A Plate-Based Assay to Measure Cellular ERK Substrate Phosphorylation: Utility for Drug Discovery of the MAPK-Signaling Cascade

Abstract

The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.

Introduction

The Ras/MAP kinase pathway is involved in regulating many cellular processes including proliferation, survival, and motility.¹ The pathway is activated by external stimuli from growth factors, hormones, neurotransmitters, and mutation, and its deregulation is a major contributor to cancer. Ras mutation is present in 15% of all cancers while BRaf mutations have been observed in melanoma (45%), thyroid (25%), ovarian (14%), and colon (12%) cancers making the members of this signaling pathway attractive targets for cancer therapy.^2,3 There has been great interest in developing specific inhibitors targeting different components of the pathway and several molecules have been pre-clinically characterized including the Raf inhibitor GDC-0879⁴ and the mitogen-activated protein kinase kinase (MEK) inhibitor PD0325901,^5,6 while a small number of extracellular signal-regulated kinase (ERK) inhibitors have been reported.^7,8

The terminal mediator of the MAPK pathway, ERK, has >100 known substrates including p90RSK (RPS6KA1) as one the earliest identified.¹ ERK phosphorylates amino acid residue Thr573 in the activation loop of the C-terminal kinase domain of p90RSK leading to subsequent autophosphorylation at Thr359/Ser363 and Ser380 located between the N-terminal and C-terminal kinase domains. Autophosphorylation of these residues results in the activation of the N-terminal kinase domain of p90RSK and thereby induces downstream signaling events. In addition to being useful for performing structure activity design of pathway inhibitors in a cellular context, measurement of the phosphorylation status of a downstream effector such as p90RSK can also serve as a pharmacodynamic marker of efficacy of pathway inhibitors in vivo. Currently, phosphorylation of p90RSK in cells or tumor lysates is measured by western blotting, high content profiling,⁹ or the bead-based Luminex technology, all requiring extensive sample preparation and time commitment, making them unfavorable techniques for routine compound investigation.

Meso Scale Discovery (MSD^®) utilizes proprietary microplates to generate an electrochemiluminescent signal that enables high-sensitivity detection of binding events measured via CCD camera.¹⁰ This technology has been well described as a high-throughput technique to monitor phosphorylation events in vitro and in vivo. ^11,12 The capacity to custom-spot plates or to utilize less specific coating strategies such as goat anti-mouse (GAM)-coated plates has prompted us to custom-build our own assay capable of monitoring MAPK pathway inhibition. In this article, we describe the development and utility of an assay utilizing the MSD technology platform to measure p90RSK phosphorylation (Thr359/Ser363) levels from both in vitro and in vivo samples. The protocol was optimized to detect changes in p90RSK phosphorylation levels upon treatment with specific pathway inhibitors. We evaluated this effect in different cancer cell types including those with constitutive activity as a result of mutation (HCT116 and LoVo harboring the KRas mutation (G13D and G12D, respectively); A375 harboring the BRafV600E mutation) or in a nonmutated cell line (HEK293) stimulated by exogenous growth factors. This is the first plate-based mechanistic assay described that can be used to assess the potency of specific inhibitors of ERK as well as Raf and MEK in a single experimental system. This article demonstrates a universal assay platform that is readily adaptable to measure the extent of p90RSK phosphorylation in a variety of cellular backgrounds and has been proven to efficiently measure phospho-p90RSK in lysates of tumor xenografts, offering utility as a pharmacodynamic marker during preclinical development of inhibitors.

Materials and Methods

Inhibitors

GDC-0879, and MEK and ERK inhibitors were provided by the Medicinal Chemistry Department at Genentech, Inc.

Cell Lines

All cell lines were obtained from ATCC (Manassas, VA) and maintained under normal culture conditions in a humidified incubator at 37°C with 5% CO₂ and were split twice weekly. A375, H2122, and LoVo cell lines were cultured in RPMI medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, and 10 mM HEPES (pH 7.2). HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% heat-inactivated FBS. HEK293 and Panc1 cells were cultured in high glucose DMEM medium containing 10% heat-inactivated FBS. All tissue culture reagents were purchased from Invitrogen (Carlsbad, CA).

Western Immunoblotting

A375, H2122, LoVo, Panc1, HCT116, and HEK293 cells were plated at a density of 1 × 10⁶ cells/well in 6-well tissue culture-treated plates (Corning Costar, Lowell, MA) and permitted to adhere overnight. The following day cells were treated with 1 μM MEK inhibitor (PD0325901) or DMSO vehicle control (0.2%) in culture medium containing 0.1% FBS for the indicated time. HEK293 cells were serum-starved for a 24-h period prior to 2-h treatment with compound followed by stimulation with 20 ng/mL EGF (Cat# AFL236; R&D systems, Minneapolis, MN) for 5 min. Cells were lysed with 150 μL NP40 cold cell lysis buffer (Invitrogen, Carlsbad, CA) supplemented on the day of the assay with complete protease inhibitor cocktail tablet (Roche, Indianapolis, IN) and 1 mM PMSF (Sigma-Aldrich, St. Louis, MO). The protein concentrations of the cell lysates were measured using a bicinchoninic acid assay (BCA; Pierce, Rockford, IL) using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA) and 25 μg of each cellular lysate was subjected to SDS-PAGE using a 4%–12% Bis–Tris gel (Invitrogen) for 2 h at 150 V. Protein transfer from gel to 0.45 μm nitrocellulose membrane (Invitrogen) was performed in semi-dry format using a Transblot semi-dry electrophoretic transfer cell (BioRad, Hercules, CA). Following a 1-h block of nonspecific-binding sites with Odyssey blocking buffer (LiCor, Lincoln, NE), the nitrocellulose membrane was incubated with 1:1,000 dilution of rabbit anti-phospho-p90RSK polyclonal antibody (Cat# 04-419; Millipore, Billerica, MA) and mouse anti-total p90RSK (Cat# AHO1472; Invitrogen) in Odyssey block buffer with constant shaking at 4°C overnight. The following day, the membrane was washed 4 times for 5 min with Tris-buffered saline (Invitrogen) containing 0.05% Tween-20 (TBST) (Fisher Scientific, Pittsburgh, PA) followed by incubation with 1:2,000 dilution of IRDye 800-conjugated affinity-purified anti-mouse IgG (Cat# 610-132-121; Rockland, Gilbertsville, PA) and Alexa Fluor 680 goat anti-rabbit IgG (Cat# A21076; Invitrogen) secondary antibodies in blocking buffer for 1 h at room temperature. Finally, the membrane was washed 4 times for 5 min with TBST and scanned using a LiCor Odyssey Infrared Imaging system (LiCor, Lincoln, NE).

Preparation of Anti-p90RSK MSD Plates

The 96-well GAM MSD plates (Cat# L41MA-1; Meso Scale Discovery, Gaithersburg, MD) were prepared by incubation with 100 μL/well of 3% Blocker Buffer A (Meso Scale Discovery) in PBS for 1 h. The plate was washed 4 times with Tris Wash buffer (Meso Scale Discovery) followed by a 1 h incubation with 1 μg/mL of mouse-anti-p90RSK antibody (Invitrogen) prepared in 2% Blocker Buffer A in PBS. Antibody-coated plates were then aspirated, washed, and used immediately.

Phospho-p90RSK Meso Scale Assay

Cells were seeded at a density of 45,000 cells per well (or indicated cell density) in 96-well clear tissue culture-treated plates (Corning Costar) and permitted to adhere overnight in a humidified incubator. The following day, cell culture medium was removed and replaced with medium containing 0.1% FBS and the indicated concentration of test compound with a final DMSO concentration of 0.2%. DMSO treatment alone was used as a control to establish baseline phospho-p90RSK levels (hereafter referred to as “signal”). Cells were incubated with test compound or DMSO under normal culture conditions for 2 h, washed once with PBS, and lysed with 45 μL of Tris lysis buffer (Meso Scale Discovery) containing 1× phosphatase inhibitors I, II, III (Calbiochem, Gibbstown, NJ), 0.1% SDS, and 1 mM PMSF. HEK293 cells were serum-starved for a 24-h period prior to 2-h treatment with compound followed by stimulation with 20 ng/mL EGF for 5 min. Twenty-five microliters of cellular lysate was transferred from each well of the 96-well tissue culture plate to the anti-total p90RSK-coated GAM plates and permitted to incubate 16 h at 4°C on a plate shaker. The following day the MSD plates were washed 4 times with 1× Tris Wash buffer (Meso Scale Discovery) then incubated with 25 μL of 0.12 μg/mL anti-phospho-p90RSK (Thr359/Ser 363) (Cat# 04-419; Millipore) antibody in 1% Blocker Buffer A for 3 h. The MSD plates were washed 4 times with 1 × Tris Wash buffer followed by 1-h incubation with 25 μL of 0.5 μg/mL anti-rabbit sulfo-tagged antibody (Cat# R32AB-5; Meso Scale Discovery) made in 1% Blocker A in PBS. One hundred and fifty microliters of 1 × Read Buffer T with surfactant (Meso Scale Discovery) was added to the plate and the relative light units (RLU) were recorded in a Sector Imager 6000 (Meso Scale Discovery).

Phospho-ERK and Phospho-MEK Meso Scale Assay

MSD assays to monitor ERK or MEK phosphorylation were performed essentially as described for detection of phospho-p90RSK with the following exceptions: A375 cells were plated at a density of 25,000 cells per well, test compound was added in serum free medium with 0.4% DMSO for 3 h of incubation, and 100% of cellular lysate was transferred to the appropriate MSD plate (Cat# K11107D-2 for phospho-ERK and Cat# K11129A-3 for phospho-MEK). Cell lysis was achieved with Tris lysis buffer (Cat# 9803; Cell Signaling) containing 250 mM NaCl, 0.1% SDS, and 1 mM PMSF. Values determined represent the ratio of phosphoprotein to total protein since these kits permit multiplexed readout of both protein species simultaneously.

Data Analysis

The signal in each well was plotted as a function of the concentration of test compound and the 50% inhibition (IC₅₀) values were determined using a nonlinear least squares fit of the data to a 4-parameter equation using Graph Prism software (San Diego, CA).

Preparation of Human Tumor Xenografts

Female nu/nu mice (Harlan Laboratories, Indianapolis, IN) were subcutaneously implanted with 1-mm³ A375 tumor fragments in the flank. Animals were treated with 3 mg/kg of MEK inhibitor P.O. QD or vehicle containing 0.5% methylcellulose and 0.2% Tween 80 in deionized water and tumors were collected 2 and 16 h post last dose (8 days). Excised tumor pieces were placed in Lysing Matrix A tubes (MP Biomedicals, Solon, OH) containing protein lysis buffer (Invitrogen) supplemented with a protease inhibitor tablet (Roche), 1× PMSF (Sigma-Aldrich), and 1× phosphatase inhibitor cocktails 1 and 2 (Sigma) and were homogenized with a Fast Prep 24 instrument (MP Biochemicals). Pre-cleared lysates were assayed for protein concentration using BCA kit. Tumor lysates were added (30 μg protein/well) to freshly prepared anti-total p90RSK MSD plates and plates developed as described for the in vitro assay earlier.

Results and Discussion

A375 cells harboring the constitutively active V600E BRAF mutation are known to contain high levels of phosphorylated MEK and ERK as a consequence of tonic activation of the MAPK pathway. For this reason, this cell lineage is commonly used to probe for the inhibitory capability of specific Raf or MEK inhibitors.^4,13 In order to develop a universal cellular assay platform capable of monitoring the inhibition of these 2 nodes in the MAPK pathway, as well as ERK, we probed A375 cell lysates with specific phospho and total antibodies for known ERK substrates. Of the various antibodies tested (those directed against ELK, c-fos, and p90RSK), only the p90RSK antibodies (total and phospho (Thr359/Ser363)) identified appreciable levels of phosphorylated substrate with specific immunostaining (western blot data not shown). In order to assess whether the constitutive levels of phosphorylated p90RSK can be decreased by inhibition of the MAPK-signaling pathway, we incubated A375 cells with the MEK inhibitor PD0325901 for 5 min or 2 h prior to collecting lysates and probing for phospho-and total p90RSK. As depicted in Figure 1A lanes 2–4, the extent of immunostaining for phospho-p90RSK is appreciably decreased after only 5 min of compound incubation (39% as determined by Odyssey v3.0) and reduced by 90% after 2 h of inhibitor incubation, similar to reports published with this inhibitor on the reduction of phospho-ERK.¹⁴ A more exhaustive time course of MEK inhibition in A375 cells demonstrates near-maximal decrease of phospho-p90RSK levels (relative to DMSO) after 2 h of incubation and is maintained for up to 6 h of incubation (Fig. 1B). We examined the cellular lysates of 4 cell lines harboring the KRas mutation for their levels of phospho- and total p90RSK staining as well as the extent of decrease of phosphorylated substrate upon 2-h incubation with the MEK inhibitor. Two hours of inhibitor treatment reduced the levels of phospho-p90RSK staining in H2122, Panc1, LoVo, and HCT116 cell lysates by 100%, 70%, 99%, and 95%, respectively (Fig. 1A, lanes 5–12). HEK293 cells (wild-type KRas and BRAF) have low levels of basal phospho-p90RSK. MAPK pathway activation is efficiently induced by EGF stimulation for 5 min as evidenced by increased phosphorylation of p90RSK, and this level of phosphorylation is reduced in response to MEK inhibition by 98% (Fig. 1A, lanes 13–15).

Fig. 1.

Western blot detection of phospho- and total P90RSK from a panel of cancer cell lines. (A) A375 (BRAF mutant) and H2122, Panc1, LoVo, and HCT116 (KRas mutant) cells were treated with 1 μM mitogen-activated protein kinase kinase (MEK) inhibitor (PD0325901) or 0.2% DMSO for the indicated time prior to cell lysis. HEK293 cells (wild type) were stimulated for 5 min with EGF after the same inhibitor treatment. (B) A375 cells incubated with DMSO or 1 μM MEK inhibitor for the indicated period of time prior to cell lysis. Cellular lysates were probed for phosphorylated (top panel) or total (bottom panel) p9oRSK via western blotting as described in Material and Methods. Molecular weight markers are located in lane 1.

Having identified specific phospho- and total p90RSK antibodies, we aimed to develop a 96-well plate-based assay utilizing the MSD technology platform and A375 cells. Adherent cells were incubated for 2 h in 0.1% FBS containing medium with 0.2% DMSO or 1 μM MEK inhibitor (so as to determine signal and background, respectively) followed by collection of cellular lysate. Previous reports of the biochemical (K_i = 1 nM)¹⁵ and cellular potency when measuring phospho-ERK (IC₅₀ = 0.33 nM)⁶ indicated that 1 μM MEK inhibitor was sufficient to maximally inhibit this node in the MAPK-signaling cascade. Transfer of lysate to GAM MSD plates coated with mouse anti-total p90RSK antibody followed by incubation with rabbit anti-phospho-p90RSK and anti-rabbit sulfo-tagged antibody served to assess the relative amounts of phosphorylated p90RSK. In A375 cells, this level increases in proportion to the cellular density with no apparent increase in background levels indicating that 45,000 cells/well was suitable for continued assay development yielding a signal/background ratio of 6.9 and Z′ of 0.7 (Fig. 2A). Although a larger assay might be attainable with further increases in cell density, cell confluency was approaching 100% and was considered imprudent to surpass. We further optimized the detection of this assay by varying the concentration of the anti-phospho-p90RSK and sulfo-tagged secondary antibodies. The best signal/background ratio was found using 0.12 μg/mL phospho-p90RSK antibody and 0.25 μg/mL sulfo-tagged antibody (Fig. 2B). Relative light units were determined by performing the experiment in the absence of cellular lysate and in all cases this value and background were comparable. It should be noted that subsequent experiments were performed with 0.5 μg/mL sulfo-tagged antibody as indicated in Materials and Methods because of the higher overall signal. Antibodies directed against phospho (Thr573) on p90RSK (Erk phosphorylation site) did not provide a robust assay signal in this MSD assay format (data not shown), and the assay as it stands might have the capability to detect inhibitors of p90RSK activity, but further testing is required.

Fig. 2.

Optimization of phospho-p9oRSK meso scale discovery (MSD^®) assay. (A) A375 cells plated at the indicated densities were treated with MEK inhibitor (PD0325901) or 0.2% DMSO for 2 h prior to measuring phospho-p9oRSK. (B) Freshly prepared anti-total p9oRSK-coated MSD plates were used to capture A375 cellular lysate prior to incubation with the indicated dilution of antiphospho-p9oRSK (Thr359/Ser363) and anti-rabbit sulfo-tagged antibody. In all cases, 1 μM MEK inhibitor was used to determine background while DMSO control sample was used to quantify the baseline signal. Relative light units from wells incubated without lysate were comparable with background. Each assay was performed in quadruplicate and the mean and standard error of the mean are reported.

We utilized this plate-based assay to determine the IC₅₀ value of PD0325901 in a panel of cell lines with various mutational backgrounds. A375 (BRaf mutant), LoVo and HCT116 (KRas mutant), HEK293 (wild type) were incubated with the indicated concentrations of MEK inhibitor for 2 h (HEK293 were stimulated with EGF to activate the MAPK pathway) prior to phospho-p90RSK quantitation in this MSD assay (Fig. 3A). The potency of PD0325901 was consistent for the MAPK constitutively active cell lines with IC₅₀ values of 0.54 ± 0.09 nM, 0.40 ± 0.14 nM, and 1.1 ± 0.2 nM for A375, HCT116, and LoVo cells, respectively. The IC50 of PD0325901 in EGF-stimulated HEK293 cells was 4.2 ± 1.0 nM. This pharmacology agrees with previous reports of PD0325901 potency in these and other cellular backgrounds when measuring phosphorylated ERK levels (0.33 nM⁶ and <2.0 nM¹⁶). HCT116 cellular lysates have a lower level of basal phospho-p90RSK relative to the other cell lines tested, yet the assay robustness is sufficient to evaluate the effect of MAPK pathway inhibitors with this sensitive assay. For HCT116, LoVo, and HEK293 cells, typical signal/background and Z′ values were 1.7, 4.7, 4.2 and 0.7, 0.9, 0.7, respectively.

Fig. 3.

Effect of MAPK pathway inhibitors on the phosphorylation of p90RSK in cells and tumors. (A) A375 (•), HCT116 (▪), LoVo (▴), and HEK293 (▾) cell lines were treated with the indicated concentration of MEK inhibitor (PD0325901) for 2 h prior to measurement of phospho-p9oRSK levels. HEK293 cells were stimulated with EGF for 5 min after inhibitor treatment. (B) A375 were treated with the indicated concentration of GDC-0879 (•), MEK inhibitor (▪), or extracellular signal-regulated kinase (ERK) inhibitor (▴) for 2 h and analyzed for phospho-p9oRSK levels. (C) Four groups of athymic female nu/nu mice were treated with either vehicle or 3 mg/kg of PD0325901 as described in Materials and Methods. Tumors excised 2 and 16 h after the last dose (Day 8) were analyzed for phospho-p90RSK levels. Concentration inhibition curves in (A) and (B) were performed in duplicate and represent 1 of at least 2 experiments with similar results. The assay in (C) was performed in triplicate one time. Statistical significance was determined using unpaired f-test. In all cases the mean and standard error of the mean are reported.

As the MAPK pathway is generally considered linear in nature with Raf upstream of MEK upstream of ERK,³ we sought to examine whether inhibition of each node on this pathway could be quantified by measuring p90RSK phosphorylation as a terminal readout. Specific and potent inhibitors of Raf (GDC-0879), MEK (PD0325901), and ERK (Example #336 from International patent application publication number WO2007/070398⁷) were incubated with A375 cells for 2 h and potency values were determined (Fig. 3B). Specific inhibition of Raf and ERK resulted in decreases in the levels of phospho-p90RSK similar to that obtained by MEK inhibition with PD0325901. The IC₅₀ value of GDC-0879 (38 ± 4.1 nM) is consistent with published reports for inhibition of Raf as determined by measuring phospho-MEK levels.⁴ The cellular potency of the ERK inhibitor exemplified in the patent application⁷ was determined to be 40 ± 3.6 nM. It is evident that this assay can be readily used as a universal system to monitor inhibition of each of the members of the MAPK pathway.

The potency of these inhibitors was also evaluated in A375 cells utilizing MSD assays to monitor phospho-ERK or phospho-MEK as readouts (Table 1). The cellular potency of GDC-0879 when monitoring phosphorylated MEK or ERK is highly correlative with potency data obtained in this phospho-p90RSK assay. The MEK and ERK inhibitors also exhibit similar potencies when comparing phospho-ERK and phospho-p90RSK readouts, but both are inactive when monitoring phospho-MEK, as would be expected for this type of inhibitor. Interestingly, this particular ERK inhibitor robustly inhibited the phospho-ERK readout thus warranting future studies of the mechanism of pathway inhibition. That the ERK and MEK assays are commercially available well-established tools for interrogating the MAPK pathway, and that results correlate so convincingly with this custom p90RSK assay, serves to further confirm this assay as a valid readout.

Table 1.

Potency of MAPK Pathway Inhibitors on the Phosphorylation of p90RSK, ERK, and MEK in A375 Cells

	Mean IC₅₀ ± SD (nM)
Sample	p90RSK (n = 2)	ERK (n = 2)	MEK (n = 2)
GDC-0879	38 ± 4.1	41 ± 8.2	33 ± 2.3
MEK inhibitor	0.54 ± 0.09	0.44 ± 0.12	Inactive
ERK inhibitor	40 ± 3.6	42 ± 0.14	Inactive

Finally, we sought to ascertain whether this assay could be used as a pharmacodynamic readout to measure phospho-p90RSK levels from in vivo tumor lysates. A375 tumor xenografts from female athymic nu/nu mice treated with the MEK inhibitor (3 mg/kg) were collected 2 and 16 h after treatment and probed for the level of phospho-p90RSK using this assay (Fig. 3C). A statistically significant decrease in p90RSK phosphorylation (P < 0.05; unpaired t-test) was observed as early as 2 h with a slight but statistically insignificant increase in phosphorylation status compared with vehicle at 16 h. This increase is likely due to feedback inhibition commonly observed with MEK inhibitors (for more detail on this mechanism please refer to refs. ^17,18). These data matched western blot data probing against phospho-ERK and phospho-p90RSK (data not shown). We indicate here that this assay can be used as a rapid measure of a pharmacodynamic readout from in vivo studies.

In summary, we have developed a novel phospho-p90RSK 96-well microplate format assay using Meso Scale technology. This assay format is highly robust in evaluating the inhibitory effect of MAPK inhibitors on p90RSK phosphorylation and it translates the results from the traditional western blotting approach. MSD technology is amenable to 384-well format and could be expanded for multiplexing the measurement of additional proteins. Further robotic automation, incorporation of multiplexed measures, and reformatting into higher density are clear steps that can be investigated to significantly increase the assay throughput. We believe that this is the first report where the MSD platform has been used to evaluate inhibition of each node of the MAPK pathway by monitoring the phosphorylation status of p90RSK from in vitro and in vivo samples.

Footnotes

Acknowledgments

We thank Georgia Hatzivassiliou, Klaus Hoeflich, Jocelyn Chan, Leanne Berry, and Bonnie Liu for providing tumor lysates and Dan Burdick for synthesizing Example #336 from WO2007/070398.⁷ We also thank Maureen Beresini, Cristina Lewis, and Anne Van Abbema for critically evaluating this manuscript.

Author Disclosure Statement

No competing financial interests exist.

Abbreviations

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