The genetic bases of Alzheimer\'s disease (AD) and frontotemporal dementia (FTD) have been comprehensively studied, which is not the case for atypical cases not classified into these diagnoses. In the present study, we aim to contribute to the molecular understanding of the development of non-AD and non-FTD dementia due to hyperammonemia caused by mutations in urea cycle genes. The analysis was performed by pooled whole-exome sequencing (WES) of 90 patients and by searching for rare pathogenic variants in autosomal genes for enzymes or transporters of the urea cycle pathway. The survey returned two rare pathogenic coding mutations leading to citrullinemia type I: rs148918985, p.Arg265Cys, C>T; and rs121908641, p.Gly390Arg, G>A in the argininosuccinate synthase 1 (ASS1) gene. The p.Arg265Cys variant leads to enzyme deficiency, whereas p.Gly390Arg renders the enzyme inactive. These variants found in simple or compound heterozygosity can lead to the late-onset form of citrullinemia type I, associated with high ammonia levels, which can lead to cerebral dysfunction and thus to the development of dementia. The presence of urea cycle disorder-causing mutations can be used for the early initiation of antihyperammonemia therapy in order to prevent the neurotoxic effects.

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.

Argininosuccinate synthase (ASS1), a critical enzyme in the urea cycle, acts as a tumor suppressor in many cancers. To date, the anticancer mechanism of ASS1 has not been fully elucidated. Here, we found that phosphoglycerate dehydrogenase (PHGDH), a key rate-limiting enzyme in serine synthesis, is a pivotal protein that interacts with ASS1. Our results showed that ASS1 directly binds to PHGDH and promotes its ubiquitination-mediated degradation to inhibit serine synthesis, consequently suppressing tumorigenesis. Importantly, the tumor suppressive effects of ASS1 were strongly abrogated by PHGDH knockout. In addition, ASS1 knockout and knockdown partially rescued cell proliferation when serine and glycine were depleted, while the inhibitory effect of ASS1 overexpression on cell proliferation was restored by the addition of serine and glycine. These findings unveil a novel role of ASS1 and suggest that the ASS1/PHGDH serine synthesis pathway is a promising target for cancer therapy.

Atherosclerosis is initiated with endothelial cell (EC) dysfunction and vascular inflammation under hyperlipidemia. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase. However, the specific role of endothelial SIRT3 during atherosclerosis remains poorly understood. The present study aims to study the role and mechanism of SIRT3 in EC function during atherosclerosis. Wild-type Sirt3f/f mice and endothelium-selective SIRT3 knockout Sirt3f/f; Cdh5Cre/+ (Sirt3EC-KO) mice are injected with adeno-associated virus (AAV) to overexpress PCSK9 and fed with high-cholesterol diet (HCD) for 12 weeks to induce atherosclerosis. Sirt3EC-KO mice exhibit increased atherosclerotic plaque formation, along with elevated macrophage infiltration, vascular inflammation, and reduced circulating L-arginine levels. In human ECs, SIRT3 inhibition resulted in heightened vascular inflammation, reduced nitric oxide (NO) production, increased reactive oxygen species (ROS), and diminished L-arginine levels. Silencing of SIRT3 results in hyperacetylation and deactivation of Argininosuccinate Synthase 1 (ASS1), a rate-limiting enzyme involved in L-arginine biosynthesis, and this effect is abolished in mutant ASS1. Furthermore, L-arginine supplementation attenuates enhanced plaque formation and vascular inflammation in Sirt3EC-KO mice. This study provides compelling evidence supporting the protective role of endothelial SIRT3 in atherosclerosis and also suggests a critical role of SIRT3-induced deacetylation of ASS1 by ECs for arginine synthesis.

Arginine is a semi-essential amino acid that supports protein synthesis to maintain cellular functions. Recent studies suggest that arginine also promotes wound healing, cell division, ammonia metabolism, immune system regulation, and hormone biosynthesis-all of which are critical for tumor growth. These discoveries, coupled with the understanding of cancer cell metabolic reprogramming, have led to renewed interest in arginine deprivation as a new anticancer therapy. Several arginine deprivation strategies have been developed and entered clinical trials. The main principle behind these therapies is that arginine auxotrophic tumors rely on external arginine sources for growth because they carry reduced key arginine-synthesizing enzymes such as argininosuccinate synthase 1 (ASS1) in the intracellular arginine cycle. To obtain anticancer effects, modified arginine-degrading enzymes, such as PEGylated recombinant human arginase 1 (rhArg1-PEG) and arginine deiminase (ADI-PEG 20), have been developed and shown to be safe and effective in clinical trials. They have been tried as a monotherapy or in combination with other existing therapies. This review discusses recent advances in arginine deprivation therapy, including the molecular basis of extracellular arginine degradation leading to tumor cell death, and how this approach could be a valuable addition to the current anticancer arsenal.

Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate-limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 reexpression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20.
Tumor initiation and growth rates were measured for a spontaneous Ass1 knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied in vitro and in vivo.
Conditional Ass1 KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. Ass1 KO cells grew robustly through arginine starvation in vivo, while ADI-PEG20 remained completely lethal in vitro, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Coculture with Ass1-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect in vitro and in vivo.
Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes.

BACKGROUND: Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy.
METHODS: Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic \"hits\" were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM.
RESULTS: We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model.
CONCLUSIONS: Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

Citrullinemia type 1 (CTLN1) is a rare autosomal recessive disease caused by argininosuccinate synthetase (ASS) deficiency. Manifestations vary from the acute neonatal or \"classic\" form to a milder, late-onset, or \"unconventional\" form. To date, more than 93 variants in the ASS1 gene located on chromosome 9q43.11 (OMIM #215700) are reportedly responsible for CTLN1. Their incidence and distribution vary according to geographic origins and ethnicity, and a correlation, although not clearly delineated, has been established between the genotype and the phenotype of the disease. Though, in the Middle East, national descriptions of CTLN1 are still lacking.
A total of ten unrelated Middle Eastern families, five Lebanese, two Syrians, and three Iraqis with citrullinemia index cases, were included in this study. Upon informed consent, DNA was extracted from the whole blood of the index patients as well as their parents and siblings. Genetic analysis was carried out by Sanger sequencing of the ASS1 gene.
Seven different variants were identified. Two novel variants, c.286C>A (p.(Pro96Thr), RNA not analyzed) in exon 5 and deletion c.685_688+6del(p.(Lys229Glyfs*4), RNA not analyzed) in exon 10, were found in one Lebanese and one Syrian family, respectively, and were correlated with early-onset and severe clinical presentation. Five other known variants: c.535T>C (p.(Trp179Arg), RNA not analyzed) in exon 8, c.787G>A (p.(Val263Met), RNA not analyzed) in exon 12, c.847G>A (p.(Glu283Lys), RNA not analyzed) in exon 13, c.910C>T (p.(Arg304Trp), RNA not analyzed) in exon 13, and c.1168G>A (p.(Gly390Arg), RNA not analyzed) in exon 15, were found in Lebanese, Syrian, and Iraqi families, and were associated with diverse clinical presentations.
Two novel variants and five known variants were found in a total of ten unrelated Middle Eastern families.