Abstract
The BRAF-MKK1/2-ERK1/2 pathway is constitutively activated in response to oncogenic mutations of BRAF in many cancer types, including melanoma. Although small molecules that inhibit oncogenic BRAF and MAP kinase kinase (MKK)1/2 have been successful in clinical settings, resistance invariably develops. High affinity inhibitors of ERK1/2 have been shown in preclinical studies to bypass the resistance of melanoma and colon cancer cells to BRAF and MKK1/2 inhibitors, and are thus promising additions to current treatment protocols. But still unknown is how molecular responses to ERK1/2 inhibitors compare with inhibitors currently in clinical use. Here, we employ quantitative phosphoproteomics to evaluate changes in phosphorylation in response to the ERK inhibitors, SCH772984 and GDC0994, and compare these to the clinically used MKK1/2 inhibitor, trametinib. Combined with previous studies measuring phosphoproteomic responses to the MKK1/2 inhibitor, selumetinib, and the BRAF inhibitor, vemurafenib, the outcomes reveal key insights into pathway organization, phosphorylation specificity and off-target effects of these inhibitors. The results demonstrate linearity in signaling from BRAF to MKK1/2 and from MKK1/2 to ERK1/2. They identify likely targets of direct phosphorylation by ERK1/2, as well as inhibitor off-targets, including an off-target regulation of the p38α mitogen activated protein kinase (MAPK) pathway by the MKK1/2 inhibitor, trametinib, at concentrations used in the literature but higher than in vivo drug concentrations. In addition, several known phosphorylation targets of ERK1/2 are insensitive to MKK or ERK inhibitors, revealing variability in canonical pathway responses between different cell systems. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates.
Highlights
From the ‡Department of Chemistry and Biochemistry, §BioFrontiers Institute, ¶Department of Molecular, Cellular, Developmental Biology, University of Colorado, Boulder, CO 80303
A previous phosphoproteomics study from our lab compared molecular responses to the BRAF inhibitor, vemurafinib, and MKK1/2 inhibitor, selumetinib, and demonstrated nearly complete overlap in pathway targets [5]. This suggests that mitogen activated protein kinase (MAPK) pathway signaling at the level of BRAF and MKK1/2 works in a predominantly linear manner, with little evidence for bifurcation in the path
To determine if other MKK1/2 inhibitors besides trametinib blocked phosphorylation of p38␣ MAPK, we examined the results of a previous data set measuring phosphoproteomic responses to selumetinib and the BRAF inhibitor, vemurafenib [5]
Summary
Cell Culture—WM239A metastatic melanoma cells were a kind gift of Dr Meenhard Herlyn, Wistar Institute, Philadelphia PA. For SILAC experiments comparing DMSO, SCH772984, and GDC0994, LC-MS/MS was performed with a LTQ Orbitrap Velos mass spectrometer and Waters Acquity UPLC, using gradient elution and instrument settings previously described [5]. The protein groups output file was uploaded into the Perseus and rows not quantified or designated as reverse, contaminant, and only identified by site were removed. This threshold corresponds to FDR Յ 0.01 (͉z-score Ն 3.1), which is greater than three standard deviations from the mean, as determined by a control experiment measuring SILAC ratios in a triple labeled experiment where each dish of labeled WM239A cells was treated with DMSO [5]. Luminescence was recorded using a Biotek Synergy H1 plate reader (Biotek Instruments, Winooski, VT) and curve-fitting was done using Origin software (OriginLab Corp., Northampton, MA)
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