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Abstract
Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation.
Highlights
MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation
It has been suggested that the combination of BRAF and MKK1/2 inhibitors may be more effective because it provides a barrier to mechanisms of acquired resistance (MOR) that reactivate ERK1/2 signaling downstream of B-RAFV600E (16, 18)
Responses to Combination Treatment with AZD and PLX—We examined the effects of combination treatment with B-RAFV600E and MKK1/2 inhibitors by designing a triplelabeled SILAC experiment to compare the responses of melanoma cells to AZD, PLX, or the combination of AZD ϩ PLX (Fig. 8A)
Summary
Sample was eluted from the analytical column with a 2-min ramp to the starting percentage of buffer B2, followed by a 123-min gradient. Data Analysis—Raw MS files for both phosphopeptide and total peptide data sets were uploaded together and searched against the Uniprot human proteome database (downloaded on 01/27/2014; 88,509 entries) in MaxQuant v.1.4.1.2 (31) using the Andromeda search engine (v1.4.0.0). The protein groups output file was uploaded into the Perseus and rows designated as reverse, contaminant, and “only identified by site” were removed. For the significant sites identified in all three cell lines (879 total), 691 were matched by Uniprot identifier and amino acid position, 178 were matched by gene and phosphopeptide sequence, and 10 were matched by phosphopeptide sequence alone. Antibodies—All antibodies were from Cell Signaling: ERK1/2 (#4696), phospho-ERK1/2 (#4370), MKK1/2 (#9122), phosphoMKK1/2 (#9121), and tubulin (#5346)
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