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

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.

Figure 1

Lymphoid ASS1 immunostaining and methylation profiling. (a) Representative immunohistochemical stains for ASS1 as follows: ASS1-negative tonsillar lymphocytes; ASS1-negative FL, DLBCL, CTCL (Pautrier's microabcess) biopsies with internal positive controls (i.e., endothelial and epithelial cells); and an ASS1-positive MZL biopsy. Images were obtained using a Zeiss Axiophot microscope, magnification × 200, with Axiovision image acquisition software. (b) Differential methylation of the ASS1 promoter in a panel of normal and malignant lymphoid cell lines and primary tissues. Quantitative methylation values (beta values) were obtained as previously described. Beta values are reported for two CpG loci within the ASS1 gene (373 bp 5′ to the TSS and 7 bp 3′ to the TSS). The overall incidence of CpG island methylation was increased in malignant compared with benign samples

Figure 2

ASS1 status in normal and malignant lymphoid cell lines and sensitivity to ADI-PEG20. (a) Normal immortalised lymphoblastoid and lymphoma cell lines were screened for ASS1 mRNA and protein expression by qPCR and western blotting, respectively; ASS1 methylation was assessed using MS-PCR: HRC57, NcNc (normal lymphoblastoid cell lines); DoHH2, WSU, Karpas-422, RL2261 (FL cell lines); SUDHL-6, SUDHL-8, SUDHL-16 (DLBCL cell lines); Ramos (Burkitt's lymphoma cell line); and SeAx, MyLa (CTCL cell lines). The Jurkat leukaemia cell line was used as a positive control for ASS1 expression. A good correlation was observed between the methylation status of cell lines and the corresponding levels of ASS1 mRNA and protein. (b) Cells were treated with the arginine-depleting drug ADI-PEG20 and harvested after 2 or 4 days. Cell counting and viability were determined using the Beckman Vi-Cell cell viability analyser. Increasing concentrations of ADI-PEG20 (concentration range: 0–500 ng/ml) decreased the number of ASS1-negative lymphoma viable cells with no change in the viability of ASS1-positive cell lines by day 4. For each cell line, the viability was expressed as a percentage of untreated cells at day 2; *P<0.05 represents statistical difference to the baseline untreated cells for each cell line at each time point; ^§P<0.05 represents statistical difference at day 4 compared with day 2 for each cell line at each ADI-PEG20 concentration. The data are representative of three independent experiments. Error bars represent S.D.

Figure 3

Induction of ASS1 expression in 5-Aza-dC-treated lymphoma cells confers resistance to ADI-PEG20. Identically treated and 5-Aza-dC (5 μM)-untreated MyLa and SeAx cells were split into new flasks and treated with or without 500 ng/ml of ADI-PEG20 for a further 4 days. (a) ASS1 promoter methylation was analysed using MS-PCR and PCR products resolved on a 2% agarose gel. For each treatment, unmethylated (U) and methylated (M) reactions are shown. (b) ASS1 mRNA expression was quantified by qPCR. *P<0.05 represents statistical significance compared with untreated cells for each cell line. (c) Detection of ASS1 expression by western blotting using a Jurkat cell line as an ASS1-positive control. (d) At the end of the 12-day experiment, cells were counted using the Beckman Vi-Cell cell viability analyser. *P<0.05 represents statistical significance compared with untreated cells for each cell line. Error bars represent S.D. The data are representative of three or more independent experiments

Figure 4

ADI-PEG20 induces caspase-dependent apoptosis in ASS1-methylated lymphoma cell lines. (a) The three cell lines were treated with ADI-PEG20 (0–500 ng/ml) for 4 days. Cells were then harvested and fixed in 70% ethanol for at least 24 h. The apoptotic fraction of cells, identified as the sub-G1 population of cell cycle was measured by flow cytometry with propidium iodide staining (described in Materials and Methods); *P<0.05 represents statistical significance compared with untreated cells for each cell line; protein lysates from (b) NcNc (c) Karpas-422 and (d) MyLa cells treated with ADI-PEG20 (0–500 ng/ml) were analysed by western blotting for the expression of ASS1 and the detection of cleaved PARP and caspase-3

Figure 5

ADI-PEG20-induced autophagy and its modulation by chloroquine in lymphoma cells. (a) Malignant lymphoid cells were treated with ADI-PEG20 (500 ng/ml) either alone or in combination with chloroquine (CQ, 25 μM) and assessed for LC3-I and LC3-II levels by western blotting. (b) Follicular (Karpas-422) and CTCL (MyLa) lymphoma cells were treated with ADI-PEG20 and CQ for 72 h, and analysed for the subG1 fraction corresponding to apoptotic cells by flow cytometry; *P<0.001. (c) Similarly, primary malignant B lymphoma and CTCL (Sezary syndrome) lymphoma cells were studied using the ViaLight assay. Doxorubicin was used as a positive control for lymphoma cell cytotoxicity. *P<0.05, **P<0.001. Error bars represent S.D.

Figure 6

(a) ADI-PEG20 treatment of a patient with an ASS1-methylated CTCL. (a) Baseline appearance with marked skin oedema due to CTCL (Sezary syndrome). (b) Resolution of skin oedema after a single dose of ADI-PEG20 (160 IU/m², i.m.). (c) Baseline tumour biopsy showing ASS1-negative CTCL cells, with skin appendageal and endothelial cells showing strong ASS1 expression ( × 400). (d) Methylated ASS1 in the tumour biopsy confirmed by MS-PCR