M.O.L. V600E-driven melanoma.6 Vemurafenib (PLX4032) was the first drug approved for the treatment of BRAFV600E mutant melanoma, showing improved response rates and both progression-free and overall survival NPS-1034 in the clinic.7 Unfortunately, the clinical benefits of vemurafenib are short-lived and the majority of patients relapse within 6C7 months.8 Molecular mechanisms of resistance to MAPK pathway inhibition can be MAPK-dependent (amplification of mutation, MEK (and gene NPS-1034 amplification or elevated expression (z-score?>?2) was analysed in relation to survival in a group of 469 patients. Interestingly, 5.5% of patients had tumours with amplification of or or increased expression of the mRNAs they encode. In these subjects, overall survival was significantly decreased with median survival of 85 months in unaffected patients and of 49 months in affected patients (Fig.?6a), suggesting the potential clinical relevance of our findings and indicating that PGE2 synthesis could be a promising target for combinatorial therapy. No clear correlation was found between or expression and survival in this dataset. Furthermore, gene expression analysis of pre-treatment and post-progression biopsies from a published cohort of melanoma patients treated with the BRAF inhibitors vemurafenib or dabrafenib indicated that the mRNA expression of or as well as was increased in the tumours of some patients who experienced progressive disease (Fig.?6b).23 Therefore, it is conceivable that elevated and/or expression may contribute to BRAF-inhibitor resistance in melanoma patients. Open in a separate window Fig. 6 Elevated expression of is associated with poor survival of melanoma patients and acquired resistance to BRAF inhibition. a Overall survival in 469 patients affected by melanoma tumours with or without genetic alterations (amplification or NPS-1034 mRNA overexpression) in the or genes. Alterations in or (red line, z-score?>?2) correlated with a significantly lower survival (and mRNA in pre-treatment and post-progression tumour biopsies from melanoma patients treated with vemurafenib or dabrafenib (red lines and symbols indicate increased expression in the post-progression biopsy relative to the pre-treatment biopsy). Discussion Acquired resistance to BRAF-MEK-ERK signalling inhibitors, which occurs through ERK signalling-dependent and -independent mechanisms, has been a major challenge for the treatment of synthesis and breakdown/utilisation. In contrast, the dynamic 13C NMR flux detects de novo synthesis from 13C-glucose, which may not necessarily lead to changes in the total 1H NMR-measured metabolite pool. Molecular analysis of parental and R6 cells revealed lower expression of the glucose transporter GLUT-1 and of glutaminase, a key enzyme in glutamine metabolism, consistent with lower glycolytic and glutamine metabolism in the resistant cells. An increase in PC expression was consistent with a higher anaplerotic TCA activity compared to the parental clone and this was also observed in the other two resistant clones, suggesting that it is a common feature in this model. The 13C isotopomer and molecular analyses indicated that R6 cells are less dependent on glucose and glutamine metabolism than sensitive cells. It has been reported that dependence on glycolysis and a lack of functional mitochondrial respiration increases melanoma sensitivity to BRAF inhibitors44 and that an increased dependency on mitochondria for survival is a characteristic of acquired resistance to BRAF inhibitors.45 However, in some cases dependence on increased oxidative metabolism of resistant melanoma cells is associated with a switch from glucose to glutamine metabolism.45 Here we report a metabolic shift from glycolysis to mitochondrial activation in resistant cells via anaplerotic PC NPS-1034 activity. Previous reports have linked increased PC flux in glioblastoma and non-small-cell lung BMP6 cancer NPS-1034 cells to reduced dependency on glutamine,46,47 in line with our observations. Indeed, we have previously shown that a shift from glycolysis to anaplerotic mitochondrial metabolism occurs following response to vemurafenib in in melanoma samples was associated with a significantly lower patient success, emphasising the importance of our results. Notably, provided our observation that mRNA appearance (aswell as mRNA in.