Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Delage, Barbara; Luong, Phuong; Maharaj, Lenushka; O’Riain, Ciarán; Syed, Nelofer; Crook, Tim; Hatzimichael, Eleftheria; Papoudou‐Bai, Alexandra; Mitchell, Tracey; Whittaker, Sean; Cerio, Rebecca J.; Gribben, John G.; Lemoine, Nicholas R.; Bomalaski, John S.; Li, Chunfang; Joel, S.; Fitzgibbon, Jude; Lt, Chen; Szlosarek, Peter W.

doi:10.1038/cddis.2012.83

Cited by 116 publications

(135 citation statements)

References 26 publications

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“…In contrast, in the ASS1-methylated NMFH-1 cells, 5-aza-2 0 -deoxycytidine pretreatment failed to regain ASS1 expression irrespective of combined or separate use of EZH2 knockdown and various inhibitors against formation of repressive histone codes. This finding in NMFH-1 myxofibrosarcoma cells was not in keeping with the series of ASS1-methylated T-cell lymphoma cell lines, in which the ASS1 enzyme was readily reexpressed by demethylating agents (35). Our results indicated that a combination therapy of ADI-PEG20 and demethylating agents appears without effect in myxofibrosarcomas, a subset of which may use additional mechanism(s) other than DNA methylation and/or deregulated histone modifications to abolish ASS1 expression.…”

Section: Discussioncontrasting

confidence: 64%

“…However, the mechanistic underpinning responsible for suppression or reexpression of ASS1 is yet to be elucidated, especially in cancer tissues. Until recently, DNA methylation was identified as an inactivating mechanism of ASS1 expression only in pleural mesotheliomas, lymphomas, and ovarian carcinomas (27,29,35). Nevertheless, data for ASS1 methylation in hepatocellular carcinomas or melanomas are limited, despite being 2 of the best-known neoplasms devoid of ASS1 expression, with auxotrophy for arginine and sensitivity to ADI-PEG20 (20).…”

Section: Discussionmentioning

confidence: 99%

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ASS1as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance

Huang

Wang

et al. 2013

Clinical Cancer Research

127

126

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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 0 -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 G 1 -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.

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Section: Discussioncontrasting

confidence: 64%

Section: Discussionmentioning

confidence: 99%

ASS1as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance

Huang

Wang

et al. 2013

Clinical Cancer Research

127

126

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“…The dose of ADI-PEG 20 was derived from leukemia cell line studies and prior publications. 21,23 Three days after treatment, cells were harvested and counted using a Vi-CELL Cell Viability Analyzer (Beckman).…”

Section: Methylation Of Ass1 Genementioning

confidence: 99%

Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo

Miraki-Moud

Ghazaly

Ariza‐McNaughton

et al. 2015

Blood

116

103

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Key Points• Most AMLs lack ASS1, which allows synthesis of arginine, and so depend on exogenous sources.• Depletion of arginine via ADI-PEG 20 reduces the burden of primary AML in vivo and in vitro.The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia.Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials. (Blood. 2015;125(26): 4060-4068)

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“…The demethylating agent decitabine reexpressed ASS1 protein in 253J, UMUC-3, and T24 cells, confirming epigenetic silencing as a key mechanism involved in ASS1 inactivation in bladder cancer cells (data not shown; ref. 22).…”

Section: Methylated Ass1 Is Linked To Increased Proliferation and Invmentioning

confidence: 99%

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

et al. 2014

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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. Cancer Res; 74(3); 896-907. Ó2013 AACR.

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Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Cited by 116 publications

References 26 publications

ASS1as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance

ASS1as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance

Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

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