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.
Highlights
Improved knowledge of the oncogenic events in melanoma indicates that a majority of mutations activate the mitogen-activated protein kinase (MAPK) pathway [1,2]
The only exception is the breast tumor kinase (BRK), which is inhibited at 130 nM, a one-log difference compared to the V600E mutated B-Raf kinase [9]
We describe that PLX4032 works differentially in melanoma cell lines with BRAFV600E mutations and that the positron emission tomography (PET) tracer 2-fluoro-2-deoxy-D-glucose (FDG) can be used in non-invasive PET imaging to distinguish between sensitive and resistant cell lines
Summary
Improved knowledge of the oncogenic events in melanoma indicates that a majority of mutations activate the mitogen-activated protein kinase (MAPK) pathway [1,2]. PLX4720 inhibits the mutated B-Raf kinase at 13 nM, while the wild type kinase requires tenfold higher concentration (160 nM) [9], predicting high specificity for BRAFV600E mutant cell lines. The basis of this specificity for the mutated kinase is thought to be the preferential inhibition of the active conformation of B-Raf. In addition, its access to a Raf-selective pocket accounts for the selectivity against most other non-Raf kinases, which require concentrations 100 to 1000 times higher for kinase inhibition. The only exception is the breast tumor kinase (BRK), which is inhibited at 130 nM, a one-log difference compared to the V600E mutated B-Raf kinase [9]
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