Abstract 2671: BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation
Authors: Li Ying-Ying , Wu Chunjing , Shah Sumedh , Chen Shu-Mei , Wangpaichitr Medhi , Feun Lynn , Kuo Macus , Suarez Miguel , Savaraj Niramol 
Published: July 2016

Abstract
The combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) has been FDA approved to treat melanomas harboring (V600E) mutation. Although most BRAF mutant melanomas are highly responsive to these treatments, they all relapse ultimately. In this study, we found a unique mechanism which can be used to treat BRAFi resistant patients. Five BRAFi resistant (BR) cells were established from known BRAF mutant cell lines A375, MEL-1220, A2058, UACC-62, and SK-MEL28 by long-term exposure to BRAFi. These BR cells are hypersensitive to ADI-PEG20, an enzyme which degrades arginine to citrulline. ADI-PEG20 treatment resulted in a 2-3-fold decrease in BR cell viability (MTT assay) and 10-30% increase in apoptosis (Annexin V/PI) in BR cells compared to their paternal cells. The mechanisms involved are as follows: All BR cells express very low levels or do not express argininosuccinate synthetase (ASS, a key enzyme to generate arginine from citrulline). Thus, these BR cells rely primarily on exogenous arginine. Furthermore, these BR cells also have attenuation of glucose uptake which makes them rely mainly on amino acids. Importantly, their ability to undergo autophagy upon nutritional stress is also impaired. The underlying mechanism for low levels of ASS is due to diminished c-Myc expression which is a positive regulator for ASS. Additionally, AMPK-α1, which possesses phosphorylation site at Thr172 governing autophagy and glucose uptake, was attenuated in BR cells. Overexpression of AMPK-α1 using plasmid transfection in A2058BR and MEL-1220BR cells rescued 8-28% ADI-PEG20-induced apoptosis and enhanced autophagosome formation (detected by LysoTacker). Conversely, knockdown of AMPK-α1 using siRNA significantly enhanced ADI-PEG20-reduced cell viability (compared to non-targeting siRNA) in A2058 and MEL-1220 cells through attenuation of autophagy and glucose uptake. This is evidenced by decreased GLUT1 and LC3-II expression, and low activity of 2-NBDG uptake, which also can be seen in most BR cells. The xenograft model also showed that ADI-PEG20 aborted tumor growth of A2058BR and A375BR cells but retarded growth of A2058 and A375 cells. Immunohistochemical staining further confirmed lower levels of ASS and AMPK-α1 in A2058BR xenograft and in tumor tissues from 10 BR patients relative to their parental counterparts (average H-scores of ASS and AMPK in parental tissues vs. BR tissues are 58.2 vs. 7.8, and 146 vs. 78.3, respectively, p &lt;0.03). Importantly, these findings also apply to cell lines and tumor samples from patients who failed both BRAF and MEK inhibitor. In summary, our data suggest that attenuated AMPK-α1 mediated autophagy and glucose uptake and decreased c-Myc mediated ASS re-expression sensitize BR cells to arginine deprivation. Thus, ADI-PEG20 is a potential salvage therapy for BR patients (Supported by R01CA152197).
Citation Format: Ying-Ying Li, Chunjing Wu, Sumedh Shah, Shu-Mei Chen, Medhi Wangpaichitr, Lynn Feun, Macus Kuo, Miguel Suarez, Niramol Savaraj. BRAF inhibitor resistance reprograms metabolic and survival pathways to sensitize melanoma cells to arginine deprivation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2671.