clinical cancer therapy: preclinical cancer research · (k52r; upstate) and 0.02 mci/ml 33p-gatp in...

Cancer Therapy: Preclinical

GSK1120212 (JTP-74057) Is an Inhibitor of MEK Activity and Activation withFavorable Pharmacokinetic Properties for Sustained In Vivo Pathway Inhibition

Aidan G. Gilmartin, Maureen R. Bleam, Arthur Groy, Katherine G. Moss, Elisabeth A. Minthorn, Swarupa G. Kulkarni,Cynthia M. Rominger, Symon Erskine, Kelly E. Fisher, Jingsong Yang, Francesca Zappacosta, Roland Annan,David Sutton, and Sylvie G. Laquerre

AbstractPurpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for

patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of

the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric

inhibitor of theMEK1 andMEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical

trial (ASCO 2010). Our studies characterize GSK1120212’s enzymatic, cellular, and in vivo activities,

describing its unusually long circulating half-life.

Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of

recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212

inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation.

Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in

multiple tumor xenograft models.

Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents

Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent

cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor

models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of

p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B, and

caused tumor growth inhibition inmultiple tumormodels. The largest antitumor effect was among tumors

harboring mutant BRAF or Ras.

Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering

promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors.

Clin Cancer Res; 17(5); 989–1000. �2011 AACR.

Introduction

Activating mutations in the mitogen-activated proteinkinase (p42/p44 MAPK) pathway represent an importantand unsolved therapeutic target in multiple solid tumors(1–5). The combined activating mutations in KRAS,BRAF, NRAS, and HRAS occur frequently in malignantmelanomas (64%), carcinomas of the pancreas (73%),colon (51%), and lung (19%)–estimates from COSMIC,2010.Inhibitors of MEK1 and MEK2 (MEK1/2) have long

shown promise as targeted therapies for tumors dependent

on these activating mutations in the MAPK pathway, but sofar they have disappointed with their lack of clinical activ-ity. The failure is presumed to be due to a narrow ther-apeutic window, constrained by the essential nature of thep42/p44 MAPK pathway in nontumor cells. The elusivegoal has been a clinically active MEK inhibitor that com-bines sustained high level of MEK inhibition in tumorswith limited systemic toxicity due to MEK inhibition innontargeted organs. We hypothesize that the preferredpharmacokinetic (PK) profile for a MEK inhibitor wouldcombine prolonged exposure and a low Cmax (peak con-centration) to Ctrough (trough concentration) ratio, deliver-ing sustained levels of sufficient dose and potentiallyavoiding toxicities that might result from high transientpeak exposure. With previous MEK inhibitors and severalnew MEK inhibitors currently being evaluated in earlyclinical trials, PK profiles have generally been reported tohave relatively high Cmax and short half-life in blood,requiring twice-daily dosing for activity (6–10). A repeatedhigh Cmax may be a concern for systemic toxicity, whereas ashort-circulating half-life may be insufficient to achieve thenecessary sustained pathway inhibition in tumors.

Authors' Affiliation: GlaxoSmithKline, Collegeville, Pennsylvania

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Aidan G. Gilmartin, GlaxoSmithKline, 1250 S.Collegeville Rd, Collegeville, PA 19426. Phone: 610-917-4078; Fax: 610-917-4181. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-2200

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 989

on March 13, 2020. © 2011 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 18, 2011; DOI: 10.1158/1078-0432.CCR-10-2200





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Here we describe GSK1120212 (JTP-74057), an allostericinhibitor of MEK1/2 activity that has shown promisingearly clinical activity in tumors with activatingmutations inBRAF (ASCO 2010). GSK1120212 is orally bioavailable,potent, and specific, and has a long circulating half life withlow Cmax to Ctrough ratio. We observe potent inhibition ofERK phosphorylation in all cell lines tested and cell growthinhibition among most cancer cell lines with activatingmutations in the MAPK pathway. In addition to inhibitingMEK-dependent ERK phosphorylation, GSK1120212 inhi-bits MEK1/2 activation by preventing Raf phosphorylationof MEK on S217 (for MEK1). Finally, we observed signifi-cant growth inhibition in multiple tumor xenografts, andparticularly in those harboring activating mutations inBRAF or KRAS.

Materials and Methods

MEK1 enzyme assaysInhibitors were dissolved in dimethyl sulfoxide (DMSO)

and were present at final concentrations of 0.015 to 5,000nmol/L with a final DMSO concentration 2.5%. Activateddiphosphorylated MEK (pp-MEK) assays contained40 mmol/L 33P-gATP (AppKm8.5 � 1.8 mmol/L), 0.5nmol/L human-activated MEK1 or MEK2 (Millipore), 1mmol/L kinase-dead ERK2 (K52R; Millipore; AppKm 0.73� 0.07 mmol/L). All assays were in buffer containing 20mmol/L HEPES (pH 7.2), 5 mmol/L 2-mercaptoethanol(Sigma), 0.15mg/mL bovine serum albumin (BSA), and 10mmol/L MgCl2. The final concentration of 33P-gATP (Per-kinElmer) was 0.02 mCi/mL for all assays. pp-MEK kinasereactions were stopped after 40 minutes by transferring30 mL of reaction mixture to a Durapore 0.45-mm filtersplates (Millipore) containing 12.5% Trichloroacetic acid(TCA). Filters were dried and read with liquid scintilant on

a TopCount (Perkin Elmer). Concentration response datawere analyzed by nonlinear regression using a 3-parameterequation with the bottom set to zero for IC50 estimationusing GraphPad Software.

For IC50 determination of initially unphosphorylatedMEK (u-MEK), 0.2 nmol/L recombinant human MEK1orMEK2 (GlaxoSmithKline) was preincubated with vehicleor with GSK1120212 for 40 minutes in reaction buffer.Phosphorylation/activation was then initiated by the addi-tion of 20 nmol/L final B-RafV600E (University of Dundee)and 30 mmol/L final ATP for 10 minutes. B-Raf activity wasthen quenched by addition of the B-Raf inhibitorSB590885 (100 nmol/L final, 500� ki), and MEK kinaseactivity was assayed by the addition of 1 mmol/L KD-ERK2(K52R; Upstate) and 0.02 mCi/mL 33P-gATP in reactionbuffer. The kinase reactions were stopped after 90 minutesby transferring 30 mL of reaction mixture to a Duraporefilter plate, and read as above.

Activity comparison of mono- and diphosphorylatedMEK1

u-MEK1 was incubated with vehicle or 5 mmol/L finalGSK1120212 for 30 minutes. Activation was then initiatedby the addition of 50 nmol/L final B-Raf wild type (WT;University of Dundee) and 0.5 mmol/L final ATP. Activa-tion reactions were quenched after 60 minutes with addi-tion of 1 B-Raf inhibitor SB590885. At this time, 5 mmol/LGSK1120212 was added to the vehicle-treated samples tocontrol for washout completion. Each reaction was loadedin centrifugal spin columns (10 kDMWCO; Millipore Cat)and spun 5� with 10� volume dilutions for bufferexchange to effectively remove GSK1120212 and ATP(reduced to 50 pmol/L and 5 nmol/L, respectively).SB590885 was maintained at 1 mmol/L throughout thebuffer exchange. MEK1 kinase assay was carried out in abuffer containing 20 mmol/L HEPES (pH 7.2), 0.15 mg/mL BSA, 10 mmol/L MgCl2, 0.8 mmol/L CHAPS (Sigma),and 1 mmol/L TCEP (Thermo), using kinase-dead ERK2 assubstrate with 33P-gATP detection in TCA precipitationfilter binding assay. MEK kinase reactions were stoppedafter 40 minutes by transferring 30 mL of reaction mixtureto a Durapore 0.45-mm filters plates (Millipore) containing12.5% TCA. Filters were dried and read with liquid scinti-lant on a TopCount (Perkin Elmer). Concentrationresponse data were analyzed by nonlinear regression usinga 3-parameter equation with the bottom set to zero forIC50 estimation using GraphPad Software. Rates of ERK2phosphorylation were determined as measures of relativeactivity.

ImmunoblottingCell lysates were prepared using RIPA buffer (TEKnova)

supplemented with protease (ROCHE) and phosphataseinhibitor cocktails (SIGMA), immunoblotted according tostandard procedures, andmembranes were imaged with anOdyssey imager. Primary antibodies included diphosphoERK1/2(T202/Y204), total ERK1/2 (137F5), phosphoMEK1/2, total MEK1/2, p27KIP1 and phospho C-Raf

Translational Relevance

Activating mutations in the p42/p44 MAPK pathway,particularly in Ras and BRAF, represent some of themost important unsolved therapeutic targets inmultiplesolid tumors. MEK inhibitors have long shown promiseas targeted therapies for tumors driven by mutant Rasand Raf, but have so far disappointed with their lack ofclinical activity. Here we describe GSK1120212 (JTP-74057), an allosteric inhibitor of MEK1 and MEK2(MEK1/2) activity that has shown promising early clin-ical activity in tumors with activating mutations inBRAF. We characterize the biochemical, cellular, andin vivo activities of GSK1120212 in preclinical models.We also suggest several properties of GSK1120212 thatmay translate to superior clinical activity including (i) along circulating half-life with low peak concentration totrough concentration ratio and (ii) the ability to inhibitMEK1/2 activation by preventing Raf phosphorylationof MEK on S217 (for MEK1).

Gilmartin et al.

Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research990




(ser338) (56A6; Cell Signaling Technologies). Secondaryantibodies included goat anti-mouse and anti-rabbit(Odyssey).

Cell culture and proliferation assaysCell lines were obtained from ATCC and grown in the

recommended media at 37oC, 5% CO2 in a humidifiedincubator. Cells were seeded in triplicate 96-well microtiterplates at 1,000 cells per well. GSK1120212 dissolved inDMSO or negative control (0.1% DMSO) were added thefollowing day and one plate was harvested with 50 mL ofCellTiter-Glo (Promega) for a time 0 (T¼ 0) measurement.Remaining duplicate cell plates were typically incubated for72 hours. Cells were then lysed with 50 mL CellTiter-Glo,and chemiluminescent signal was read on the WallacEnVision 2100 plate reader. All data were normalized tosignal at the time of compound addition (T ¼ 0). Curveswere analyzed using the XLfit (IDBS Ltd.) tool, fitting to a 4-parameter curve to determine the gI50 (concentration of50% growth inhibition relative to T ¼ 0 and Ymax values),the gI-Maximum (concentration giving maximum growthinhibition), and the Ymin (bottom of the 4-parameter curveat gI-Maximum). On the basis of Ymin value we defined themaximum biological response for each cell line as "cyto-toxic" (Ymin < 90% reflecting a net-cell decrease), "cyto-static" (Ymin ¼ 90%–200%, where cells undergo 1 or lesscomplete divisions), or "resistant" (Ymin > 200%).

Site-specific analysis of MEK phosphorylation by massspectrometryFor cellular studies, endogenous MEK1 was immuno-

precipitated from 1 mg of whole cell lysate with a MEK1(61B12) antibody and protein G beads. For in vitro kinasereactions, 7 mmol/L recombinant MEK1 was preincubatedwith vehicle or GSK1120212 (10 mmol/L) for 30 minutes,and treated with an equal volume containing constitutivelyactive C-Raf (50 nmol/L; Millipore) for 120 minutes inkinase buffer (20 mmol/L MOPS, 0.05 mg/mL BSA, 0.01%Tween-20, 10 mmol/L MgCl2, 5 mmol/L EGTA, 1 mmol/LDTT). Reactions were quenched by adding LDS loadingbuffer and boiling for 10minutes. The samples were loadedonto a Bis-Tris Gel and visualized by Coomassie Bluestaining.Gel bands corresponding to endogenous MEK1 (immu-

noprecipitated) or recombinant MEK1 (kinase reaction)were excised, reduced, alkylated, and digested over nightwith trypsin (Promega). After organic extraction, sampleswere injected on an Agilent 1100 nanoLC system. ThenanoLC was interfaced to a LTQ-Orbitrap-XL mass spectro-meter (ThermoFisher). Tryptic peptides were separated ona Dionex 100-mm RP-PSDVB monolithic column using agradient of 0% to 30% acetonitrile/0.2% formic acid over40 minutes. Mass spectrometry (MS)-based peptidesequencing was accomplished by tandem mass spectro-metry either using data-dependent LC/MS/MS or bytargeted MS/MS sequencing of selected precursors. Unin-terpreted tandem MS spectra were searched for proteinmatches against an in-house protein sequence database

using Mascot (Matrix Science). Alternatively, selected MS/MS spectra were manually interpreted to determine the siteof phosphorylation within a specific peptide. Apparentphosphorylation stoichiometry was determined from thearea under the LC/MS peaks corresponding to the phos-phorylated form(s) of the peptide and the unphosphory-lated counterpart. Stoichiometry was calculated using theformula:

Stoichiometry For Pn ¼ AðPnÞ þAðP3ÞAðP1Þ þ AðP2Þ þ AðP3Þ þ AðNPÞ

where A(Pn) is the abundance of either P1 or P2. A(P1) is theabundance of phosphopeptide 206–227 containingpSer217, A(P2) is the abundance of peptide 206–227containing pSer221, A(P3) is the abundance of the doublyphosphorylated peptide, and A(NP) is the abundance ofnonphosphorylated peptide 206–227.

Tumor xenograft studiesCells were implanted in Nude mice and grown as tumor

xenografts. Dosing began when tumors achieved approxi-mately 150 to 200 mm3. GSK1120212 or vehicle wasadministered by oral gavage at a dose volume of 0.2mL/20 g body weight in 0.5% hydroxypropylmethylcellu-lose (Sigma) and 0.2% Tween-80 in distilled water (pH8.0). Dosing was daily for 14 consecutive days (qdx14).Results are reported as mean tumor volume for 7 to 8mice/group. Tumors were measured twice weekly with Verniercalipers, and tumor volume was estimated from 2-dimen-sional measurements using a prolate ellipsoid equation[Tumor volume (mm3) ¼ (length � width2) � 0.5]. Themaximum tolerated dose (MTD) was defined as the highestdose that produced less than 20% mortality and less than20% weight loss (�4 g). Antitumor activity was defined astumor growth inhibition (TGI), partial regression (PR), orcomplete regression (CR). TGI represents the percentvolume differential between the treated and control tumorsat the time vehicle tumors exceeded a volume of 1,000mm3. PR was defined as a 50% decrease in an individualtumor from the initial starting volume for at least 1 week(3 consecutive measurements). CR was defined as a greaterthan 93% decrease in an individual tumor volume for atleast 1 week. All studies were conducted after review by theGSK Institutional Animal Care and Use Committee and inaccordance with the GSK Policy on the Care, Welfare andTreatment of Laboratory Animals.

Pharmacokinetic analysisFor mouse PK studies, tumor-bearing mice were eutha-

nized at specified timepoints postdose and blood wasdrawn and combined immediately with an equal volumewater to hemolyze the sample. Brains were harvested, flashfrozen on dry ice, and weighed (with minimal handling toprevent tissues from thawing). Required water volume wasthen calculated on the basis of the brain weight and addedto the frozen tissues (4 volumes of water per volumetissue), and samples were homogenized using a Polytronhomogenizer. Aliquots of the homogenized brain and

Preclinical Characterization of MEK1/2 Inhibitor GSK1120212

www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 991




blood were flash frozen and subsequently evaluated forGSK1120212 concentration by HPLC/MS/MS analysis. Inboth mouse and rat studies, compound half-life in blood isevaluated after multiple oral doses, and the "effective half-life" is calculated as previously defined (11, 12).

For rat PK studies, Sprague–Dawley rats (3 females) wereorally dosed for 3 weeks (21 days) at a constant dosevolume of 40 mL/m2/day to a dose of 1.0 mg/m2, wherebody surface area (m2) was estimated as (0.105) � (bodyweight in kg). On day 21, serial blood samples werecollected into tubes containing EDTA as an anticoagulantat times 0 (predose day 21), 0.5, 1, 2, 4, 8, and 24 hourspostdose. Plasma was isolated by centrifugation, and allresulting samples were snap frozen on dry ice and thenstored at �20�C until analyzed for GSK1120212 by HPLC/MS/MS analysis.

Pharmacodyamic (PD) measurement of p-ERK levelsin blood and brain tissues

Tissues were harvested and homogenized using Medi-machine (BD Bioscience) with 1 mL of lysis buffer[25 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA (pH8.0), 2 mmol/L EGTA (pH 8.0), 1% Triton X-100, 0.1%SDS, 50 mmol/L Disodium b-glycerol phosphate, 2 mmol/L Na3VO4, 4 mmol/L Na-pyrophosphate, 2� phosphataseinhibitor cocktail], and kept on ice. Following homoge-nization of all samples, homogenates were centrifuged at14,000 rpm for 15 minutes at 4�C. Clarified lysates wereflash frozen for later analysis. All samples were analyzedwith a duplex ELISA (MescoScale Discovery) measuringtotal ERK1/2 and phospho ERK1/2 according to themanufacturer’s instructions. Plates were read on MSD.SI6000.

ImmunohistochemistryXenografted A-375PF11 tumor tissue was harvested 4

hours postdose with GSK1120212 or vehicle after dose 1 ordose 4. Tumor tissue cut to approximately 100 mm3

samples was fixed in 10% formalin (neutral-buffered;VWR) for 24 hours, and then transferred to 70% ethanolprior to paraffin embedding. Paraffin-embedded blockswere shipped to Mosaic Laboratories for sectioning (to4–5 mm), immunostaining, and imaging. Primary antibo-dies detected p-ERK1/2-YT (Sigma), Ki67 (Dako), andp27Kip1 (Dako).

Results

GSK1120212 inhibits MEK1 and MEK2 in enzymaticand cellular assays

GSK1120212 (JTP-74057; Fig. 1A) was identified duringan extensive lead optimization campaign of hits froman earlier cellular screen for inducers of p15INK4B andp27Kip1/CDKN1B; GSK1120212 and the related JTP-70902were subsequently confirmed to be a MEK1/2 inhibitorychemotype (Takayuki Yamaguchi, Reina Kakefuda,Nobuyuki Tajima, Yoshihiro Sowa, Toshiyuki Sakai, sub-mitted manuscript; refs. 13, 14). GSK1120212 is a DMSO

solvate compound (ratio 1:1) with potent and ATP non-competitive inhibition of MEK1/2 (SupplementaryFig. 1A). Specificity of GSK1120212 for MEK1/2 was con-firmed against a panel of more than 180 kinases includingB-Raf, C-Raf, and MEK5 the closest kinase homolog(Takayuki Yamaguchi, Reina Kakefuda, Nobuyuki Tajima,Yoshihiro Sowa, Toshiyuki Sakai, submitted manuscript;and data not shown). Mutual exclusivity studies withanother allosteric inhibitor of MEK, PD0325901, con-firmed that GSK1120212 binds MEK in the same commonbinding domain (Supplementary Fig. 1B), adjacent to theactive site and defined on one side by the activation loop(15). We observed that GSK1120212 showed betterpotency against u-MEK1 or MEK2 when combined priorto activation by C-Raf (u-MEK1 mean IC50: 0.7 nmol/L)compared with preactivated pp-MEK1 (mean IC50: 14.9nmol/L; Fig. 1B; Supplementary Fig. 1C). These resultssuggested that u-MEK affords a higher affinity binding sitefor GSK1120212 than the pp-MEK.

In all cancer cell lines tested, GSK1120212 inhibits theMEK1/2-dependent activating dual phosphorylation ofERK1/2 on both T202 and Y204. (Unless otherwisenoted, p-ERK1/2 indicates the doubly phosphorylatedERK1/2 proteins.) Treatment of SK-MEL-28, A-375P(F11), A549, or HCT-116 cells with 250 nmol/LGSK1120212 caused sustained inhibition of ERK1/2phosphorylation up to 72 hours after compound addi-tion (Fig. 1C). In addition, MEK1/2 inhibition triggers aconsequent increase of p27Kip1/CDKN1B from 6 to 48 hoursposttreatment, indicative of cells exiting the cell cycle.Notably, inhibition of p-ERK1/2 is observed in cellsirrespective of their response to GSK1120212 in a 3-day proliferation assay (Supplementary Fig. 2); this indi-cates that differential antiproliferative activity is due todifferences in the cellular dependence on MEK1/2 signal-ing rather than on cellular differences in the inhibition ofthe MEK1/2 by GSK1120212.

We also observe that GSK1120212 induces a decrease inMEK1/2 phosphorylation (phosphoMEK) in several tumorlines (Fig. 1C). Although decreased phospho MEK is sus-tained in the BRAF-mutant SK-MEL-28 cells, in the A-375P(F11) and the KRAS-mutant HCT-116, the decrease inphospho MEK is transient, with levels rebounding by 48hours. Similar inhibition of MEK phosphorylation hasbeen reported for some other MEK inhibitors (16;17), Incontrast, phospho MEK levels are not decreased in A549cells, but rather show a time-dependent increase thatcorresponds with an increase in an activating phosphor-ylation on C-Raf (S338) kinase upstream of MEK1/2 (Sup-plementary Fig. 3).The gradual increase in MEK1/2 and Rafphosphorylation, evident at later timepoints, and detectedby immunoblotting is consistent with reports for otherallosteric MEK1/2 inhibitors and is attributed to inhibitionof a negative feedback mechanism involving Sprouty andDual Specificity Phosphatases (18). Taken together, theseresults suggest that besides inhibiting MEK phosphorylat-ing ERK1/2, in some cells, GSK1120212 is partially inhibit-ing the activating phosphorylation of MEK by Raf.

Gilmartin et al.





GSK1120212 inhibits activating phosphorylation ofMEK on S217We further investigated the effect of GSK1120212 on the

activation of MEK in an in vitro assay, examining MEK1phosphorylation using mass spectrometry (MS). Previousstudies indicated that MEK1 is maximally activated on dualphosphorylation of S217 and S221, with the monopho-sphorylated proteins being only fractionally activated (19).Phospho-specific antibodies to MEK1/2 generally are notsuitable to resolve the monophosphorylated MEK (p-MEK)from pp-MEK(S217/S221), and are therefore inadequatefor the current detailed analysis. In contrast, mass spectro-metry, when combined with high resolution liquid chro-matography, allowed development of an assay permittingdiscrimination between all 3 forms of a tryptic phospho-peptide containing phosphoserines 217 and/or 221. TheMS-based analysis of in vitro kinase reactions showed that

GSK1120212 completely prevents phosphorylation ofMEK1(S217) by either C-Raf (Fig. 2A) or B-Raf (data notshown) but had no effect on S221 phosphorylation.

We then attempted to determine the relative activity of p-MEK1(S221) compared with the pp-MEK1. MEK1 wasactivated with B-Raf in the presence or absence ofGSK1120212 for 2 hours, compound was removed byrepeated washes (in the presence of a B-Raf inhibitorSB590885A to prevent further activation), a fraction ofprotein was set aside for MS-analysis confirmation ofphosphorylation, and finally the relative MEK1 kinaseactivities were compared for the rate of ERK2 phosphoryla-tion. These results confirmed that p-MEK1(S221) was 32-fold more active than u-MEK, but only fractionally as active(1/83-fold) as pp-MEK1 (Supplementary Fig. 4A). Signifi-cantly, we also observed that the IC50 for GSK1120212 wasat least 6-fold lower for purified p-MEK1(S221) than for

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Figure 1. GSK1120212 biochemical and cellular activity. A, GSK1120212 structure. B, GSK1120212 inhibition of MEK1, treated preactivation versuspostactivation (graph of representative assays); assays were run by pretreating u-MEK (u-MEK1) with drug and then activating with C-Raf, or alternatively bytreating a preactivated enzyme (pp-MEK1) with drug. Mean IC50s reflect more than 10 assays. C, GSK1120212 causes sustained inhibition of ERK1/2phosphorylation, and differential effects on MEK phosphorylation. SK-MEL-28 and A-375P(F11) (BRAF-mutant) or HCT-116 and A549 (KRAS-mutant) celllines were treated with 250 nmol/L GSK1120212 and harvested at the indicated timepoints.






pp-MEK1 (Supplementary Fig. 4B and C); an accurate IC50

could not be determined for u-MEK due to its low kinaseactivity. This suggests that the additional phosphorylation

of S217 on the MEK1 activation loop reduces the bindingaffinity of MEK1 for GSK1120212 presumably by alteringthe compound binding site.

To investigate whether GSK1120212 inhibited MEK1phosphorylation at S217 in cells, A549 and A-375P(F11)cells were treated with 250 nmol/L compound for 0, 6, 24,48, and 72 hours. MEK1 was immunoprecipitated andphosphorylation on S217 and S221 was monitored bymass spectrometry. As we observed by immunoblot ana-lysis, phospho S221 levels gradually increased in bothA549 and A-375P(F11). In contrast, S217 phosphorylationdecreased on treatment with GSK1120212 and showed noincrease over time. (Fig. 2C and D). These results areconsistent with both our observation of a feedbackresponse increase in MEK phosphorylation and the in vitrobiochemical assay findings that GSK1120212 decreasedphosphorylation of MEK1 at S217. We conclude thatGSK1120212 fully inhibits MEK activity and partially inhi-bits MEK activation by Raf. Because GSK1120212 does notdirectly inhibit either C-Raf or B-Raf activity in enzymaticassays, we believe that GSK1120212must bind to MEK in away that specifically blocks the accessibility of S217 to Rafkinases.

Profiling cells sensitive to GSK1120212To evaluate the therapeutic utility of GSK1120212 in

cancers with various mutational profiles, we conducted 72-hour proliferation assays on a panel of cell lines. For ouranalysis we considered the curve parameters gI50 (concen-tration resulting in 50% growth inhibition) indicating theeffective biological drug concentration and the Ymin (% cellnumber at the maximum effective dose relative to that attime 0) indicating the achievable biological effect. Thegrowth and apoptotic response of 2 cell lines, SK-MEL-28 (BRAFV600E) and A549 (KRASG13D), were compared inmore detail with daily cell growth curves (CellTiter-Glo). InSK-MEL-28 cells (Fig. 3A), GSK1120212 caused minimaleffect on growth within 24 hours, but caused a clear net celldecrease (Ymin¼�70% at 72 hours) at 48 and 72 hours. Bycomparison, in A549 cells (Fig. 3B), GSK1120212 causedprimarily growth inhibition (Ymin ¼ 182% at 72 hours).These results indicate that MEK inhibition can produce atime-dependent delay in growth and that the effect on netcell survival can vary across cell lines according to theirintrinsic sensitivity.

We applied this analysis of proliferation assay curvesacross 94 cancer cell lines, evaluating sensitivity toGSK1120212 on the basis of gI50 and Ymin. The latterparameter was used to define cell response as cytotoxic(net cell loss), cytostatic (<1 net cell doubling), or less thancytostatic (>1 net cell doubling). Among the cell linestested, those with the BRAFV600E mutation representedthe most sensitive group of cells. Of the BRAFV600-mutantcells, 10 of 10 had gI50 less than 50 nmol/L GSK1120212(Fig. 3C), and 7 of 10 cells had cytotoxic response (Fig. 3D).Among the other cell lines that showed the greatest sensi-tivity to GSK1120212, the predominant determinants wereactivating mutations in KRAS or NRAS. Among cell lines

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Figure 2. GSK1120212 partially inhibits Raf phosphorylation activation ofMEK. A, 120 minutes in vitro incubation of C-Raf with MEK1 causes amajority of u-MEK (gray) to be phosphorylated on S221, S217 (black), orboth (white); inclusion of GSK1120212 (10 mmol/L) preventsphosphorylation of MEK1 on S217 but not on S221 (MS analysis). B and C,MS analysis of cellular MEK1 phosphorylation in (B) A-375P(F11) and (C)A549 cells treated with 250 nmol/L GSK1120212 shows phospho S221(black) levels increased in both cell lines at later timepoints; in contrast,phospho S217 (gray) levels decreased to limit of detection.

Gilmartin et al.





with activating KRAS mutation (specifically alterations inG12, G13, or Q61), 16 of 25 (64%) had gI50 less than 50nmol/L GSK1120212 and 19 of 25 (76%) cell lines hadcytotoxic or cytostatic responses. Of the 4 cell lines withactivating NRAS mutations (Q61L/R), 2 showed sensitivityto GSK1120212 (gI50 < 10 nmol/L; cytotoxic to cytostaticresponse). Taken together, these data suggest that activatingBRAF and Ras mutations are determinant of sensitivity toGSK1120212.

The pharmacokinetic profile and pharmacodynamicactivity of GSK1120212 following oral administrationThe capability of GSK1120212 to inhibit ERK1/2 phos-

phorylation (p-ERK) in vivo was assessed in A-375P(F11)(melanoma encoding BRAFV600E mutation) xenograftmodel. GSK1120212 (3 mg/kg), the 14-day MTD in nudemice, was administrated daily by oral gavage. Blood, brain,and tumor were harvested at 2, 4, 8, and 24 hours afterday 1 and day 7 administration. Concentrations weredetermined from blood and tissue homogenates by LC/MS/MS analysis, whereas inhibition of p-ERK was mea-sured in tumor and brain lysates by ELISA (Fig. 4A; Sup-plementary Fig. 5). The data show that on day 1 a singledose of GSK1120212 significantly reduces ERK phosphor-ylation for more than 8 hours. We further observe thatblood levels of GSK1120212 accumulate following multi-

ple drug administrations (with steady state being reachedon day 4; data not shown). As a consequence of the highersteady state levels of GSK1120212, inhibition of p-ERK isgreater following the day 7 dose with reduced p-ERK levelssustained over 24 hours. The sustained inhibition of ERKphosphorylation is consistent with the PK profile in blood,which was characterized by a gradual increase inGSK1120212 blood concentration to the Cmax (Cmax aver-age ¼ 1,410 nmol/L at 4.0 hours day 7), followed by agradual decline in concentrations, resulting in a long effec-tive half-life (day 7 estimated mean T1/2 ¼ 33 hours). Brainsamples showed no inhibition of ERK phophorylation,with low levels of GSK1120212 detected at day 7 in thebrain (Supplementary Fig. 5).

By immunohistochemistry (IHC) of the tumors, weconfirmed both the immediate inhibition of ERK phos-phorylation as well as the longer-term antiproliferativeactivity. Consistent with the p-ERK ELISA results, IHCstaining for ERK phosphorylation (diphospho ERK1/2)confirmed significant inhibition 4 hours after doses 1and 4 with 3 mg/kg GSK1120212 (Fig. 5). Additionally,IHC staining for levels of Ki67 and p27Kip1/CDKN1B (mar-kers of cell proliferation and cell cycle arrest, respectively)confirmed inhibition of cell proliferation (reduced Ki67)and G1 cell cycle arrest (elevated p27Kip1/CDKN1B) following4 days GSK1120212 treatment.

Figure 3. Profiling cellularresponse to GSK1120212. A, cellswere treated with titrations ofGSK1120212 to conduct 72 hourscell growth assays using CellTiter-Glo reagent. In highly sensitiveSKMEL-28 cells (BRAFV600E),GSK1120212 initially causes aslight growth delay within 24hours, with increased net celldeath evident by 48 and 72 hours.B, in the intermediate-sensitiveA549 cells (KRASG13D),GSK1120212 causes little effect in24 hours, with growth inhibitionand a slight decrease in net cellsoccurring at 48 and 72 hours. Alldata are normalized to the cellsignal at time 0 (dashed gray line).Characterization of the cellresponse for 94 cell lines arebased on the gI50. C and D,characterization of the cellresponse for 94 cell lines based on(C) the gI50 and (D) the maximumdrug effect (Ymin of the 4-parameter growth inhibition curve)confirms that activating mutationsin BRAF and KRAS predisposecells to sensitivity toGSK1120212.

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To evaluate the PK profile of GSK1120212 inmore detail,repeat dose PK studies were also conducted in rats. Femalerats were orally dosed with 1.0 mg/kg GSK1120212 for3 weeks (n¼ 3 rats). Following the day 1 and day 21 doses,blood was drawn serially at timepoints extending to 24hours (Fig. 4B). Consistent with the mouse data, on eachday we observed a gradual increase of GSK1120212 con-centrations in plasma to the Cmax (average: 42.4 nmol/L) at4 hours Tmax followed by a gradual decline in concentra-tions. As observed in mouse studies, there was drug accu-mulation following repeated administration, andsubsequent studies confirmed that steady state is achievedby day 4 (data not shown). On the basis of these PK profilesand the drug accumulation observed following repeat dos-ing (increase in area under curve of 2.5- to 3-fold) an

effective half-life of approximately 36 hours is estimated.This profile of sustainedplasma concentrationswithnarrow"peak/trough" ratio (�1.6–2.8) after single or repeat dosingis consistent with our observations in mice of sustainedinhibition of ERK phosphorylation.

GSK1120212 shows broad antitumor activity inmultiple tumor xenograft models

In all tumor xenograft studies, nude mice harboringxenografted tumors (approximate tumor volume 200mm3) were administered GSK1120212 by oral gavage dailyfor 14 days. Dosing in the BRAF-mutant A-375P(F11)xenograft model caused a dose-dependent TGI from 0.1to 3.0 mg/kg GSK1120212 (MTD; Fig. 6A). Althoughtreatment with GSK1120212 caused significant tumor

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Figure 4. GSK1120212 causessustained inhibition of ERK1/2phosphorylation. A, daily oraldosing in mice with 3 mg/kgGSK1120212 caused bloodcompound accumulation andsustained inhibition of pERK in A-375P(F11) tumor xenografts.Blood levels increased after day 1,reaching steady-state levels byday 4 (data not shown). In mice,the mean apparent half-life inblood was approximately 33hours. B, more detailed PKanalysis of plasma levelsGSK1120212 in female SD ratsdosed daily at 1.0 mg/m2 (n ¼ 3per day). Serial draws on day 1 andday 21 reveals a gradual increasein plasma concentration, shallowCmax, and gradual decrease,resulting in drug accumulation.The mean apparent half-life in ratswas approximately 36 hours.

Gilmartin et al.





growth delay and some regressions, A-375P(F11) tumorsgenerally resumed growth when drug treatment was inter-rupted (data not shown). Similarly in the KRAS-mutantHCT 116 xenograft model, GSK1120212 caused significantTGI at 1.0 or 3.0 mg/kg doses (Fig. 6B).GSK1120212 was tested for activity in multiple addi-

tional tumor xenograft models with activatingmutations inBRAF, KRAS, or neither (Supplementary Fig. 6) and showedantitumor activity in all models. In all experiments, 3.0mg/kg GSK1120212 was orally administered daily for 14 days,and TGI was determined in comparison to the vehicletreated tumors at the time when mean tumor size exceeded1,000 mm3. Among the xenograft lines tested, the BRAF-mutant Colo205, A-375P(F11), and HT29 models showedthe most significant mean TGI (85%, 80%, and 87% TGI)with multiple complete and partial tumor regressions. TwoKRAS-mutant xenograft models, HCT-116 and A549, alsoshowed significant TGI (83% and 75% TGI) but withoutsignificant tumor regressions. As predicted by cell prolif-eration assays, tumor xenograft lines with WT RAF/RAS(PC3, BxPC3, and BT474) were relatively much less sensi-tive, showing only modest TGI with no tumor regressions(46%, 45%, and 44% TGI).

Discussion

Past studies of other allosteric MEK inhibitors haveexpressed conflicting results as to their ability to inhibitRaf phosphorylation of MEK (7,16,17,20). Although thismay reflect real differences in the mechanism of action fordifferent compounds, it also likely reflects the technique

used for measuring MEK activation. Most commercialphospho MEK1/2 antibodies do not differentiate betweenp-MEK and pp-MEK, but these represent significantly dif-ferent activation states (19). GSK1120212 inhibition ofS217 phosphorylation contributes to the overall inhibitionof MEK. At high concentration (5 mmol/L), both AZD6244and PD0325901A also inhibited S217 phosphorylation invitro (data not shown), but we have not investigated theircellular inhibition of MEK activation nor how phosphor-ylation affects the binding affinity of MEK for compoundsother than GSK1120212.

In enzymatic assays we observed that GSK1120212 inhi-bits the initially u-MEK1/2 with IC50s approximately 1/20th or 1/12th that of pp-MEK1 and pp-MEK2, respec-tively. Furthermore, we observed that GSK1120212 is atleast a 6-fold more potent inhibitor of kinase activity for p-MEK1(S221) compared with pp-MEK1. This suggests thatphosphorylation of S217 (and likely S221 as well) reducesthe affinity of the compound for MEK1, likely by alteringthe adjacent activation loop spanning (for MEK1)L206CDFGVSGQLIDSMANSFVGTR227 that partiallydefines the compound binding pocket. This explanationis consistent with structural observations that an earlierMEK inhibitor, PD318088, stabilized the activation loop inan inactive conformation while simultaneously shifting acatalytically critical helix domain (15). Notably, multiplestudies have now identified single mutations that conferresistance to allosteric MEK inhibitors by both reducing thebinding affinity and simultaneously increasing the basalactivity of MEK (20, 21; our unpublished results). Ourdata suggest that by partially inhibiting MEK activating

Figure 5. GSK1120212 effects ontumor biomarkers. Mice harboringA-375P(F11) tumors were treatedwith 3 mg/kg GSK1120212 po for4 days. Immunohistochemicalstaining of formalin-fixed paraffinembedded A-375P(F11) tumorsections on day 1 no treatment, orday 1 and day 4 (4 hoursposttreatment, 3 mg/kgGSK1120212) indicates earlydecrease in ERK phosphorylationand longer-term decreased Ki67and increased p27Kip1 expression.

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phosphorylation, GSK1120212 sustains its binding toMEKand inhibits the accumulation of fully activated pp-MEKthat is triggered in some tumors by pathway feedbackmechanisms.

In profiling cell lines for response to GSK1120212, weclearly observe the elevated sensitivity of cell lines depen-dent on activating mutations in the MAPK pathway, parti-cularly BRAF and KRAS. Although there were insufficientnumbers for statistical significance, we also observed inseveral cell lines that activating mutations in PIK3CAcorrelate negatively with sensitivity in KRAS-mutant celllines, consistent with recently published observations for

MEK inhibitors (22). By far the most sensitive cellularsubset, BRAF-mutant cell lines respond to low concentra-tion GSK1120212 (low gI50) with increased net celldeath in a 72 hours proliferation assay; in contrast, mostKRAS-mutant cell lines respond to low concentra-tion GSK1120212 (low gI50), but the net cell response ispredominantly cytostasis or only growth delay. Theseresults suggest that a MEK inhibitor may have the strongestsingle-agent therapeutic effect in BRAF-mutant tumors.Although our data show that there should be single-agentactivity of GSK1120212 in some KRAS-mutant tumors, forother KRAS tumors MEK inhibition may be insufficient;

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Figure 6. GSK1120212 antitumoractivity A.B. 14-day daily (QDx14)oral (po) dosing of GSK1120212causes dose-dependent TGI in (A)A-375P(F11) (BRAFV600E) and (B)HCT 116 (KRASG13N) tumorxenograft models.

Gilmartin et al.





these tumors may require combination with inhibitors ofthe adjacent PI3K signal transduction pathway (e.g., inhi-bitors of PI3K, AKT, mTOR).Recent clinical results with the B-Raf inhibitor PLX4032

have revealed promising activity in melanoma patients(23). In addition to showing therapeutic validity of target-ing the MAPK pathway, these results suggest two potentialbenefits from a combination of B-Raf and MEK inhibitors.First, the combination approach has the potential to staveoff drug resistance arising from individual mutations thatrepresent an Achilles heel to most kinase inhibitors. Sec-ond, the combination may abrogate a reported toxicity ofB-Raf inhibitors, increased frequency of squamous celllesions, attributed to a mechanism in which one B-Rafinhibitor–bound member of a Raf dimer transactivates theother member resulting in down-stream phosphorylationof MEK (24). Combination with a MEK inhibitor might beexpected to block this effect in nontumor cells.Multiple MEK inhibitors have now failed to show sig-

nificant efficacy in clinical trials. CI-1040 produced a rangeof now recognizable on-target adverse events includingskin rash, edema, and diarrhea (4), but unpromising activ-ity. PD0325901 was discontinued due in part to toxicitiesthat included retinal vein occlusions and neuropathy thatapparently reflected intolerable drug levels passing theblood barriers of the retina and central nervous system;these toxicities were subsequently modeled in animalmodels where PD0325901 caused retinal vein occlusionin rabbits (25) and inhibition of p-ERK in the brain (10).We showed that GSK1120212 does not significantly pene-trate intact brain nor inhibit brain p-ERK, suggesting thattoxicity observed for PD0325901 might be avoided. BothPD0325901 and, more recently, AZD6244 (Arry-142886)showed promising inhibition of ERK phosphorylation in asubset of melanoma patients, but these failed to translateinto improved patient outcome in single agent trials (26–29). One interpretation is that previous MEK inhibitorcompounds have not achieved the drug properties required

to hit a pharmacokinetic "sweet spot," producing in vivoconcentrations that remain within the narrow therapeuticwindow, raising the question of whether this goal is in factachievable.

GSK1120212 is orally bioavailable, exceptionallypotent and specific, and has a long half-life with a shallowCmax to Ctrough PK profile. These characteristics suit theprofile for an inhibitor of tumors dependent on activatingmutations in the MAPK pathway, especially those harbor-ing activating mutations in KRAS and BRAF. Becausegrowth inhibition by a MEK inhibitor is generally fullyreversible on drug removal, the presumed optimal profileis a sustained steady-state drug concentration in excess ofthe dose required for activity. In contrast to multipleother MEK inhibitors that required twice-daily dosingin mice to achieve sustained or near-sustained MEKinhibition, GSK1120212 dosed daily at the repeat-doseMTD achieves steady state blood levels that remain morethan 250 nmol/L (5� our defined "sensitive" concentra-tion in cells) for 24 hours, causing sustained inhibition ofp-ERK. We showed that GSK1120212 does not signifi-cantly penetrate intact brain nor inhibit brain p-ERK,suggesting that brain penetration–related toxicities maybe avoidable by a favorable drug distribution profile.Recent phase I clinical data for GSK1120212 indicatesthat it has shown favorable early activity in melanomapatients (30) and is currently being evaluated in phase IIclinical trials.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Received August 16, 2010; revised January 5, 2011; accepted January 9,2011; published OnlineFirst January 18, 2011.

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Correction

Correction: GSK1120212 (JTP-74057) Is anInhibitor of MEK Activity and Activation withFavorable Pharmacokinetic Properties forSustained In Vivo Pathway Inhibition

In this article (Clin Cancer Res 2011;17:989–1000), which was published inthe March 1, 2011, issue of Clinical Cancer Research (1), Fig. 4A was incor-rectly labeled due to the following production error: The sequence for Day 1and Day 7 was labeled "2h, 4h, 4h, 24h," instead of "2h, 4h, 8h, 24h." Thecorrected figure appears below. The online version has been corrected and nolonger matches the print version.

Reference1. Gilmartin AG, Bleam MR, Groy A, Moss KG, Minthorn EA, Kulkarni SG, et al. GSK1120212

(JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokineticproperties for sustained in vivo pathway inhibition. Clin Cancer Res 2011;7:989–1000.

Published OnlineFirst April 2, 2012.doi: 10.1158/1078-0432.CCR-12-0606�2012 American Association for Cancer Research.

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ClinicalCancer

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2011;17:989-1000. Published OnlineFirst January 18, 2011.Clin Cancer Res Aidan G. Gilmartin, Maureen R. Bleam, Arthur Groy, et al.

Pathway InhibitionIn VivoSustained Activation with Favorable Pharmacokinetic Properties for GSK1120212 (JTP-74057) Is an Inhibitor of MEK Activity and

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http://clincancerres.aacrjournals.org/content/17/5/989.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/17/5/989


clinical cancer therapy: preclinical cancer research · (k52r; upstate) and 0.02 mci/ml 33p-gatp in...

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