BRAF Inhibitor (Vemurafenib) Resistance Confers Sensitivity to Arginine Deprivation in Melanoma

Melanomas harboring BRAF mutation (V600E) have been reported to recur frequently following treatment with BRAF inhibitors (BRAFi) despite a high initial response rate. Combination of MEK inhibitor (MEKi) and BRAFi significantly improves the progression free survival but patients eventually relapse. Hence, there is an urgent need for second line treatment for BRAF (V600E) mutant melanoma patients. In this study, we propose arginine deprivation to treat BRAFi-resistant (BR) melanoma cells. Around 70% of melanomas do not express argininosuccinate synthase (ASS) to generate arginine through urea cycle, and hence require exogenous arginine to compensate for this defect. Arginine deprivation is achieved by adding arginine deiminase (ADI-PEG20), an enzyme from mycoplasma which converts arginine to citrulline. ADI-PEG20 has been shown to inhibit tumor growth in treatment naïve melanomas. However, ADI-PEG20 induces autophagy and ASS re-expression which have been proven to impair anti-tumor efficacy. When melanoma cells become BR cells, they are hypersensitive to arginine deprivation. Mechanistically, we demonstrated that BRAFi resistance attenuates c-Myc expression (a positive regulator of ASS expression) through active ubiquitin-proteasome system (UPS). Downregulated c-Myc leads to less binding to the promoter of ASS and hence is unable to initiate ASS transcription. Thus, dampening the ability to re-express ASS is one of the mechanisms leading to increased vulnerability to arginine deprivation in BR cells. Impaired ability to undergoing autophagy due to downregulation of Atg5 and AMPK-α1 is another major contributory factor to hypersensitivity to ADI-PEG20 in BR cells. Overexpression of Atg5 or AMPK-α1 using plasmids renders BR cells resistant to ADI-PEG20 through an increase in autophagic flux. Conversely, knockdown of AMPK-α1 in parental cells increases sensitivity to ADI-PEG20 due to impairment to undergo autophagy, and resulted in metabolic switching from glucose to arginine addiction, which can be seen in BR cells. We further discovered that AMPK-α1 degradation through UPS in BR cells is more active compared to parental cells. Our results uncovered that the novel E3 ubiquitin ligase RNF44 is responsible for promoting AMPK-α1 degradation in BR cells. Elevated RNF44 expression and down-regulated AMPK-α1 are also found in BR and BRAFi/MEKi double resistant (BMR) melanoma cell lines and tumor samples from BR and BMR patients. These BMR cells are also sensitive to arginine deprivation as well as BR cells. Overall, our results suggest that BRAFi-resistance drives proteasomal degradation of AMPK-α1 and c-Myc. These are two crucial factors which contribute to vulnerability of arginine deprivation in BR melanoma cells. Thus, depleting exogenous arginine can be considered for novel salvage treatment in melanoma patients who failed BRAFi or BRAFi/MEKi treatment