Abstract 3701: BRAF inhibitor (vemurafenib) resistance confers sensitivity to arginine deprivation in melanoma
Authors: Li Ying-Ying , Wu Chunjing , Chen Shu-Mei , Wangpaichitr Medhi , You Min , Spector Seth , Feun Lynn G. , Kuo Macus T. , Savaraj Niramol 
Published: May 2015

Abstract
The BRAF inhibitor, vemurafenib, has been proven to be efficacious for the treatment of melanomas harboring (V600E) mutation. While the response rate is high, resistance is inevitable. The response to salvage therapy is poor. While the MEK inhibitor may be able to enhance anti-tumor efficacy of vemurafenib, but patients eventually relapse. Hence, there is an urgent need for second line treatment for melanoma patients who fail vemurafenib treatment.
In this study, we discovered that BRAF inhibitor-resistant (BR) melanoma cells are exquisitely sensitive to arginine deprivation. Arginine deprivation was achieved using arginine degrading enzyme (pegylated arginine deiminase, ADI-PEG20, kindly provided by Polaris). ADI-PEG20 has been shown to induce autophagy in primary melanoma culture derived from patients whose tumors do not express argininosuccinate synthetase (ASS). Apoptosis does occur when these cells are exposed longer (5-7 days) and at higher doses. Inducible ASS expression upon arginine deprivation also impedes the antitumor effect of ADI-PEG20. We have found that BR cells from MEL-1220 (derived from patient with V600E and ASS (-)), A375, and A2058 cells (inducible ASS) cultured in the presence of vemurafenib at IC50 over 20 weeks had non-inducible ASS expression. MTT and Annexin V/PI analyses showed that ADI-PEG20 induced 3-6 fold increase in apoptosis and growth inhibition in BR cells compared to parental cells following 3-day incubation. These results were further confirmed by increased active (cleaved) form of caspase-3 as detected by western blotting after the addition of ADI-PEG20. The mouse xenograft model also verified that ADI-PEG20 abolished tumor growth of A2058BR cells while only retardation of growth seen in A2058 cells (the reduction rate of tumor volume is 92% vs. 63%). We found that two vital autophagic proteins (Beclin1 and ATG5) decreased in resistant cells after treatment with ADI-PEG20, which might explain the inability to undergo autophagy upon arginine deprivation as a survival mechanism and re-directing the cells toward apoptosis. The other mechanism leading to non-inducible ASS expression is c-Myc degradation. It has been proven that c-myc is a positive regulator in ASS transcription, and c-Myc stability is governed by ubiquitination. By immunoprecipitation assay, we found that BR cells had lower levels of deubiquitinating enzyme USP28, and hence cannot prevent c-Myc from ubiquitin-dependent prosteasomal degradation. Therefore, our results demonstrated that BRAF inhibitor resistance suppresses c-Myc-mediated ASS transcription and in turn increases sensitivity to ADI-PEG20.
In conclusion, ADI-PEG20 appears to be a good candidate for targeted therapy for melanoma patients who have tumor recurrence or disease progression after vemurafenib treatment. Supported by R01CA152197
Citation Format: Ying-Ying Li, Chunjing Wu, Shu-Mei Chen, Medhi Wangpaichitr, Min You, Seth Spector, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. BRAF inhibitor (vemurafenib) resistance confers sensitivity to arginine deprivation in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3701. doi:10.1158/1538-7445.AM2014-3701