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.

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.

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.

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.

[Detection of CPS1 gene mutation in a neonate with carbamoyl phosphate synthetase I deficiency].

OBJECTIVE: To explore the genetic basis for a neonate featuring hyperammonemia. METHODS: The patient was examined and tested by tandem mass spectrometry and next generation sequencing (NGS). Suspected mutations were confirmed by Sanger sequencing of the proband and her parents. Potential impact of the mutation was predicted with SIFT, PolyPhen-2 and MutationTaste software. RESULTS: Plasma ammonia and alanine were significantly increased in the proband, while serum citrulline was decreased. The neonate was found to harbor compound heterozygous mutations of the CPS1 gene [c.1631C>T(p.T544M) and c.1981G>T(p.G661C)], which were respectively inherited from her father and mother. CONCLUSION: The carbamoyl phosphate synthetase I deficiency of the proband can probably be attributed to the mutations of the CPS1 gene. Above finding has expanded the spectrum of CPS1 mutations in association with carbamoyl phosphate synthetase I deficiency.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy.

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

Population screening in a Druze community: the challenge and the reward.

PURPOSE: The Druze community is characterized by consanguinity and endogamy, and by reluctance to genetic testing and technological interventions for the prevention of birth defects. Multiple patients with four rare and severe inborn errors of metabolism cerebrotendinous xanthomatosis, prolidase deficiency, argininosuccinate lyase deficiency, and carbamyl phosphate synthetase I deficiency were identified in an isolated Druze village in northern Israel. The aims of this study were to identify couples at risk for four inherited diseases, and to prevent birth defects in a community presenting religious and cultural obstacles to genetic testing. METHODS: A genetic screening and counseling program in a high-risk community. RESULTS: The 1425 residents who attended group genetic counseling sessions between 2003 and 2007 consented to genetic testing. We identified 217 carriers for either one or two disease causing mutations. High carrier frequencies for cerebrotendinous xanthomatosis, prolidase deficiency, argininosuccinate lyase deficiency, and carbamyl phosphate synthetase I deficiency were identified as 1:11, 1:21, 1:41, and 1:95, respectively. Fifty-eight percent (125) of the carriers' spouses agreed to genetic counseling and testing. Ten couples at risk for affected offspring were identified and offered prenatal genetic counseling and diagnosis. CONCLUSIONS: The genetic screening program, the first of its kind reported in a Druze community, was well received. We expect this program to increase awareness of genetic counseling, to contribute to disease prevention, and to serve as a model for other isolated communities.

Understanding carbamoyl phosphate synthetase deficiency: impact of clinical mutations on enzyme functionality.

Carbamoyl phosphate synthetase I (CPSI) deficiency, a recessively inherited error of the urea cycle, causes life-threatening hyperammonaemia. CPSI is a multidomain 1500-residue liver mitochondrial matrix protein that is allosterically activated by N-acetyl-l-glutamate, and which synthesises carbamoyl phosphate (CP) in three steps: bicarbonate phosphorylation by ATP, carbamate synthesis from carboxyphosphate and ammonia, and carbamate phosphorylation by ATP. Several missense mutations of CPSI have been reported in patients with CPSI deficiency, but the actual pathogenic potential and effects on the enzyme of these mutations remain non-characterised. Since the structure of Escherichia coli CPS is known and systems for its overexpression and purification are available, we have constructed and purified eight site-directed mutants of E.coli CPS affecting the enzyme large subunit (A126M, R169H, Q262P, N301K, P360L, V640R, R675L, S789P) that are homologous to corresponding missense mutations found in patients with CPSI deficiency, studying their stability and their ability to catalyse the CPS reaction as well as the partial reactions that reflect the different reactional steps, and analysing the substrate kinetics for the overall and partial reactions. The results show that all the mutations significantly decrease CP synthesis without completely inactivating the enzyme (as reflected in the catalysis of at least one partial reaction), that one of these mutations (Q262P) causes marked enzyme instability, and validate the use of E.coli CPS as a pathogenicity testing model for CPSI deficiency. The causality of the reported clinical mutations is supported and the derangements caused by the mutations are identified, revealing the specific roles of the residues that are mutated. In particular, the findings highlight the importance for carbamate phosphorylation and for allosteric activation of a loop that coordinates K(+), stress the key role of intersubunit interactions for CPS stability, and suggest that lid opening at both phosphorylation sites is concerted.

Environmentally determined genetic expression: clinical correlates with molecular variants of carbamyl phosphate synthetase I.

Carbamyl phosphate synthetase I (CPSI) determines the rate-limiting entry of free ammonia into the urea cycle. Disruption of CPSI affects the liver's ability to remove waste nitrogen and produce arginine, citrulline, and urea. Arginine is the necessary precursor for the critical biomolecule, nitric oxide (NO). We have studied the classic model of CPSI deficiency, which results in severe hyperammonemia, and identified a large number of molecular defects. A number of CPSI polymorphisms have been found that appear to result in functional consequences. We have examined the association of these polymorphisms with various environmental stress conditions and found that certain CPSI alleles are associated with clinical outcome. We refer to these associations as environmentally determined genetic expression (EDGE) affects. In addition to studies of classic CPSI deficiency, we have developed data for the EDGE concept in post-cardiac surgery-related pulmonary hypertension, hepatic veno-occlusive disease after bone marrow transplantation, and persistent pulmonary hypertension of the newborn. We have linked these outcomes and genotypes to the availability of the urea cycle intermediates, citrulline and arginine, and their role in NO synthesis. We hypothesize that these polymorphisms affect the functional efficiency of CPSI and thus the entire urea cycle and as such, the availability of the NO substrates. By piecing together the various functional aspects of the urea cycle changes we have seen, we can better understand the clinical vulnerabilities of patients in environmentally stressful situations. This knowledge should allow us to design intervention strategies to either predict or modify the associated adverse outcomes.