Allele-Specific Mechanisms of Activation of MEK1 Mutants Determine Their Properties

Abstract

Mutations at multiple sites in MEK1 occur in cancer, suggesting that their mechanisms of activation might be different. We analyzed 17 tumor-associated MEK1 mutants and found that they drove ERK signaling autonomously or in a RAS/RAF-dependent manner. The latter are sensitive to feedback inhibition of RAF, which limits their functional output, and often cooccur with RAS or RAF mutations. They act as amplifiers of RAF signaling. In contrast, another class of mutants deletes a hitherto unrecognized negative regulatory segment of MEK1, is RAF- and phosphorylation-independent, is unaffected by feedback inhibition of upstream signaling, and drives high ERK output and transformation in the absence of RAF activity. Moreover, these RAF-independent mutants are insensitive to allosteric MEK inhibitors, which preferentially bind to the inactivated form of MEK1. All the mutants are sensitive to an ATP-competitive MEK inhibitor. Thus, our study comprises a novel therapeutic strategy for tumors driven by RAF-independent MEK1 mutants.Significance: Mutants with which MEK1 mutants coexist and their sensitivity to inhibitors are determined by allele-specific properties. This study shows the importance of functional characterization of mutant alleles in single oncogenes and identifies a new class of MEK1 mutants, insensitive to current MEK1 inhibitors but treatable with a new ATP-competitive inhibitor. Cancer Discov; 8(5); 648-61. ©2018 AACR.See related commentary by Maust et al., p. 534This article is highlighted in the In This Issue feature, p. 517.

Figure 1. MEK1 mutants are affected differently by S218/S222 phosphorylation

A, Diagram of MEK1 mutations identified in human cancer from Cbioportal genomic database. B, 293H cells were transfected with vector, wild-type FLAG-MEK1, or 17 FLAG-MEK1 Mutants along with HA-ERK2. Western blot analysis was performed using antibodies against p-MEK, p-ERK, FLAG and total ERK. The relative p-ERK and p-MEK levels from western blot were determined by densitometry analysis using Image J. C–E, Ser218 and Ser222 of WT or mutant MEK1 were mutated into alanines (S218A+S222A), then WT or mutant MEK1 with or without S218A+S222A mutation along with HA-ERK2 were transfected into 293H cells. Western blot analysis was performed using antibodies against p-MEK, p-ERK, FLAG and total ERK.

Figure 2. Activity of MEK1 mutants is differently regulated by RAF kinase

A, Purified GST fusion WT or mutant MEK1 proteins were incubated with recombinant inactive ERK2 K52R in the absence or presence of recombinant BRAF V600E at 30 °C for 15 min. Western blot analysis was performed using antibodies against p-ERK, ERK, p-MEK, MEK and BRAF. The relative p-ERK and p-MEK levels from western blot were determined by densitometry analysis using Image J. Representative data of western blot were shown from three independent experiments. Data are shown as mean(SD). T-test, *p<0.05, **P<0.01, ***p<0.001. B, K97R kinase dead mutation was introduced to WT or mutant MEK1. Purified GST fusion WT or mutant MEK1 proteins with or without kinase dead mutation were incubated with recombinant inactive ERK2 K52R as a substrate. Western blot analysis was performed using antibodies against p-MEK, p-ERK and total protein. C, Two possible models for auto-phosphorylation of MEK1 mutants. Active MEK1 mutant protein was labeled with FLAG tag, kinase dead MEK1 mutant protein was labeled with GST tag. Then MEK1 proteins labeled with two different tags were incubated together. If phosphorylation happens in trans, kinase dead one will be phosphorylated by th active one; if it’s in cis, only the active one will be phosphorylated. D, FLAG tagged active MEK1 mutant protein was purified through immunoprecipitation with an anti-FLAG antibody from 293H cells expressing mutant MEK1; active or kinase dead MEK1 mutant protein labeled with GST tag were purified from BL21(DE3); then MEK1 proteins labeled with two different tags were incubated either alone or together. Phosphorylation of different tagged MEK1 proteins were examined by immunoblotting. E, A-Raf^lox/lox; B-Raf^lox/lox;c-Raf^lox/lox;RERT^ert/ert MEF cells that inducibly express WT or mutant MEK1 were infected with Adeno-CRE virus and cultured in medium with 1 μM 4-Hydroxytamoxifen for a week. 300 ng/ml Doxycycline was added to the cells to induce the expression of those MEK1 proteins, cells were then collected after 24 hrs. Whole cell lysates were prepared and examined by Western blot.

Figure 3. Loss of RAF dependency predicts enhanced ERK signaling output by MEK1 mutants

A, NIH 3T3 cells stably expressing doxycycline-inducible WT or indicated mutant MEK1 were treated with doxycycline (300 ng/ml) for 24 hr. Expression and phosphorylation of the indicated proteins was assayed by western blot. B, ERK signaling ouptput was analyzed by real-time quantitative RT-PCR of nine validated ERK downstream target genes in NIH 3T3 cells inducibly expressing WT or mutant MEK1. The boxplot shows the log fold changes of gene expression (y axis) between 3 classes of MEK1 mutants (grouped in × axis and filled with three colors) and WT, which is overlaid with detailed changes of each gene plotted as colored points. Significant increasing changes were evaluated by T-tests among three groups. C, Co-mutation of MEK1 with RTK/RAS/RAF/NF1 in MEK1 mutant cancer patients. The data were collected from https://cbioportal.mskcc.org. Upper histogram showing RAF independent activity ratio (RAF independent in vitro kinase activity divided by kinase activity when RAF is added to the assay) RAF independent in vitro kinase activity was estimated by pERK in the absence of RAF, pERK in the presence of RAF was quantified as total MEK activity from Figure 2A. D, Weighted Pearson correlation between frequency of co-mutation and RAF independent activity ratio from c. The weights are based on the number of samples for each MEK1 mutation.

Figure 4. Sensitivity of MEK1 mutants to allosteric MEK inhibitors is associated with their RAF dependency

A–C, WT or mutant MEK1 tagged with V5 were expressed in NIH3T3 cells upon cultured in medium containing doxycycline (300 ng/ml) for 24 hrs. Cells were then treated for 1 hr with increasing concentrations of three different allosteric MEK1 inhibitors CH5126766 (A) or PD901 (B) or Trametinib (C). IC50 of p-ERK inhibition to all three drugs were calculated on the basis of densitometry analysis of western blot results.

Fig 5. MEK1 mutants are equally sensitive to a new ATP-competitive MEK inhibitor

A, In the in vitro kinase assay, pre-phosphorylated MEK1 protein were treated with MAP855 at increasing concentrations. Alternatively, MEK1 were pre-treated with MAP855 before incubation with activated BRAF V600E kinase. The p-ERK level was quantified by densitometry, then normalized to the p-ERK level in untreated sample. The p-ERK response curves were generated using Prism 7.01. B, WT or mutant MEK1 tagged with V5 were expressed in NIH3T3 cells upon cultured in medium containing 300 ng/ml doxycycline for 24 hrs. Cells were then treated for 1 hr with increasing concentrations of MAP855. Expression and phosphorylation of the indicated proteins was assayed by western blot. IC50 values of p-ERK inhibition were calculated based on densitometry analysis of western blot result.

Fig 6. Deletions in aa 98–104 region of MEK1 reduced its binding to BRAF kinase

A, Structure of MEK-BRAF binding complex. MEK-BRAF X-ray molecules are shown in green (MEK) and blue (BRAF), Molecular Dynamics (MD) calculated MEK is shown in gold. The allosteric MEK inhibitor G573 is shown in RED. The deletions region (magenta loop) can be seen in direct contact with BRAF. B, Close-up view of the interaction space between BRAF(blue) and the deletion region (magenta). Within this region GLU102 and LYS104 form polar interactions with an associated BRAF protein. LYS104 interacts with GLU side chain and a backbone carbonyl of an ILE in BRAF. GLU102 interacts with a TYR side chain in BRAF. C, NIH-3T3 cells inducibly expressing V5 tagged WT MEK1 or mutant MEK1 were exposed to 300ng/ml doxycycline for 24 hrs. Then cells were collected and subjected to immunoprecipitation with anti-V5 antibody. The input and pull-down proteins were assayed by Western blot. D, 293H cells stably expressing V5 tagged BRAF were transfected with FLAG tagged WT or mutant MEK1 with or without S218A+S222A mutation, 24 hrs after transfection, cells were collected and subjected to immunoprecipitation with anti-FLAG antibody. The input and pull-down proteins were assayed by Western blot.