Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated

Abstract

Background: Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.Methods: DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.Results: Three cell lines 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 they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. Conclusion:Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

Cutaneous Melanoma Classification: The Importance of High-Throughput Genomic Technologies

“…The initiation and progression of cutaneous melanoma are finely driven by specific genomic alterations (20,21). Although hundreds of genes can be found mutated in a single case of cutaneous melanoma, only some mutations are true "drivers" of the tumor, either as gain-of-function (GOF)/activating or lossof-function (LOF)/deleterious mutations.…”

“…Activating mutations occur in oncogenes. The two most frequent alterations, commonly mutually exclusive both in cell lines and tumors, have been described in the kinase domain of B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF), encoded by exons 11 and 15, and in exons 2, 3 and 4 of Neuroblastoma RAS Viral Oncogene Homolog (NRAS) gene, with a frequency of 50-70% and 15-30%, respectively (20,21,(27)(28)(29)(30)(31).…”

“…More than 90% of BRAF gene mutations occur at codon 600 of exon 15, within the activation segment of the kinase, by substitution of a single nucleotide (GTG to GAG), which results in a single amino acid substitution from valine (V) to glutamic acid (E) (BRAF-V600E). The BRAF V600E mutation has been described to confer a 400-fold increased activity to the protein (20,31). Another prevalent BRAF mutation at the same residue, accounting for 10-30% of all BRAF V600 -mutated melanomas, is V600K mutation (BRAF-V600K) in which the valine residue (V) is replaced by a lysine (K) through a two nucleotides substitution (GTG to AAG) (32).…”

“…Although NRAS mutations associated with malignant transformation have been predominantly detected in codons 12, 13 (exon 2), and 61 (exon 3), the most common NRAS gene mutation in cutaneous melanoma occurs at position 61, where glutamine (Q) is substituted by arginine (R), lysine (K), or leucine (L) (NRAS-Q61R/K/L). NRAS mutations lead to the reduction of the intrinsic GTPase activity of NRAS and its constitutive activation, with consequent growth factorindependent melanocyte proliferation and ultimately melanomagenesis (20,30,31,36).…”

“…The last decade has witnessed a rapid increase in the number of next-generation sequencing (NGS) technologies implemented, with entire genome sequencing producing gigabases of reads on a daily basis (124,(132)(133)(134). The application of NGS technologies is currently providing a more comprehensive understanding of the mutational landscape of cancer and as a consequence, a better understanding of its pathogenesis (20,21,(135)(136)(137).…”