The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate–Lactate Exchange in BRAF-Mutant Human Melanoma Cells

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.1 H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAF WT (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.13 C 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 BRAF WT 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 13 C-pyruvatelactate 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);

Metabolic imaging using hyperpolarized 13C‐pyruvate to assess sensitivity to the B‐Raf inhibitor vemurafenib in melanoma cells and xenografts

“…16 Our in vitro hyperpolarization results confirm and extend the work of Beloueche-Babari and colleagues: they observed a BRAFi-mediated drop in the HP pyruvate-to-lactate exchange in the BRAF-mutated cell line WM266.4, but not in cells harbouring wild-type BRAF, therefore, intrinsically resistant to BRAFi. 17 By using a BRAFiresistant clone derived from the parental cell line, 30 we have shown that the effect in term of reduced glycolysis and label exchange is specific to sensitive cells and may not be present in BRAF-mutated F I G U R E 5 BRAFi reduced hypoxia in vivo, without affecting the vessel density. A, Representative EPR spectra obtained from a mouse prior treatment (top) and after 5 d of treatment with vemurafenib (bottom).…”

“…17 Within the scope, immunocompetent mice would allow to obtain a more thorough picture of the cross-talk between cancer cells and immune cells. 17 Within the scope, immunocompetent mice would allow to obtain a more thorough picture of the cross-talk between cancer cells and immune cells.…”

“…[16][17][18][19][20] Resistance to BRAFi seems to be accompanied by precise metabolic changes as well: higher reliance on oxidative phosphorylation, [21][22][23][24] glutamine dependency 21,25,26 and up-regulated serine metabolism. Apart from a small subset of melanomas, BRAF mutations are associated with increased glycolysis and attenuated oxidative phosphorylation, and such balance is reversed upon treatment with BRAFi.…”

Overcoming resistance to BRAF inhibitors

“…The interplay between cancer cell metabolism and response to targeted therapies too warrants further research. A growing number of reports are linking nutrient metabolism (glucose, glutamine) and melanoma cells responsiveness to BRAF inhibition while at the same time it is clear that the MAPK pathway regulates glycolysis and oxidative phosphorylation (16,(135)(136)(137)(138)(139)(140)(141). All these gaps in our understanding of melanoma biology and the potential of managing MAPK inhibitor schedules with drug-holidays or combination with immunotherapies shows that there is room for improvement to better manage advanced melanoma.…”

Disruption of Redox Balance Enhances the Effects of BRAF-inhibition in Melanoma Cells

“…Such a quantitative approach can be utilized to predict global metabolic states of cancer cells under various conditions. Melanoma cells upon BRAF-inhibition have been shown to induce an enhanced oxidative phosphorylation (Delgado-Goni et al, 2016;Haq et al, 2013;Smith et al, 2016) . Byproducts of augmented mitochondrial activity are reactive oxygen species (ROS), and this metabolic switch promotes oxidative stress in cells (Cesi et al, 2017;Zorov et al, 2014) .…”