Differential sensitivity of melanoma cell lines with BRAF V600E mutation to the specific Raf inhibitor PLX4032

Abstract

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

Streamlining Molecular Pathobiology of Malignant Melanoma

“…By contrast, p53 mutations are more common in lentigo maligna and CMM developed on chronically sun-exposed sites of older patients exhibiting prominent actinic damages [86]. Distinct somatic gene mutations such as at the cKIT locus were identified in other CMM, particularly those on the mucosa, palms and soles and in the hyperpigmented CMM variant [82].…”

“…CMM with multiple melanocytic nevi and BRAF mutation occur more frequently at a younger age and on intermittently sun-exposed sites [86]. By contrast, p53 mutations are more common in lentigo maligna and CMM developed on chronically sun-exposed sites of older patients exhibiting prominent actinic damages [86].…”

“…Thus, PCR amplification of BRAF exon 15 followed by conventional dideoxy sequencing is commonly used for the identification of BRAF mutations. However, this method suffers from limited sensitivity for detecting BRAF mutations that are present in low percentages (<20 %) in a sample [86]. This could occur when a specimen is heavily contaminated by non-CMM tissues or due to the intratumor heterogeneity in BRAF V600 mutations [86].…”

“…However, this method suffers from limited sensitivity for detecting BRAF mutations that are present in low percentages (<20 %) in a sample [86]. This could occur when a specimen is heavily contaminated by non-CMM tissues or due to the intratumor heterogeneity in BRAF V600 mutations [86]. Thus, to avoid false-negative results by sequencing, microdissection of formalin-fixed paraffinembedded sections to enrich CMM cells before DNA extraction is recommended.…”

“…V600 is also possibly, mutated in other parts including V600K (≈ 16%) and V600R or V600D (≈ 3%). Rare mutations of neighbouring codons in BRAF exon 16 are also identified [86]. Thus, PCR amplification of BRAF exon 15 followed by conventional dideoxy sequencing is commonly used for the identification of BRAF mutations.…”