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Uveal melanoma, involving the iris, choroid and ciliary body, is the commonest intraocular tumor in adults. Approximately half of these patients will develop metastasis with a high mortality despite currently available systemic therapies including immune checkpoint blockade. The urea cycle enzyme argininosuccinate synthetase 1 (ASS1), responsible for arginine synthesis, is downregulated in melanoma and other cancers and these are therefore sensitive to arginine deprivation therapy. In early phase trials of the arginine depletor pegylated arginine deiminase, ADI-PEG20, uveal melanoma, in particular, was identified with potential for further therapeutic development. Here, we tested uveal melanoma cell lines for sensitivity to ADI-PEG20 and analyzed enucleated tumors for ASS1 expression to assess the extent of ASS1 deficiency. Methods: ASS1 gene and protein expression were assessed in three uveal melanoma cell lines (OMM1, OMM2.5 and Mel270) by real-time quantitative PCR (qPCR) and western blot analysis, respectively. Sensitivity to ADI-PEG20 was performed using the cell viability MTS assay. We screened 102 enucleated choroidal and ciliary body melanomas for ASS1 protein using red chromogen for the immunohistochemistry (IHC) and selected the most positive area for scoring. Results: The uveal melanoma cell lines expressed negligible ASS1 mRNA with a complete absence of ASS1 protein. All three ASS1 negative uveal melanoma cell lines were sensitive to ADI-PEG20 by day 6 of the MTS assay, whereas an ASS1-expressing positive control cell line was resistant. There was a convincing lack of ASS1 expression ( Conclusion: ASS1 is absent in the tested uveal melanoma cell lines and predisposes to arginine sensitivity with ADI-PEG20 in vitro. Most primary uveal melanomas have a marked deficiency of ASS1, representing a good target for exploring arginine deprivation further in the clinic, either alone or in combination with rationally selected agents. A trial of ADI-PEG20 combined with carboplatin and paclitaxel is planned in metastatic malignant melanoma with an expanded cohort in patients with uveal disease. Citation Format: Ramsay S. Khadeir, Melissa M. Phillips, Mandeep S. Sgoo, Amit Arora, Victoria Cohen, Caroline Thaung, Peter W. Szlosarek. Widespread deficiency of ASS1 in uveal melanoma and sensitivity to pegylated arginine deiminase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2015-1156