Abstract 2900: Biochemical alterations in BRAF inhibitor (BRAFi) resistant melanoma cells increase their vulnerability to arginine deprivation
Authors: Li Ying-Ying , Savaraj Niramol , Wu Chunjing , Chen Shumei , Wangpaichitr Medhi , Kuo Macus T. , Feun Lynn G. 
Published: August 2015

Abstract
The BRAFi, has been applied to treat melanomas harboring (V600E) mutation. While the response rate is high, resistance is inevitable. Despite combination with MEK inhibitor which enhances the antitumor efficacy as well as increases duration of response, patients ultimately relapse. Therefore, there is an urgent need for second line treatment for melanoma patients who are resistant to BRAFi treatment.
In this study, we found that BRAFi-resistant (BR) melanoma cells are susceptible to arginine deprivation due to their inability to turn on argininosuccinate synthetase (ASS), a key enzyme for arginine synthesis as well as their inability to undergo autophagy. Thus, arginine deprivation using pegylated arginine deiminase (ADI-PEG20, supplied by Polaris Inc.) which degrades arginine to citrulline results in cell death. We established 6 BR cell lines from BRAF mutant (V600E) cell lines including ASS-negative cell lines such as A375 and MEL-1220 (derived from a patient) and ASS-positive and/or inducible cell lines include UACC-62, SK-MEL28, A2058, and MEL-GP (derived from a patient) by continuous exposure to vemurafenib at IC50 over 30 weeks. MTT, caspase activity, and Annexin V/PI analyses showed that treatment with ADI-PEG20 (100ng/ml) for 72 hr induced 2-3 fold increase in growth inhibition and 10-30% increase in apoptosis in BR cells compared to their parental cells counterpart. The xenograft mouse model also confirmed that ADI-PEG20 abrogated tumor growth of A2058BR cells but only retarded growth seen in A2058 cells (tumor growth inhibition (T/C ratio) in A2058 and A2058BR is 38.7% vs. 8.3%; T/C&lt;42% indicates significant inhibition). The results observed by cyto-ID staining combined with fluorescent microscope and electron microscope also exhibited 40-80% decrease in autophagosome formation in BR cells compared to parental cells after treatment with ADI-PEG20.
We further found that BRAFi-resistance attenuated autophagy and ASS re-expression due to loss of Atg5 and c-Myc. Atg5-overexpressed plasmid rescued 12-35% apoptosis in MEL-1220BR and A2058BR cells through autophagic process as evidenced by more autophagosome formation following treatment with ADI-PEG20. The other mechanism leading to attenuated ASS re-expression is decreased c-Myc (positive regulator of ASS transcription). It is known that c-Myc stability is governed by ubiquitination-dependent proteasomal degradation. Using immunoprecipitation assay, our results showed that the levels of deubiquitinating enzyme, USP28, were too low to prevent c-Myc from ubiquitination. Overall, our data suggest that attenuated Atg5 which halted autophagy and decreased c-Myc which prevented re-expression of ASS made the BR cells vulnerable to cell death upon arginine deprivation. Thus, ADI-PEG20 is a potential candidate for salvage therapy in BR patients (Supported by R01CA152197).
Citation Format: Ying-Ying Li, Niramol Savaraj, Chunjing Wu, Shumei Chen, Medhi Wangpaichitr, Macus T. Kuo, Lynn G. Feun. Biochemical alterations in BRAF inhibitor (BRAFi) resistant melanoma cells increase their vulnerability to arginine deprivation. [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 2900. doi:10.1158/1538-7445.AM2015-2900