Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Integration of transcriptomics and metabolomics provides metabolic and functional insights into reduced insulin secretion in MIN6 ß-cells exposed to deficient and excessive arginine.

Previous work demonstrated that arginine is one of the strongest insulin secretagogues. However, knowledge of the mechanisms linking chronic arginine metabolism with ß-cell function and insulin secretion is relatively limited. After preliminary selection of concentration according to the cell proliferation, the MIN6 pancreatic ß-cells were randomly assigned to culture in 0.04 mM (low-arginine, LA), 0.4 mM (standard-arginine, SA), or 8 mM arginine (high-arginine, HA) for 24 h. Following the treatment, a combination of transcriptomics and metabolomics, together with a series of molecular biological tests were performed to investigate the responses of ß-cells to varied arginine availability. Our results showed that HA treatment reduced the chronic insulin releases, and LA and HA treatments decreased the glucose-stimulated insulin secretions (GSIS) of ß-cells relative to the SA group (p < .05). Transcriptomics analysis indicated that LA administration significantly inhibited oxidative phosphorylation and ATP metabolic process but promoted DNA repair and mRNA processing in ß-cells, while HA administration affected ammonium ion metabolic process and mRNA export (p < .05). Both LA and HA regulated the expressions of genes involved in DNA replication, cell-cycle phase transition, and response to oxidative stress (p < .05). Protein-protein interaction and transcription factor analyses suggested that Trp53 and Nr4a2 genes may play key roles during arginine stimulation. On the contrary, metabolomics analysis demonstrated that the differentially expressed metabolites (DEM) of MIN6 ß-cells induced by LA were mainly enriched in glycerophospholipid metabolism, linoleic acid metabolism, and purine metabolism, while most DEMs between LA vs. SA comparison belonged to amino acid metabolism. When combined the three groups, co-expression analysis suggested that insulin secretions had strong associations with L-pyroglutamic acid, L-glutamate, and creatine concentrations, while intracellular insulin contents were mainly correlated to L-arginine, argininosuccinic acid, and phosphorylcholine. At last, integrated analysis of transcriptomics and metabolomics showed that glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids, and amino acid metabolism were the most relevant pathways in ß-cells exposed to abnormal arginine supply. This descriptive bioinformatics analysis suggested that the disturbed carbohydrate, lipid, and amino acid metabolisms, as well as the increased apoptosis and elevated oxidative stress, contributed to the reduced insulin secretion and lower GSIS in ß-cells induced by LA or HA treatments, while some underlying mechanisms need to be further explored.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Enhancing the Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Signaling and Arginine Deprivation in Melanoma.

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed.

Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

Dysregulation of homocysteine homeostasis in acute intermittent porphyria patients receiving heme arginate or givosiran.

Acute intermittent porphyria (AIP) is a rare metabolic disease caused by mutations within the hydroxymethylbilane synthase gene. Previous studies have reported increased levels of plasma total homocysteine (tHcy) in symptomatic AIP patients. In this study, we present long-term data for tHcy and related parameters for an AIP patient cohort (n = 37) in different clinical disease-states. In total, 25 patients (68%) presented with hyperhomocysteinemia (HHcy; tHcy > 15 µmol/L) during the observation period. HHcy was more frequent in AIP patients with recurrent disease receiving heme arginate, than in nonrecurrent (median tHcy: 21.6 µmol/L; range: 10-129 vs median tHcy: 14.5 µmol/L; range 6-77). Long-term serial analyses showed a high within-person tHcy variation, especially among the recurrent patients (coefficient of variation: 16.4%-78.8%). HHcy was frequently associated with low blood concentrations of pyridoxal-5'-phosphate and folate, while cobalamin concentration and the allele distribution of the methylene-tetrahydrofolate-reductase gene were normal. Strikingly, 6 out of the 9 recurrent patients who were later included in a regime of givosiran, a small-interfering RNA that effectively reduced recurrent attacks, showed further increased tHcy (median tHcy in 9 patients: 105 µmol/L; range 16-212). Screening of amino acids in plasma by liquid-chromatography showed co-increased levels of methionine (median 71 µmol/L; range 23-616; normal <40), suggestive of acquired deficiency of cystathionine-ß-synthase. The kynunerine/tryptophan ratio in plasma was, however, normal, indicating a regular metabolism of tryptophan by heme-dependent enzymes. In conclusion, even if HHcy was observed in AIP patients receiving heme arginate, givosiran induced an aggravation of the dysregulation, causing a co-increase of tHcy and methionine resembling classic homocystinuria.

Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Roles of nitric oxide and polyamines in brain tumor growth.

Nitric oxide (NO) and polyamines: putrescine, spermidine and spermine, are key arginine metabolites in mammalian tissues that play critical roles i.a. in regulation of vascular tone (NO), and cell cycle regulation (polyamines). In the brain, both classes of molecules additionally have neuromodulatory and neuroprotective potential, and NO also a neurotoxic potential. Here we review evidence that brain tumors use the NO- and polyamine-synthesizing machineries to the benefit of their differentiation and growth from healthy glia and neurons. With a few exceptions, brain tumors show increased activities of one or all of the three arginine (Arg) to NO-converting nitric oxide synthase (NOS) isoforms (iNOS, eNOS, nNOS), but also elevated activities of polyamines-generating and modifying enzymes: arginase I/II, ornithine decarboxylase and spermidine/spermine N1-acetyltransferase. The degree of stimulation of NO- and polyamine synthesis often correlates with brain tumor malignancy. Excess NO, but also spermine, spermidine and their N1-acetylated forms, are tumor- and context-dependently involved in angiogenesis, tumor initiation and growth, and resistance to chemo- or radiotherapy. Hypothetically, increased demand for NO and/or polyamines is likely to contribute to Arg auxotrophy of malignant brain tumors, albeit the causal nexus awaits experimental verification.

Arginine deprivation as a strategy for cancer therapy: An insight into drug design and drug combination.

Extensive studies have shown that cancer cells have specific nutrient auxotrophy and thus have much a higher demand for certain nutrients than normal cells. Amino acid deprivation has attracted much attention in cancer therapy with positive outcomes from clinical trials. Arginine, as one of the conditionally essential amino acids, plays a pivotal role in cellular division and metabolism. Since many types of cancer cells exhibit decreased expression of argininosuccinate synthetase and/or ornithine transcarbamylase, they are auxotrophic for arginine, which makes arginine deprivation an accessible choice for cancer treatment. Arginine deiminase (ADI) and human arginase (hArg) are the two major protein drugs used for arginine deprivation and are undergoing many clinical trials. However, the clinical application of ADI and hArg is facing some common problems, including their short half-lives, immunogenicity and inconsistent production, which underlines the importance of improving these drugs using protein engineering techniques. Thus, we systematically review the latest studies of protein engineering and anti-cancer studies based on in vitro, in vivo and clinical models of ADI and hArg, and we include the latest studies on drug combinations consisting of ADI/hArg with chemotherapeutic drugs.

Indospicine combined with arginine deprivation triggers cancer cell death via caspase-dependent apoptosis.

Arginine-deprivation therapy is a rapidly developing metabolic anticancer approach. To overcome the resistance of some cancer cells to this monotherapy, rationally designed combination modalities are needed. In this report, we evaluated for the first time indospicine, an arginine analogue of Indigofera plant genus origin, as potential enhancer compound for the metabolic therapy that utilizes recombinant human arginase I. We demonstrate that indospicine at low micromolar concentrations is selectively toxic for human colorectal cancer cells only in the absence of arginine. In arginine-deprived cancer cells indospicine deregulates some prosurvival pathways (PI3K-Akt and MAPK) and activates mammalian target of rapamycin, exacerbates endoplasmic reticulum stress and triggers caspase-dependent apoptosis, which is reversed by the exposure to translation inhibitors. Simultaneously, indospicine is not degraded by recombinant human arginase I and does not inhibit this arginine-degrading enzyme at its effective dose. The obtained results emphasize the potential of arginine structural analogues as efficient components for combinatorial metabolic targeting of malignant cells.

Rational design, engineer, and characterization of a novel pegylated single isomer human arginase for arginine depriving anti-cancer treatment.

AIMS: Arginine depleting enzymes are found effective to treat arginine-auxotrophic cancers and therapy-resistant malignancies, alone or in combination with cytotoxic agents or immune checkpoint inhibitors. We aim to select and validate a long-lasting, safe and effective PEGylated and cobalt-chelated arginase conjugated at the selective cysteine residue as a therapeutic agent against cancers. MAIN METHODS: Exploring pharmacokinetic and pharmacodynamic properties of the three arginase conjugates with different PEG modality (20 kDa linear as A20L, 20 kDa branched as A20Y, and 40 kDa branched as A40Y) by cell-based and animal studies. KEY FINDINGS: Arginase conjugates showed comparable systemic half-lives, about 20 h in rats and mice. The extended half-life of PEGylated arginase was concurrent with the integrity of conjugates of which PEG and protein moieties remain attached in bloodstream for 72 h after drug administration. Arginase modified with a linear 20 kDa PEG (A20L) was chosen as the lead candidate (PT01). In vitro assays confirmed the very potent cytotoxicity of PT01 against cancer cell lines of breast, prostate, and pancreas origin. In MIA PaCa-2 pancreatic and PC-3 prostate tumor xenograft models, weekly infusion of the PT01 at 5 and 10 mg/kg induced significant tumor growth inhibition of 44-67%. All mice experienced dose-dependent but rapidly reversible weight loss following each weekly dose, suggesting tolerable toxicity. SIGNIFICANCE: These non-clinical data support PT01 as the lead candidate for clinical development that may benefit cancer patients by providing an alternative cytotoxic mechanism.

Arginine depletion as a therapeutic approach for patients with COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a source of significant morbidity and death worldwide, and effective treatments are urgently needed. Clinical trials have focused largely on direct antiviral therapies or on immunomodulation in patients with severe manifestations of COVID-19. One therapeutic approach that remains to be clinically investigated is disruption of the host-virus relationship through amino acid restriction, a strategy used successfully in the setting of cancer treatment. Arginine is an amino acid that has been shown in nonclinical studies to be essential in the life cycle of many viruses. Therefore, arginine depletion may be an effective therapeutic approach against SARS-CoV-2. Several arginine-metabolizing enzymes in clinical development may be a viable approach to induce a low arginine environment to treat COVID-19 and other viral diseases. Herein, we explore the rationale for arginine depletion as a therapeutic approach for COVID-19.

Dual role of ER stress in response to metabolic co-targeting and radiosensitivity in head and neck cancer cells.

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.

Activation of autophagy following [HuArgI (Co)-PEG5000]-induced arginine deprivation mediates cell death in colon cancer cells.

Deregulating cellular energetics by reprogramming metabolic pathways, including arginine metabolism, is critical for cancer cell onset and survival. Drugs that target the specific metabolic requirements of cancer cells have emerged as promising targeted cancer therapeutics. In this study, we investigate the therapeutic potential of targeting colon cancer cells using arginine deprivation induced by a pegylated cobalt-substituted recombinant human Arginase I [HuArgI (Co)-PEG5000]. Four colon cancer cell lines were tested for their sensitivity to [HuArgI (Co)-PEG5000] as well as for their mechanism of cell death following arginine deprivation. All four cell lines were sensitive to arginine deprivation induced by [HuArgI (Co)-PEG5000]. All cells expressed ASS1 and were rescued from arginine deprivation-induced cytotoxicity by the addition of excess L-citrulline, indicating they are partially auxotrophic for arginine. Mechanistically, cells treated with [HuArgI (Co)-PEG5000] were negative for AnnexinV and lacked caspase activation. Further investigation revealed that arginine deprivation leads to a marked and prolonged activation of autophagy in both Caco-2 and T84 cell lines. Finally, we show that [HuArgI (Co)-PEG5000] causes cell death by sustained activation of autophagy as evidenced by the decrease in cell cytotoxicity upon treatment with chloroquine, an autophagy inhibitor. Altogether, these data demonstrate that colon cancer cells are partially auxotrophic for arginine and sensitive to [HuArgI (Co)-PEG5000]-induced arginine deprivation. They also show that the activation of autophagy does not play protective roles but rather, induces cytotoxicity and leads to cell death.

Phase II Study of Arginine Deprivation Therapy With Pegargiminase in Patients With Relapsed Sensitive or Refractory Small-cell Lung Cancer.

BACKGROUND: Pre-clinical studies indicated that arginine-deprivation therapy using pegylated arginine deiminase (pegargiminase, ADI-PEG 20) may be effective in patients with argininosuccinate synthetase 1 (ASS1)-deficient small-cell lung cancer (SCLC). PATIENTS AND METHODS: Patients were enrolled into either a 'sensitive' disease cohort (≥ 90 days response to first-line chemotherapy) or a 'refractory' disease cohort (progression while on chemotherapy or < 90 days afterwards or ≥ third-line treatment). Patients received weekly intramuscular pegargiminase, 320 IU/m2 (36.8 mg/m2), until unacceptable toxicity or disease progression. The primary endpoint was tumor response assessed by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 with secondary endpoints including tolerability, pharmacodynamics, and immunogenicity. RESULTS: Between January 2011 and January 2014, 22 patients were enrolled: 9 in the sensitive disease cohort and 13 in the refractory disease cohort. At a pre-planned interim analysis, the best overall response observed was stable disease in 2 patients in each cohort (18.2%). Owing to the lack of response and slow accrual in the sensitive disease cohort, the study was terminated early. Pegargiminase treatment was well-tolerated with no unexpected adverse events or discontinuations. CONCLUSION: Although pegargiminase monotherapy in SCLC failed to meet its primary endpoint of RECIST-confirmed responses, more recent molecular stratification, including MYC status, may provide new opportunities moving forward.

Kinetics of leukemic cells in 3D culture with stromal cells and with arginine deprivation stress.

Previously, we established a three-dimensional (3D) bone marrow culture system that maintains normal hematopoiesis, including prolongation of hematopoietic stem cell proliferation and differentiation. To analyze the role of bone marrow stromal cells that compose the microenvironment, the growth of a leukemic cell line (K562) in the 3D condition and with arginine deprivation stress was compared with two-dimensional stromal cell monolayers (2D) and suspension cultures without stromal cells (stroma (-)). Arginine is essential for the proliferation and differentiation of erythrocytes. The proliferation and differentiation of K562 cells cultured in the 3D system were stabilized compared with cells in 2D or stroma (-). Furthermore, the number of K562 cells in the G0/G1 phase in 3D was increased significantly compared with cells grown in 2D or stroma (-). Interestingly, the mRNA expression of various hematopoietic growth factors of stromal cells in 3D was not different from 2D, even though supportive activity on K562 cell growth was observed in the arginine deprivation condition. Thus, the hematopoietic microenvironment involves multi-dimensional and complex systems including biochemical and physiochemical factors that regulate quiescence, proliferation, activation, and differentiation of normal hematopoietic cells and cloned leukemic cells. Our 3D culture system may be a valuable new tool for investigating leukemic cell-stromal cell interactions in vitro.

A modified arginine-depleting enzyme NEI-01 inhibits growth of pancreatic cancer cells.

Arginine deprivation cancer therapy targets certain types of malignancies with positive result in many studies and clinical trials. NEI-01 was designed as a novel arginine-depleting enzyme comprising an albumin binding domain capable of binding to human serum albumin to lengthen its half-life. In the present work, NEI-01 is shown to bind to serum albumin from various species, including mice, rat and human. Single intraperitoneal administration of NEI-01 to mice reduced plasma arginine to undetectable level for at least 9 days. Treatment of NEI-01 specifically inhibited cell viability of MIA PaCa-2 and PANC-1 cancer cell lines, which were ASS1 negative. Using a human pancreatic mouse xenograft model, NEI-01 treatment significantly reduced tumor volume and weight. Our data provides proof of principle for a cancer treatment strategy using NEI-01.

Effects of Arginine and Its Deprivation on Human Glioblastoma Physiology and Signaling.

The observations that numerous cancers are characterized by impairment in arginine synthesis and that deficit of exogenous arginine specifically affects their growth and viability are the ground for arginine deprivation-based anticancer treatment strategy. This review addresses molecular mechanisms of the human glioblastoma cell response to arginine deprivation. Our earlier studies have shown that arginine deprivation specifically impairs glioblastoma cell motility, adhesion and invasiveness. These changes were evoked by alterations in the actin cytoskeleton organization resulting from a decreased arginylation of ß-actin isoform. Moreover, deficit of arginine induces prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response, not leading, however, to a massive apoptosis in glioblastoma cells. Our current research indicates that cell death could be augmented by other compounds such as modulators of ER stress, for example arginine analogue of plant origin, canavanine. Implication of these studies on the development of new anti-glioma metabolic therapeutic modalities are discussed.

Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression.

Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating the global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluated the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase 2 clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage.