BackgroundMany advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20.MethodsASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR.ResultsA good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied.ConclusionASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC.

PEGylated arginine deiminase (ADI-PEG 20) is being investigated in clinical studies in arginine auxotrophic cancers and is well-tolerated. The anti-tumor properties of ADI-PEG 20 have been extensively investigated - ADI-PEG 20 inhibits the growth of auxotrophic cancers in vitro and in vivo - however, its impact on immune cells is largely unknown. Here we report the potential impact of ADI-PEG 20 on the tumor immune microenvironment. ADI-PEG 20 induced immunosuppressive programmed death-ligand 1 expression on some cancer cells in vitro, but the magnitude of the increase was cell line dependent and in most relatively small. Using healthy donor human peripheral blood mononuclear cells (PBMCs) we discovered that when present during initiation of T cell activation (but not later on) ADI-PEG 20 can inhibit their differentiation after early activation stage manifested by the expression of CD69 marker.In vivo, ADI-PEG 20 induced tumor T-cell infiltration in a poorly immunogenic syngeneic mouse melanoma B16-F10 model and reduced its growth as a single agent or when combined with anti-PD-1 mAb. It was also effective by itself or in combination with anti-PD-L1 mAb in CT26 colon carcinoma syngeneic model.

Many cancers (unlike normal cells) are deficient in argininosuccinate synthetase-1 (ASS1), a critical enzyme required for arginine synthesis from citrulline, which makes them dependent on importing extracellular arginine. Pegylated arginine deiminase (ADI-PEG 20) depletes the external supply of arginine by degrading it to citrulline thereby affecting growth and viability of cancer cells while sparing normal tissues which express ASS1. ADI-PEG 20 is being investigated in clinical studies in arginine auxotrophic cancers and is well-tolerated. Arginine is used in a number of metabolic pathways including those affecting tumor growth and immune cell regulation. The anti-tumor properties of ADI-PEG 20 have been extensively investigated, however, its impact on immune cells is largely unknown. Therefore, we have investigated potential implications of ADI-PEG 20 treatment for tumor immune environment. We studied how ADI-PEG 20 affects T cell subsets in healthy donor human peripheral blood mononuclear cells (PBMCs) under resting and activation conditions and expression of immunosuppressive programmed death-ligand 1 (PD-L1) on cancer cell lines. PBMCs were treated with ADI-PEG 20 under resting and activation conditions and were characterized by immune cell phenotyping using flow cytometry. For PBMC stimulation we used anti-CD3/CD28 beads or phytohemagglutinin (PHA). During stimulation (and not under resting conditions) ADI-PEG 20 markedly boosted CD69 expression on T cells (both CD4+ and CD8+) while moderating their exhaustion (CTLA-4 and PD-1 levels remained low, similar to that at a resting state). Moreover, ADI-PEG 20 reduced accumulation of regulatory T cells (Treg) induced by anti-CD3/CD28 beads. The effect of ADI-PEG 20 on PD-L1 expression in ASS1-low cancer cell lines was analyzed by flow cytometry and RT-qPCR. ADI-PEG 20 treatment induced PD-L1 upregulation on some cancer cell lines in vitro, the magnitude of the upregulation was cell line dependent and in most it was much lower than that induced by 200 ng/mL IFNγ. Based on our findings that ADI-PEG 20 downmodulates Treg cells we have hypothesized that ADI-PEG 20 may improve immunogenicity of non-immunogenic tumors. To test this hypothesis we used poorly immunogenic syngeneic mouse melanoma B16-F10 model. IHC analysis of the tumor sections revealed that five out of six ADI-PEG 20 treated animals had a large number of T cells in their tumors; only one ADI-PEG 20 treated mouse had very little tumor T cell infiltrate, similar to the non-treated controls. This demonstrates that ADI-PEG 20 can improve tumor immunogenicity. ADI-PEG 20 also inhibits growth of the B16-F10 tumor in vitro and in vivo. In summary, we discovered that ADI-PEG 20 increases healthy human donor effector T cell activation and blocks their exhaustion while reducing accumulation of Tregs in vitro. While ADI-PEG 20 induced PD-L1 expression in some cancer cell lines in vitro this increase was relatively small in most studied cell lines and is unlikely to significantly contribute to tumor immune evasion. Importantly, ADI-PEG 20 treatment resulted in a marked increase in tumor infiltrating T cells in a poorly immunogenic B16-F10 melanoma model. Our data suggests that in addition to the direct anti-tumor effects ADI-PEG 20 can help boost tumor immune surveillance and could be a good primer for an additional anti-tumor immune therapy. Citation Format: Elena Brin, Katherine Wu, Hsin-Tze Lu, Yudou He, Zhaoming Dai, Wei He. Arginine deiminase can boost anti-tumor immune surveillance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A31.

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