Unveiling The Metabolic Response Of Braf Mutant Melanoma Cells To Braf Inhibition

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PABackground and Purpose: BRAF-MEK1/2 signaling inhibitors have shown remarkable clinical activity in BRAF-mutant melanomas. However, responses are not durable suggesting activation of alternative tumour survival mechanisms that allow resistance to therapy.This work investigates the metabolic consequences of treatment with the BRAF inhibitor vemurafenib in BRAF mutant human melanoma cells that could potentially enable resistance to treatment.Materials and Methods: BRAFV600D WM266.4 human melanoma cells were treated with 2μM vemurafenib (5xGI50) for 24h in standard culture media. Cell extracts were prepared and analyzed by 1H NMR spectroscopy to assess the levels of the aqueous metabolites. The areas of the NMR resonances were analysed using a multivariate method (Partial Least Squares-Discriminant Analysis, PLS-DA) as well as targeted analysis in which metabolite levels were normalized to cell number and an internal standard.Target inhibition was confirmed by western blotting for P-MEK1/2 and P-ERK1/2 protein levels while treatment-induced changes in metabolic enzyme expression were assessed by qRT-PCR and western-blotting.Results: As expected, exposure to vemurafenib led to a fall in P-MEK1/2 and P-ERK1/2 levels, consistent with BRAF inhibition. PLS-DA analyses revealed a good separation of control and treated 1H NMR spectra (correlation coefficient, R2 = 100%, predicted variation, Q2 = 96.5%) mainly based on differences in lactate, myo-inositol, glycine and acetate. This correlated well with the values obtained after targeted analysis of the 1H NMR spectra, which showed a significant reduction in intracellular lactate (to 62±9%) and acetate (to 62±9%), and an increase in glycine (to189±62%,) and myo-inositol (to 251±25%) in vemurafenib-treated cells relative to controls (p≤0.03). These changes are consistent with inhibition of the glycolytic pathway, confirmed by a reduction in hexokinase II and lactate dehydrogenase A expression, both detected by qRT-PCR and western-blotting. qRT-PCR also detected a fall in phosphoglycerate dehydrogenase expression indicating reduced flux to serine-glycine synthesis and suggesting that glycine accumulation may be due to decreased utilization. Reduced pyruvate dehydrogenase kinase 1 expression was also observed indicating enhanced TCA cycle activity.Conclusions: Our data show that BRAF inhibition with vemurafenib affects various metabolic pathways in BRAF mutant human melanoma cells inducing a shift in cellular energetic profile that may enable survival following treatment.Citation Format: Teresa Delgado-Goni, Slawomir Wantuch, Paul Workman, Richard Marais, Martin Leach, Mounia Beloueche-Babari. Unveiling the metabolic response of BRAF mutant melanoma cells to BRAF inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2015-1130