oa Differential Responsiveness to Braf Inhibitors of Melanoma Cell Lines Braf V600e-Mutated

Background

Melanoma is an aggressive neoplasm characterized by a complex etiology. Several molecular alterations occur during melanoma progression. The most commonly mutated pathway is the mitogen-activated protein kinases (MAPK)/ERK cascade. The activation of the MAPK/ERK signaling occurs either through gain-of-function mutations in BRAF and NRAS gene or through autocrine growth factor stimulation. Documented mutations have been found in the kinase domain of BRAF gene encoded by exon 11 and 15 with a frequency of 50–70%. The majority of these mutations affect one critical amino acid, resulting in the V600E substitution which account for more than 90% of all BRAF mutations. Given the high incidence of BRAF V600E mutation in melanoma, patient management is based on the use of specific inhibitors when patients carry BRAF V600E mutation.

By comparing RNA-seq and DNA Sanger sequencing data, we found that among 15 melanoma cell lines 3 were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that these cell lines express very low level of BRAF RNA and the expression may be in favor of the WT allele.

Given the low BRAF V600E RNA expression, we tested in this study whether the three discordant cell lines may respond differently to BRAF-specific inhibitors compared to the concordant BRAF WT and BRAF V600E control cell lines.

Methods

The three discordant cell lines, one BRAF V600E and one BRAF WT control cell lines were treated with three BRAF inhibitors, including two BRAF V600E specific (vemurafenib and PLX4720) and one aspecific (sorafenib).

Measurement of cell proliferation was performed by MTT assay. Quantitative real time PCR and Western Blot were also performed to detect BRAF V600E RNA and protein expression and to assess MAPK pathway activation.

Results

The three discordant cell lines showed BRAF V600E expression both at the RNA and protein level and MAPK pathway activation although at a lower level as compared to the BRAF V600E control cell line. The proliferation rate of the discordant cell lines decreased after treatment with vemurafenib and PLX4720 but was not affected by treatment with sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the BRAF V600E control.

All together these data suggest that the cell lines carrying BRAF V600E mutations at the DNA level may respond differently to BRAF targeted treatment and the differential responsiveness may be related to a lower BRAF V600E RNA and protein expression.