), to reduce intracellular ammonia toxicity. To leverage ammonia combating BC, here, an intravesically perfusable nanoporter-encased hydrogel system is reported. A biomimetic fusogenic liposomalized nanoporter (FLNP) that is decorated with urea transporter-B (UT-B) is first synthesized with protonated chitosan oligosaccharide for bladder tumor-targeted co-delivery of urease and small interfering RNA targeting CPS1 (siCPS1). Mussel-inspired hydrogel featured with dual functions of bio-adhesion and injectability is then fabricated as the reservoir for intravesical immobilization of FLNP. It is found that FLNP-mediated UT-B immobilization dramatically induces urea transportation into tumor cells, and co-delivery of urease and siCPS1 significantly boosts ammonia accumulation in tumor inducing cell apoptosis. Treatment with hybrid system exhibits superior anti-tumor effect in orthotopic bladder tumor mouse model and patient-derived xenograft model, respectively. Combined with high-protein diet, the production of urinary urea increases, leading to an augmented intracellular deposition of ammonia in BC cells, and ultimately an enhanced tumor inhibition. Together, the work establishes that cascade modulation of ammonia in tumor cells could induce tumor apoptosis and may be a practical strategy for eradication of TP53-mutated bladder cancer.

p53 inhibits the Urea cycle and represses polyamine biosynthesis in glioma cell lines.

Glioma is the most common malignant tumor of the central nervous system. The urea cycle (UC) is an essential pathway to convert excess nitrogen and ammonia into the less toxic urea in humans. However, less is known about the functional significance of the urea cycle in glioma. p53 functions as a tumor suppressor and modulates several cellular functions and disease processes. In the present study, we aimed to explore whether p53 influences glioma progression by regulating the urea cycle. Here, we demonstrated the inhibitory impact of p53 on the expression of urea cycle enzymes and urea genesis in glioma cells. The level of polyamine, a urea cycle metabolite, was also regulated by p53 in glioma cells. Carbamoyl phosphate synthetase-1 (CPS1) is the first key enzyme involved in the urea cycle. Functionally, we demonstrated that CPS1 knockdown suppressed glioma cell proliferation, migration and invasion. Mechanistically, we demonstrated that the expression of ornithine decarboxylase (ODC), which determines the generation of polyamine, was regulated by CPS1. In addition, the impacts of p53 knockdown on ODC expression, glioma cell growth and aggressive phenotypes were significantly reversed by CPS1 inhibition. In conclusion, these results demonstrated that p53 inhibits polyamine metabolism by suppressing the urea cycle, which inhibits glioma progression.

Deficiency of Carbamoyl Phosphate Synthetase 1 Engenders Radioresistance in Hepatocellular Carcinoma via Deubiquitinating c-Myc.

PURPOSE: Tumor radiation resistance is the main obstacle to effective radiation therapy for patients with hepatocellular carcinoma (HCC). We identified the role of urea cycle key enzyme carbamoyl phosphate synthetase 1 (CPS1) in radioresistance of HCC and explored its mechanism, aiming to provide a novel radiosensitization strategy for the CPS1-deficiency HCC subtype. METHODS AND MATERIALS: The expression of CPS1 was measured by western blot and immunohistochemistry. Cell growth assay, EdU assay, cell apoptosis assay, cell cycle assay, clone formation assay, and subcutaneous tumor assay were performed to explore the relationship between CPS1 and radioresistance of HCC cells. Lipid metabonomic analysis was used for investigating the effects of CPS1 on lipid synthesis of HCC cells. RNA sequencing and coimmunoprecipitation assay were carried out to reveal the mechanism of CPS1 participating in the regulation of HCC radiation therapy resistance. Furthermore, 10074-G5, the specific inhibitor of c-Myc, was administered to HCC cells to investigate the role of c-Myc in CPS1-deficiency HCC cells. RESULTS: We found that urea cycle key enzyme CPS1 was frequently lower in human HCC samples and positively associated with the patient's prognosis. Functionally, the present study proved that CPS1 depletion could accelerate the development of HCC and induce radiation resistance of HCC in vitro and in vivo, and deficiency of CPS1 promoted the synthesis of some lipid molecules. Regarding the mechanism, we uncovered that inhibition of CPS1 upregulated CyclinA2 and CyclinD1 by stabilizing oncoprotein c-Myc at the posttranscriptional level and generated radioresistance of HCC cells. Moreover, inactivation of c-Myc using 10074-G5, a specific c-Myc inhibitor, could partially attenuate the proliferation and radioresistance induced by depletion of CPS1. CONCLUSIONS: Our results recapitulated that silencing CPS1 could promote HCC progression and radioresistance via c-Myc stability mediated by the ubiquitin-proteasome system, suggesting that targeting c-Myc in CPS1-deficiency HCC subtype may be a valuable radiosensitization strategy in the treatment of HCC.

Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer.

OBJECTIVE: The sensitivity and specificity of current biomarkers for gastric cancer were insufficient. The aim of the present study was to screen novel biomarkers and determine the diagnostic values of ornithine aminotransferase (OAT) and carbamoyl phosphate synthetase 1 (CPS1) for detecting gastric cancer. METHODS: With stable isotope tags, we labelled an initial discovery group of four paired gastric cancer tissue samples and identified with LC-ESI-MS/MS. A validation group of 159 gastric cancer samples and 30 healthy controls were used to validate the candidate targets. GSEA was used to explore the pathways activated in gastric cancer. RESULTS: Four hundred and thirty one proteins were found differentially expressed in gastric cancer tissues. Of these proteins, OAT and CPS1 were found over-expressed in gastric cancer patients, with sensitivity of 70.4% (95% CI: 63.3%-77.6%) and specificity of 80.5% (95% CI: 74.3%-86.7%) for ornithine aminotransferase, and with sensitivity of 68.6% (95% CI: 61.3%-75.8%) and specificity of 73% (95% CI: 66%-79.9%) for carbamoyl phosphate synthetase 1. The co-expression of OAT and CPS1 in gastric cancer tissues has a sensitivity of 81% (95% CI: 73.2%-88.8%) and specificity of 89% (95% CI: 83%-95%). Furthermore, both OAT and CPS1 were overexpressed in patients with local invasion T3 and T4 stages than those in patients with T1 and T2 stages. The co-expression of OAT and CPS1 was strongly correlated with histological grade I 68% (95% CI: 58.7%-77.3%) and TNM stage I/II 52% (95% CI: 42%-62%). The areas under ROC curves were up to 0.758 for the co-expression of OAT and CPS1 in gastric cancer. GSEA results showed that two gene sets and 30 gene sets were activated in OAT high- and CPS1 high-expression patients with gastric cancer, respectively. CONCLUSIONS: The present findings indicated a tight correlation between the co-expression of OAT and CPS1 and the histological grade, local invasion, and TNM stages of gastric cancer. Therefore, OAT and CPS1 might be predictors for gastric cancer invasion and potential targets for anticancer drug design for gastric cancer.

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation.

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr -/-). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m2), diabetes mellitus and ALT were found to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.

CRISPR-Mediated Genomic Addition to CPS1 Deficient iPSCs is Insufficient to Restore Nitrogen Homeostasis.

CPS1 deficiency is an inborn error of metabolism caused by loss-of-function mutations in the CPS1 gene, catalyzing the initial reaction of the urea cycle. Deficiency typically leads to toxic levels of plasma ammonia, cerebral edema, coma, and death, with the only curative treatment being liver transplantation; due to limited donor availability and the invasiveness and complications of the procedure, however, alternative therapies are needed. Induced pluripotent stem cells offer an alternative cell source to partial or whole liver grafts that theoretically would not require immune suppression regimens and additionally are amenable to genetic modifications. Here, we genetically modified CPS1 deficient patient-derived stem cells to constitutively express human codon optimized CPS1 from the AAVS1 safe harbor site. While edited stem cells efficiently differentiated to hepatocyte-like cells, they failed to metabolize ammonia more efficiently than their unedited counterparts. This unexpected result appears to have arisen in part due to transgene promoter methylation, and thus transcriptional silencing, in undifferentiated cells, impacting their capacity to restore the complete urea cycle function upon differentiation. As pluripotent stem cell strategies are being expanded widely for potential cell therapies, these results highlight the need for strict quality control and functional analysis to ensure the integrity of cell products.

Association between genetic variations in carbamoyl-phosphate synthetase gene and persistent neonatal pulmonary hypertension.

Persistent pulmonary hypertension of the new-borns (PPHN) is one of the main etiologies of morbidity as well as mortality in neonates. Previous studies found that genetic polymorphisms in urea cycle enzymes are associated with PPHN. Few of the genetic polymorphisms in neonates have been recognized with PPHN. We aimed to find out the prevalence of the CPS-I gene polymorphism and to correlate the genotype with the serum nitric oxide (NO) levels in Egyptian neonates with idiopathic PPHN. We included neonates diagnosed with PPH (n = 150) while the control group included healthy neonates with matched age and sex (n = 100). The CPS-I gene polymorphism: A/C, trans-version substitution, rs4399666 genotype was identified using TaqMan-based quantitative PCR. The results revealed that the CPS-I A/C rs4399666 gene polymorphism and lower serum NO levels were significantly associated with idiopathic PPHN in neonates. In addition, serum NO level was significantly associated with an rs4366999 A/C variant gene in idiopathic PPHN (p = 0.001). Univariable regression analysis demonstrated that there was a significant association between CPS-I A/C rs4399666 CC and increased risk of PPHN (odd ratio, 95% CI of 1.8 (0.78 to 1.75), p-value = 0.04).Conclusion: We concluded that mutant CPS-I A/C rs4399666 minor variant especially the homozygous CC genotype is frequently distributed among the PPHN group. This demonstrates that the presence of mutant CPS-I rs4399666 does not necessarily predispose to the development of PPHN in neonates, but nonetheless, if the C allele is inherited in the homozygous CC genotype, it is associated with a higher risk of PPHN. What is Known: • Prior studies found that polymorphisms in urea cycle enzyme genes are associated with PPHN. • Association between CPS-1 gene polymorphisms is significantly associated with PPHN. What is New: • The prevalence of CPS-1, A/C trans-version substitution, rs4399666 gene polymorphism in Egyptian neonates presented with idiopathic PPHN. • Mutant CPS-I A/C rs4399666 especially the homozygous CC genotype is more frequently distributed among PPHN, and it is significantly associated with low serum nitric oxide level.

Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells.

Upregulation of the neuropeptide neurotensin (NTS) in a subgroup of lung cancers has been linked to poor prognosis. However, the regulatory pathway centered on NTS in lung cancer remains unclear. Here we identified the NTS-specific enhancer in lung adenocarcinoma cells. The AF4/FMR2 (AFF) family protein AFF1 occupies the NTS enhancer and inhibits NTS transcription. Clustering analysis of lung adenocarcinoma gene expression data demonstrated that NTS expression is highly positively correlated with the expression of the oncogenic factor CPS1. Detailed analyses demonstrated that the IL6 pathway antagonizes NTS in regulating CPS1. Thus, our analyses revealed a novel NTS-centered regulatory axis, consisting of AFF1 as a master transcription suppressor and IL6 as an antagonist in lung adenocarcinoma cells.

The Role of TRIP6, ABCC3 and CPS1 Expression in Resistance of Ovarian Cancer to Taxanes.

The main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in ABCC3, CPS1, and TRIP6 gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

Small Molecule Inhibition of CPS1 Activity through an Allosteric Pocket.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

Caspase recruitment domain family member 10 regulates carbamoyl phosphate synthase 1 and promotes cancer growth in bladder cancer cells.

Bladder cancer, which can be divided into non-muscle-invasive and muscle-invasive bladder cancer, is the most common urinary cancer in the United States. Caspase recruitment domain family member 10 (CARD10), also named CARD-containing MAGUK protein 3 (CARMA3), is a member of the CARMA family and may activate the nuclear factor kappa B (NF-κB) pathway. We utilized RNA sequencing and metabolic mass spectrometry to identify the molecular and metabolic feature of CARD10. The signalling pathway of CARD10 was verified by Western blotting analysis and functional assays. RNA sequencing and metabolic mass spectrometry of CARD10 knockdown identified the metabolic enzyme carbamoyl phosphate synthase 1 (CPS1) in the urea cycle as the downstream gene regulated by CARD10. We confirmed that CARD10 affected cell proliferation and nucleotide metabolism through regulating CPS1. We indicated that CARD10 promote bladder cancer growth via CPS1 and maybe a potential therapeutic target in bladder cancer.

Expression and clinical significance of CPS1 in glioblastoma multiforme.

Carbamoyl phosphate synthetase-1 (CPS1), the first rate-limiting mitochondrial enzyme in the urea cycle, regulates proliferation and differentiation during tumor progression. However, the detailed function of CPS1 in glioblastoma Multiforme (GBM) is still unclear. Here, we highlight mechanisms for CPS1 upregulation and the effects of upregulated CPS1 on GBM tumorigenesis. The transcriptome data from several public databases, such as Oncomine and GEPIA, revealed that CPS1 transcriptional level was significantly upregulated in GBM tissues and cells. Moreover, CPS1 was hypomethylated in GBM tissues. The Wanderer database, linked to the Cancer Genome Atlas (TCGA), showed the association between CPS1 expression or its methylation values and the clinicopathological parameters in GBM patients. Our work fully demonstrated that CPS1 expression was upregulated in GBM and this gene could be used as a potential diagnostic and prognosis indicator for GBM.

Non-alcoholic fatty liver disease alters expression of genes governing hepatic nitrogen conversion.

BACKGROUND & AIMS: We recently showed that the functional capacity for ureagenesis is deficient in non-alcoholic fatty liver disease (NAFLD) patients. The aim of this study was to assess expression of urea cycle-related genes to elucidate a possible gene regulatory basis to the functional problem. METHODS: Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis [NASH]) and 12 obese and 14 lean healthy individuals. Sixteen NAFLD patients were included for gene expression validation. Relationship between gene expressions and functional capacity for ureagenesis was described. RESULTS: Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs both control groups; markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (~3.5-fold, P < .0001). In NASH, CPS1 downregulation paralleled the deficit in ureagenesis (P = .03). Additionally, expression of several genes involved in amino acid uptake and degradation, and the glucagon receptor gene, were downregulated in NAFLD. Conversely, glutamine synthetase (GS) expression increased >1.5-fold (P ≤ .03), inversely related to CPS1 expression (P = .004). CONCLUSIONS: NAFLD downregulated the expression of urea cycle-related genes. Downregulation of urea cycle flux-generating CPS1 correlated with the loss of functional capacity for ureagenesis in NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed related to a fatty liver as such rather than NASH or obesity. The findings support gene regulatory mechanisms involved in the deficient ureagenesis of NAFLD, but it remains unexplained how hepatocyte fat accumulation exerts these effects.

Differentially Expressed Mitochondrial Proteins in Human MCF7 Breast Cancer Cells Resistant to Paclitaxel.

Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis.

Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.

Urea Cycle Sustains Cellular Energetics upon EGFR Inhibition in EGFR-Mutant NSCLC.

Mutations in oncogenes and tumor suppressor genes engender unique metabolic phenotypes crucial to the survival of tumor cells. EGFR signaling has been linked to the rewiring of tumor metabolism in non-small cell lung cancer (NSCLC). We have integrated the use of a functional genomics screen and metabolomics to identify metabolic vulnerabilities induced by EGFR inhibition. These studies reveal that following EGFR inhibition, EGFR-driven NSCLC cells become dependent on the urea cycle and, in particular, the urea cycle enzyme CPS1. Combining knockdown of CPS1 with EGFR inhibition further reduces cell proliferation and impedes cell-cycle progression. Profiling of the metabolome demonstrates that suppression of CPS1 potentiates the effects of EGFR inhibition on central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism, coinciding with reduced glycolysis and mitochondrial respiration. We show that EGFR inhibition and CPS1 knockdown lead to a decrease in arginine levels and pyrimidine derivatives, and the addition of exogenous pyrimidines partially rescues the impairment in cell growth. Finally, we show that high expression of CPS1 in lung adenocarcinomas correlated with worse patient prognosis in publicly available databases. These data collectively reveal that NSCLC cells have a greater dependency on the urea cycle to sustain central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism to meet cellular energetics upon inhibition of EGFR. IMPLICATIONS: Our results reveal that the urea cycle may be a novel metabolic vulnerability in the context of EGFR inhibition, providing an opportunity to develop rational combination therapies with EGFR inhibitors for the treatment of EGFR-driven NSCLC.

Hepatic glutamine synthetase augmentation enhances ammonia detoxification.

The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.