Abstract 3642: Relationship of argininosuccinate synthetase (ASS) and PD-L1 expression in arginine enriched or depleted environment in melanoma
Authors: Wu Chunjing , Li Ying-Ying , Wangpaichitr Medhi , Feun Lynn G. , Kuo Macus T. , Savaraj Niramol 
Published: August 2018

Abstract
While treatment with BRAF/MEK inhibitors represents a breakthrough for melanoma with 80% response rate, recurrences often occur. Salvage treatment with check point inhibitors yield 40% response rate, but usually time to response is long and not suitable for aggressive disease. In this report, we investigate a new modality to improve treatment outcome in melanoma. We have previously shown that BRAF inhibitor resistant (BR) and combined BRAF and MEK resistant (BMR ) melanomas express low levels of argininosuccinate synthetase (ASS1-) compared to their parental cells (Oncotarget 7:17665 ,2016) . Treatment with pegylated arginine deiminase (ADI-PEG20 from Polaris Inc), which degrades arginine (Arg) to citrulline results in growth inhibition and cell death. However, its effect on check point inhibitor ligand PD-L-1 is not known. In this report, we studied PD-L1 gene expression upon arginine deprivation in parental, and their respective BR and BMR cells as well as BRAF wild type cell. We have analyzed PD-L1 expression by flow cytometry as well as immunoblot in 4 pairs of melanoma cells (A2058 ,A375, Mel1220 and UACC62) and their BR and BMR counterpart . Our results showed a 5-20% increase in PD-L1 expression in both BMR and BR cells. BMR cells showed higher levels ( 5-10%) more compared to BR cells. The highest levels of increase in BR and BMR cells are found in A2058. Interestingly, this cell line is known to have inducible ASS upon arginine deprivation. The induction can be found at 72 hr. post ADI-PEG20 treatment. Next, we treated this panel of cell lines with ADI-PEG20 for three days and assessed the PD-L1 expression. The results showed that PD-L1 expression increased slightly (&lt;5% ) in parental cells but more in BR and BMR cells (10-20% ). Interestingly, A-2058 R, a cell line made resistant to ADI-PEG20 with high levels of ASS expression, expressed the highest amount of PD-L1 (approximately 50 times higher). In contrast, the ASS transfected cell lines (A2058ass, A375ass , Mel1220ass) showed no changes in PD-L1 compared to their parental cells. This indicates that ASS does not influence PD-L1 expression. However, Arg deprivation in (ASS-) cells is the factor influencing PD-L1 expression. Interestingly, we also found increase in PD-L1 expression in tumor samples obtained from patients who failed BRAF and MEK inhibitor. On the other hand, melanoma cells with wild type BRAF and low ASS expression (one cell line and 5 primary cultures ) showed variable amounts of PD-L1 expression with minor increase upon ADI-PEG 20 treatment. There is no relationship with mTOR activity or pERK with PD-L1 expression in these panels of cell lines. The mechanism(s) involving HIF1 alpha is being investigated. Overall, our data suggest that combination with ADI-PEG20 and PD-L1 inhibitor, sequentially or concomitantly, should be beneficial for BR and BMR patients. Supported by VA Merit Review Award (1BX003328).
Citation Format: Chunjing Wu, Ying-Ying Li, Medhi Wangpaichitr, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. Relationship of argininosuccinate synthetase (ASS) and PD-L1 expression in arginine enriched or depleted environment in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3642.