It is estimated that ~50% of patients with melanoma harbour B-Raf (BRAF)V600 driver mutations, with the most common of these being BRAFV600E, which leads to the activation of mitogen-activated protein kinase proliferative and survival pathways.,BRAF inhibitors are used extensively to treat BRAF-mutated metastatic melanoma; however, acquired resistance occurs in the majority of patients.,The effects of long-term treatment with PLX4032 (BRAFV600 inhibitor) were studied in vitro on sensitive V600E BRAF-mutated melanoma cell lines.,After several weeks of treatment with PLX4032, the majority of the melanoma cells died; however, a proportion of cells remained viable and quiescent, presenting senescent cancer stem cell-like characteristics.,This surviving population was termed SUR cells, as discontinuing treatment allowed the population to regrow while retaining equal drug sensitivity to that of parental cells.,RNA sequencing analysis revealed that SUR cells exhibit changes in the expression of 1,415 genes (P<0.05) compared with parental cells.,Changes in the expression levels of a number of epigenetic regulators were also observed.,These changes and the reversible nature of the senescence state were consistent with epigenetic regulation; thus, it was investigated as to whether the senescent state could be reversed by epigenetic inhibitors.,It was found that both parental and SUR cells were sensitive to different histone deacetylase (HDAC) inhibitors, such as SAHA and MGCD0103, and to the cyclin-dependent kinase (CDK)9 inhibitor, CDKI-73, which induced apoptosis and reduced proliferation both in the parental and SUR populations.,The results suggested that the combination of PLX4032 with HDAC and CDK9 inhibitors may achieve complete elimination of SUR cells that persist after BRAF inhibitor treatment, and reduce the development of resistance to BRAF inhibitors.
The detection of V600E BRAF mutation in melanoma is fundamental since here BRAF inhibitors represent an effective treatment.,Non-V600E BRAF mutations that may also respond are not detected by certain screening methods.,Thus, knowledge about detection of these mutations is needed.,A total of 276 tumour samples from 174 melanoma patients were investigated for BRAF mutations by pyrosequencing.,Rare mutations were confirmed by capillary sequencing and compared with findings from COBAS test and immunohistochemistry using a novel BRAF antibody.,Melanoma type, localisation, and survival were summarised.,BRAF mutations were found in 43% of patients (124 tumours in 75 patients).,Among those, 14 patients (18.7%) exhibited rare mutations.,The V600EK601del and V600DK601del mutations have never been described before in melanoma.,Furthermore, V600K, V600E2, and V600D, V600G, V600R, and L597S mutations were detected.,Mutations were not detected by COBAS test in 7 out of these 14 patients and immunohistochemistry only reliably detected patients with the V600E2 and V600EK601del mutation.,Accurate diagnosis of rare BRAF mutations is crucial.,We show that pyrosequencing is accurate, highly sensitive, reliable, and time saving to detect rare BRAF mutations.,Missing these rare variant mutations would exclude a subset of patients from available effective BRAF-targeting therapy.