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    A Phase I Study of Pegylated Arginine Deiminase (Pegargiminase), Cisplatin, and Pemetrexed in Argininosuccinate Synthetase 1-Deficient Recurrent High-grade Glioma. Hall Peter E,Lewis Rachel,Syed Nelofer,Shaffer Richard,Evanson Jane,Ellis Stephen,Williams Matthew,Feng Xiaoxing,Johnston Amanda,Thomson Jim A,Harris Fiona P,Jena Raj,Matys Tomasz,Jefferies Sarah,Smith Kate,Wu Bor-Wen,Bomalaski John S,Crook Timothy,O'Neill Kevin,Paraskevopoulos Dimitris,Khadeir Ramsay S,Sheaff Michael,Pacey Simon,Plowman Piers N,Szlosarek Peter W Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:Patients with recurrent high-grade gliomas (HGG) are usually managed with alkylating chemotherapy ± bevacizumab. However, prognosis remains very poor. Preclinically, we showed that HGGs are a target for arginine depletion with pegargiminase (ADI-PEG20) due to epimutations of argininosuccinate synthetase () and/or argininosuccinate lyase (). Moreover, ADI-PEG20 disrupts pyrimidine pools in ASS1-deficient HGGs, thereby impacting sensitivity to the antifolate, pemetrexed. PATIENTS AND METHODS:We expanded a phase I trial of ADI-PEG20 with pemetrexed and cisplatin (ADIPEMCIS) to patients with ASS1-deficient recurrent HGGs (NCT02029690). Patients were enrolled (01/16-06/17) to receive weekly ADI-PEG20 36 mg/m intramuscularly plus pemetrexed 500 mg/m and cisplatin 75 mg/m intravenously once every 3 weeks for up to 6 cycles. Patients with disease control were allowed ADI-PEG20 maintenance. The primary endpoints were safety, tolerability, and preliminary estimates of efficacy. RESULTS:Ten ASS1-deficient heavily pretreated patients were treated with ADIPEMCIS therapy. Treatment was well tolerated with the majority of adverse events being Common Terminology Criteria for Adverse Events v4.03 grade 1-2. The best overall response was stable disease in 8 patients (80%). Plasma arginine was suppressed significantly below baseline with a reciprocal increase in citrulline during the sampling period. The anti-ADI-PEG20 antibody titer rose during the first 4 weeks of treatment before reaching a plateau. Median progression-free survival (PFS) was 5.2 months (95% confidence interval (CI), 2.5-20.8) and overall survival was 6.3 months (95% CI, 1.8-9.7). CONCLUSIONS:In this recurrent HGG study, ADIPEMCIS was well tolerated and compares favorably to historical controls. Additional trials of ADI-PEG20 in HGG are planned. 10.1158/1078-0432.CCR-18-3729
    Rewiring of cisplatin-resistant bladder cancer cells through epigenetic regulation of genes involved in amino acid metabolism. Yeon Austin,You Sungyong,Kim Minhyung,Gupta Amit,Park Myung Hee,Weisenberger Daniel J,Liang Gangning,Kim Jayoung Theranostics Alterations in DNA methylation are important epigenetic markers in bladder cancer (BC). These epigenome modifications may drive the mechanisms of aggressive chemo-resistant BC. Clinicopathological biomarkers that indicate chemotherapeutic resistance are critical for better assessing treatment strategies for individual patients. Thus, in this study, we aimed to determine whether DNA methylation of certain metabolic enzymes is significantly altered in cisplatin-resistant BC cells. To characterize CpG methylation and nucleosome accessibility in cisplatin-resistant BC cells, the Illumina Infinium HM450 DNA methylation assay was performed. Perturbed gene expression was found to be associated with cisplatin resistance, and the biological roles of spermidine/spermine N-acetyltransferase (SAT1) and argininosuccinate synthase 1 (ASS1) were further studied using qRT-PCR analysis and various cell biology assays, including western blot. and , genes for amino acid and polyamine metabolism catalysts, respectively, were found to be vastly hypermethylated, resulting in greatly downregulated expression. ASS1 expression is of particular interest because prior studies have demonstrated its potential association with BC stage and recurrence. In regard to chemoresistance, we found that aberrant expression or induced stimulation of SAT1 restored cisplatin sensitivity in the cell culture system. We also found that the addition of exogenous arginine deiminase through administration of ADI-PEG 20 (pegylated arginine deiminase) increased ASS1 expression and enhanced cisplatin's apoptotic effects. Our study demonstrates a novel mechanistic link between the epigenetic perturbation of SAT1 and ASS1 and cancer metabolism in cisplatin-resistant bladder cancer cells. These findings suggest potential utility of and as predictive biomarkers in re-sensitizing bladder cancer to chemotherapy and personalizing therapy. 10.7150/thno.25130
    Arginine Depletion Therapy with ADI-PEG20 Limits Tumor Growth in Argininosuccinate Synthase-Deficient Ovarian Cancer, Including Small-Cell Carcinoma of the Ovary, Hypercalcemic Type. Ji Jennifer X,Cochrane Dawn R,Tessier-Cloutier Basile,Chen Shary Yutin,Ho Germain,Pathak Khyatiben V,Alcazar Isabel N,Farnell David,Leung Samuel,Cheng Angela,Chow Christine,Colborne Shane,Negri Gian Luca,Kommoss Friedrich,Karnezis Anthony,Morin Gregg B,McAlpine Jessica N,Gilks C Blake,Weissman Bernard E,Trent Jeffrey M,Hoang Lynn,Pirrotte Patrick,Wang Yemin,Huntsman David G Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. EXPERIMENTAL DESIGN:We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed using cell lines and patient-derived xenograft mouse models representing SCCOHT. RESULTS:Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype . Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth . CONCLUSIONS:Preclinical and studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT. 10.1158/1078-0432.CCR-19-1905
    Prognostic and therapeutic impact of argininosuccinate synthetase 1 control in bladder cancer as monitored longitudinally by PET imaging. Allen Michael D,Luong Phuong,Hudson Chantelle,Leyton Julius,Delage Barbara,Ghazaly Essam,Cutts Rosalind,Yuan Ming,Syed Nelofer,Lo Nigro Cristiana,Lattanzio Laura,Chmielewska-Kassassir Malgorzata,Tomlinson Ian,Roylance Rebecca,Whitaker Hayley C,Warren Anne Y,Neal David,Frezza Christian,Beltran Luis,Jones Louise J,Chelala Claude,Wu Bor-Wen,Bomalaski John S,Jackson Robert C,Lu Yong-Jie,Crook Tim,Lemoine Nicholas R,Mather Stephen,Foster Julie,Sosabowski Jane,Avril Norbert,Li Chien-Feng,Szlosarek Peter W Cancer research Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response. 10.1158/0008-5472.CAN-13-1702
    Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis. Delage B,Luong P,Maharaj L,O'Riain C,Syed N,Crook T,Hatzimichael E,Papoudou-Bai A,Mitchell T J,Whittaker S J,Cerio R,Gribben J,Lemoine N,Bomalaski J,Li C-F,Joel S,Fitzgibbon J,Chen L-T,Szlosarek P W Cell death & disease Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma. 10.1038/cddis.2012.83
    Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression. Kim Stephanie S,Xu Shili,Cui Jing,Poddar Soumya,Le Thuc M,Hayrapetyan Hovhannes,Li Luyi,Wu Nanping,Moore Alexandra M,Zhou Lei,Yu Alice C,Dann Amanda M,Elliott Irmina A,Abt Evan R,Kim Woosuk,Dawson David W,Radu Caius G,Donahue Timothy R Theranostics 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. 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. 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 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. 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. 10.7150/thno.40195
    A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas. Bean Gregory R,Kremer Jeff C,Prudner Bethany C,Schenone Aaron D,Yao Juo-Chin,Schultze Matthew B,Chen David Y,Tanas Munir R,Adkins Douglas R,Bomalaski John,Rubin Brian P,Michel Loren S,Van Tine Brian A Cell death & disease Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology. 10.1038/cddis.2016.232
    ASS1 as a novel tumor suppressor gene in myxofibrosarcomas: aberrant loss via epigenetic DNA methylation confers aggressive phenotypes, negative prognostic impact, and therapeutic relevance. Huang Hsuan-Ying,Wu Wen-Ren,Wang Yu-Hui,Wang Jun-Wen,Fang Fu-Min,Tsai Jen-Wei,Li Shau-Hsuan,Hung Hsiao-Chin,Yu Shih-Chen,Lan Jui,Shiue Yow-Ling,Hsing Chung-His,Chen Li-Tzong,Li Chien-Feng Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:The principal goals were to identify and validate targetable metabolic drivers relevant to myxofibrosarcoma pathogenesis using a published transcriptome. EXPERIMENTAL DESIGN:As the most significantly downregulated gene regulating amino acid metabolism, argininosuccinate synthetase (ASS1) was selected for further analysis by methylation-specific PCR, pyrosequencing, and immunohistochemistry of myxofibrosarcoma samples. The roles of ASS1 in tumorigenesis and the therapeutic relevance of the arginine-depriving agent pegylated arginine deiminase (ADI-PEG20) were elucidated in ASS1-deficient myxofibrosarcoma cell lines and xenografts with and without stable ASS1 reexpression. RESULTS:ASS1 promoter hypermethylation was detected in myxofibrosarcoma samples and cell lines and was strongly linked to ASS1 protein deficiency. The latter correlated with increased tumor grade and stage and independently predicted a worse survival. ASS1-deficient cell lines were auxotrophic for arginine and susceptible to ADI-PEG20 treatment, with dose-dependent reductions in cell viability and tumor growth attributable to cell-cycle arrest in the S-phase. ASS1 expression was restored in 2 of 3 ASS1-deficient myxofibrosarcoma cell lines by 5-aza-2'-deoxycytidine, abrogating the inhibitory effect of ADI-PEG20. Conditioned media following ASS1 reexpression attenuated HUVEC tube-forming capability, which was associated with suppression of MMP-9 and an antiangiogenic effect in corresponding myxofibrosarcoma xenografts. In addition to delayed wound closure and fewer invading cells in a Matrigel assay, ASS1 reexpression reduced tumor cell proliferation, induced G1-phase arrest, and downregulated cyclin E with corresponding growth inhibition in soft agar and xenograft assays. CONCLUSIONS:Our findings highlight ASS1 as a novel tumor suppressor in myxofibrosarcomas, with loss of expression linked to promoter methylation, clinical aggressiveness, and sensitivity to ADI-PEG20. 10.1158/1078-0432.CCR-12-2641
    Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors. Prudner Bethany C,Rathore Richa,Robinson Anthony M,Godec Abigail,Chang Samuel F,Hawkins William G,Hirbe Angela C,Van Tine Brian A Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:The response to acute and long-term arginine starvation results in a conditional adaptive metabolic reprogramming that can be harnessed for therapeutic opportunities in ASS1-negative tumors. Here, we investigate the underlying biology of priming ASS1 tumors with arginine deiminase (ADI-PEG20) before treatment with gemcitabine (GEM) and docetaxel (DTX) in sarcoma, pancreatic cancer, and melanoma cell lines. EXPERIMENTAL DESIGN:ASS1 tumor cell lines were treated to create LTAT (long-term ADI treated) cell lines (ASS1) and used for drug combination studies. Protein expression of ASS1, dCK, RRM2, E2F1, c-MYC, and hENT1 was measured. c-MYC activity was determined, live-cell immunofluorescent studies for hENT1, uptake assays of FITC-cytosine probe, and rescue studies with a c-MYC inhibitor were all determined in the presence or absence of the ADI-PEG20:GEM:DTX. RESULTS:In examining modulations within the pyrimidine pathway, we identified that the addition of DTX to cells treated with ADI-PEG20 resulted in translocation of stabilized c-Myc to the nucleus. This resulted in an increase of hENT1 cell-surface expression and rendered the cells susceptible to GEM. studies demonstrate that the combination of ADI-PEG20:GEM:DTX was optimal for tumor growth inhibition, providing the preclinical mechanism and justification for the ongoing clinical trial of ADI-PEG20, GEM, and DTX in sarcoma. CONCLUSIONS:The priming of tumors with ADI-PEG20 and DTX results in the stabilization of c-MYC potentiating the effect of GEM treatment via an increase in hENT1 expression. This finding is applicable to ASS1-deficient cancers that are currently treated with GEM. 10.1158/1078-0432.CCR-19-0206
    Arginine starvation impairs mitochondrial respiratory function in ASS1-deficient breast cancer cells. Qiu Fuming,Chen Yun-Ru,Liu Xiyong,Chu Cheng-Ying,Shen Li-Jiuan,Xu Jinghong,Gaur Shikha,Forman Henry Jay,Zhang Hang,Zheng Shu,Yen Yun,Huang Jian,Kung Hsing-Jien,Ann David K Science signaling Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer biosamples, suggesting that patients with such tumors could be candidates for arginine starvation therapy. 10.1126/scisignal.2004761
    Androgen receptor regulates ASS1P3/miR-34a-5p/ASS1 signaling to promote renal cell carcinoma cell growth. Wang Kefeng,Sun Yin,Guo Changcheng,Liu Tao,Fei Xiang,Chang Chawnshang Cell death & disease Recent studies have demonstrated that the androgen receptor (AR) could play important roles to promote renal cell carcinoma (RCC) cell proliferation, and other studies have also indicated that suppressing the argininosuccinate synthase 1 (ASS1) could promote proliferation of various tumors. The potential of AR promoting cell proliferation in RCC via altering ASS1, however, remains unclear. Here we found that the expression of ASS1 was lower in RCC tissues than in adjacent normal renal tissues, and a lower ASS1 expression was linked to a worse prognosis in RCC patients. Mechanism dissection showed that AR could decrease ASS1 expression to promote RCC cell proliferation via ASS1P3, a pseudogene of ASS1. The results of RIP assay and AGO2 assay revealed that AR could bind ASS1P3 to increase RCC cell proliferation via altering miR-34a-5p function, which could bind to the 3'UTR of ASS1 to suppress its protein expression. ASS1P3 could function as a miRNA decoy for miR-34a-5p to regulate ASS1 in RCC. Preclinical study also supports the in vitro data. Together, these results demonstrated that ASS1P3 could function as a competing endogenous RNA to suppress RCC cell progression, and targeting this newly identified AR-mediated ASS1P3/miR-34a-5p/ASS1 signaling might help in blocking proliferation. 10.1038/s41419-019-1330-x
    Acid-Induced Downregulation of ASS1 Contributes to the Maintenance of Intracellular pH in Cancer. Silberman Alon,Goldman Omer,Boukobza Assayag Odeya,Jacob Adi,Rabinovich Shiran,Adler Lital,Lee Joo Sang,Keshet Rom,Sarver Alona,Frug Julia,Stettner Noa,Galai Sivan,Persi Erez,Halpern Keren Bahar,Zaltsman-Amir Yehudit,Pode-Shakked Ben,Eilam Raya,Anikster Yair,Nagamani Sandesh C S,Ulitsky Igor,Ruppin Eytan,Erez Ayelet Cancer research Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) by either promoter methylation or by HIF1α is associated with increased metastasis and poor prognosis in multiple cancers. We have previously shown that in normoxic conditions, ASS1 downregulation facilitates cancer cell proliferation by increasing aspartate availability for pyrimidine synthesis by the enzyme complex CAD. Here we report that in hypoxia, ASS1 expression in cancerous cells is downregulated further by HIF1α-mediated induction of miR-224-5p, making the cells more invasive and dependent on upstream substrates of ASS1 for survival. ASS1 was downregulated under acidic conditions, and ASS1-depleted cancer cells maintained a higher intracellular pH (pHi), depended less on extracellular glutamine, and displayed higher glutathione levels. Depletion of substrates of urea cycle enzymes in ASS1-deficient cancers decreased cancer cell survival. Thus, ASS1 levels in cancer are differentially regulated in various environmental conditions to metabolically benefit cancer progression. Understanding these alterations may help uncover specific context-dependent cancer vulnerabilities that may be targeted for therapeutic purposes. SIGNIFICANCE: Cancer cells in an acidic or hypoxic environment downregulate the expression of the urea cycle enzyme ASS1, which provides them with a redox and pH advantage, resulting in better survival. http://cancerres.aacrjournals.org/content/canres/79/3/518/F1.large.jpg. 10.1158/0008-5472.CAN-18-1062