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

B Delage,L-T Chen,C-F Li,P Luong,N Lemoine,R Cerio,C O’Riain,T Crook,S Joel,T J Mitchell,J Fitzgibbon,N Syed,A Papoudou-Bai,E Hatzimichael,L Maharaj,P W Szlosarek,S J Whittaker,J Bomalaski,J Gribben

doi:10.1038/cddis.2012.83

Abstract

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.

Highlights

Work in the early 1970s revealed for the first time that arginine was required for the survival of Burkitt lymphoma cells and murine lymphosarcoma cells.[9,10] As argininosuccinate synthetase-1 (ASS1) has not previously been studied in lymphoid malignancy, we screened for ASS1 expression in a large series of primary and relapsed lymphomas with comparative studies in normal lymphoid tissues
We showed that most B-cell lymphomas were negative for ASS1 protein expression, while B35% of cutaneous T cell lymphoma (CTCL) expressed ASS1
ASS1 loss has been identified in recent years as a potential biomarker of arginine auxotrophy in cancer cells, its regulation is complex and cell type-dependent

Summary

Introduction

Work in the early 1970s revealed for the first time that arginine was required for the survival of Burkitt lymphoma cells and murine lymphosarcoma cells.[9,10] As ASS1 has not previously been studied in lymphoid malignancy, we screened for ASS1 expression in a large series of primary and relapsed lymphomas with comparative studies in normal lymphoid tissues. ASS1 protein was largely absent in both normal and malignant lymphoid tissues, ASS1 promoter methylation was identified in the latter. Arginine depletion induced caspase-dependent cell death and autophagy in tumour cell lines treated with ADI-PEG20. Blocking autophagy with the antimalarial agent chloroquine enhanced the apoptotic effect of ADI-PEG20 in malignant lymphoid cell lines and primary B and T lymphoma cells. Our data reveal that ASS1 is epigenetically regulated in malignant lymphoid cells conferring arginine auxotrophy, and provide a rationale for targeting arginine deprivation to patients with ASS1methylated lymphomas

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