Supplementary material and methods: -Western blotting -Quantitative real-time PCR (qRT-PCR) -Reactive oxygen species (ROS) production assay -NMR metabolic analyses of cells -NMR data acquisition and processing -Measurements of ADP/ATP ratios Supplementary Figures: -Figure S1: Cell proliferation is not significantly different using 25 mM or 5 mM glucose medium after 24h or 48h of treatment. -Figrue S2: BRAF mutant cells maintain their bioenergetics level under BRAF inhibition. -Figure S3: BRAF mutant WM266.4 cells show increased ROS levels under BRAF inhibition. -Figure S4: Changes in cell counts in WM266.4 and SKMEL28 control and treated samples in the 4 nutrient-restricted conditions. -Figure S5: Changes in cell cycle distribution in control and vemurafenib-treated WM266.4 cell samples in the 4 different nutrient conditions. -Figure S6: Cell lipid content detection by 1H NMR.
ARTICLE ABSTRACT
Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. 1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAFWT cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate–lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. Mol Cancer Ther; 15(12); 2987–99. ©2016 AACR.
