Using quantitative proteomic analysis to understand genotype specific intrinsic drug resistance in melanoma

Abstract

The discovery of activating BRAF V600E mutations in 50% of all melanoma patients and the development of small molecule BRAF inhibitors looks set to revolutionize the therapy of disseminated melanoma. However, in the recent clinical trial of the BRAF inhibitor, vemurafenib (PLX4032), a significant percentage of BRAF V600E mutant melanoma patients did not meet the RECIST criteria for a response. Recent work from our lab identified loss of the tumor suppressor phosphatase and tensin homolog (PTEN) as being a possible mediator of intrinsic BRAF inhibitor resistance. In this commentary, we describe the development of a novel mass spectrometry based proteomic screen of Bcl-2 family proteins that was used to delineate the PTEN-dependent differences in apoptosis signaling observed when BRAF was inhibited. We further discuss how use of these sensitive quantitative proteomic methods gives unique insights into the signaling of cancer cells that are not captured through routine biochemical techniques and how this may lead to the development of combination therapy strategies for overcoming intrinsic BRAF inhibitor resistance.

Figure 1. Liquid Chromatography Multiple Reaction Monitoring Mass Spectrometry: Principle and Practice

After reversed-phase HPLC separation, peptides are selected by their mass-to-charge ratio and dissociated by collisions with background gas before the fragment ions are mass selected to enable specific detection and quantification of individual peptides in complex mixtures (A). This technique was applied to the measurement of expression of apoptosis-regulating proteins in the Bcl-2 family to determine the mechanism for PTEN null melanoma cells' resistance to the BRAF V600E inhibitor, PLX4720 (inset). Both the heat map and the bar graph indicate that the major difference between the two cell lines was the upregulation of Bim, which caused apoptosis in PTEN positive cells. Regulation of the other Bcl-2 family proteins was similar regardless of PTEN status.