Background: Melanomas harboring BRAF mutation (V600E) is known to recur following treatment with BRAF inhibitors (BRAFi) despite a high initial response rate. While the addition of MEK inhibitor can increase both response rate and duration of response. Relapsed is still inevitable. We have found that both BRAFi-resistant melanoma (BR) and BRAFi/ MEKi resistant (BMR) cells are vulnerable to arginine depletion. This is due to the fact that they cannot readily undergo autophagy upon arginine deprivation and unable to turn on argininosuccinate synthetase 1 (ASS1) to synthesize arginine. In this report, we have studied the underlying mechanisms.
Methods: 4 pairs of melanoma cell lines (parental and its BRAFi/MEKi resistant variants): A375/A375BMR, Skmel28/Skmel28BMR, A2058/A2058BMR, and MEL1220/Mel120BMR) were used for the study. MEL1220 was established in our laboratory. BMR resistant cell line was generated by continuous exposure of parental to BRAF inhibitor (Vemurafenib) and MEK inhibitor (Trametinib).
Results/Discussion: We demonstrated that downregulation of AMPK-1 is a major factor contributing to impairment of autophagy. In addition, decreased AMPK-1 expression also results in metabolic reprogramming in BMR cells toward more dependency on arginine and less on glycolysis evidenced by upregulated arginine transporter CAT-2 and downregulated glucose transporter GLUT1. Notably, when naïve melanoma cells become BMR cells by long-term exposure to BRAFi/MEKi, a stepwise degradation of AMPK-1 is initiated via ubiquitin-proteasome system (UPS). Using proteomic analysis, we discovered that a novel E3 ligase, RING finger 44 (RNF44), can interact with AMPK-1 and promote AMPK-1 degradation in both BR (BRAF resistance) and BMR cells. Elevated RNF44 and downregulated AMPK-1 expressions are also found in both BR BMR cells. Elevated RNF44 and downregulated AMPK-1 expressions are also found in both BR and BMR and tumor samples from BR and BMR patients.
Conclusions: Our results suggest that BRAFi resistance-driven proteasomal degradation of AMPK-1 is crucial to govern vulnerability to arginine deprivation in melanoma cells; therefore, depleting exogenous arginine can be considered as a novel salvage treatment for melanoma patients who fail BRAF/MEK inhibitors. Furthermore arginine deprivation also results in an increase PD-L1 expression which make them more sensitive to check point inhibitors (supported by VA Merit Review Award (1BX003328) to Savaraj and 1R01CA152197 to Kuo, Feun and Savaraj.
