Targeting the MAPK Signaling Pathway in Cancer: Promising … · Targeting the MAPK Signaling...

Small Molecule Therapeutics

Targeting the MAPK Signaling Pathway in Cancer:Promising Preclinical Activity with the NovelSelectiveERK1/2 InhibitorBVD-523 (Ulixertinib)Ursula A. Germann1, Brinley F. Furey1,William Markland1, Russell R. Hoover1,Alex M. Aronov1, Jeffrey J. Roix2, Michael Hale1, Diane M. Boucher1,David A. Sorrell3, Gabriel Martinez-Botella1, Matthew Fitzgibbon1, Paul Shapiro4,Michael J.Wick5, Ramin Samadani4, Kathryn Meshaw6, Anna Groover2,Gary DeCrescenzo2, Mark Namchuk1, Caroline M. Emery2, Saurabh Saha2,and Dean J.Welsch2

Abstract

Aberrant activation of signaling through the RAS–RAF–MEK–ERK (MAPK) pathway is implicated in numerous cancers,making it an attractive therapeutic target. Although BRAF andMEK-targeted combination therapy has demonstrated signifi-cant benefit beyond single-agent options, the majority ofpatients develop resistance and disease progression afterapproximately 12 months. Reactivation of ERK signaling is acommon driver of resistance in this setting. Here we reportthe discovery of BVD-523 (ulixertinib), a novel, reversible,ATP-competitive ERK1/2 inhibitor with high potency andERK1/2 selectivity. In vitro BVD-523 treatment resulted inreduced proliferation and enhanced caspase activity in sensitivecells. Interestingly, BVD-523 inhibited phosphorylation of tar-get substrates despite increased phosphorylation of ERK1/2. In

in vivo xenograft studies, BVD-523 showed dose-dependentgrowth inhibition and tumor regression. BVD-523 yieldedsynergistic antiproliferative effects in a BRAFV600E-mutant mel-anoma cell line xenograft model when used in combinationwith BRAF inhibition. Antitumor activity was also demonstrat-ed in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK–targeted therapy. On thebasis of these promising results, these studies demonstrateBVD-523 holds promise as a treatment for ERK-dependentcancers, including those whose tumors have acquired resistanceto other treatments targeting upstream nodes of the MAPKpathway. Assessment of BVD-523 in clinical trials is underway(NCT01781429, NCT02296242, and NCT02608229). Mol CancerTher; 16(11); 2351–63. �2017 AACR.

IntroductionThrough aberrant activation of ERK signaling, genetic altera-

tions in RAS or RAF family members result in rapid tumor

growth, increased cell survival, and resistance to apoptosis.Activating mutations of RAS family members KRAS and NRASare found in approximately 30% of all human cancers, withparticularly high incidence in pancreatic and colorectal cancer(1, 2). Constitutively activating mutations in BRAF (codon forV600) have been observed primarily in melanoma (�48%),papillary thyroid carcinoma (�44%), colorectal cancer(�15%), hairy cell leukemia (�100%), and non–small celllung cancer (�4%; ref. 3). Although rare, cancers bearinggenetic mutations that result in changes of the downstreamcomponents ERK (MAPK1 and MAPK3) and MEK (MAP2K1and MAP2K2) have also been reported (4, 5). Furthermore,alterations known to activate the MAPK (RAS–RAF–MEK–ERK)pathway are also common in the setting of resistance totargeted therapies, for example inhibitors of BRAF, MEK, andALK (6). Thus, targeting the MAPK pathway terminal masterkinases (ERK1/2) is a promising strategy for the treatment of abroad spectrum of tumors harboring pathway-activatingalterations.

Clinical precedent for targeting the MAPK pathway alreadyexists; three MAPK pathway-targeting drugs have been approv-ed by the FDA for single-agent treatment of nonresectable ormetastatic cutaneous melanoma with BRAFV600 mutations:the BRAF inhibitors vemurafenib and dabrafenib and theMEK inhibitor trametinib (7, 8). An additional MEK inhib-itor, cobimetinib, is approved in this indication as part of a

1Vertex Pharmaceuticals Inc, Cambridge, Massachusetts. 2BioMed Valley Dis-coveries, Inc., Kansas City, Missouri. 3Horizon Discovery Ltd, Cambridge, UnitedKingdom. 4University of Maryland School of Pharmacy, Baltimore, Maryland.5START, San Antonio, Texas. 6Charles River Discovery Services, Morrisville,North Carolina.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

S. Saha and D.J. Welsch contributed equally to this article.

Current address for U.A. Germann: OnKognos Scientific Consulting, Newton,Massachusetts and InnovaTID Pharmaceuticals Incorporated, Cambridge, Mas-sachusetts; current address for J.J. Roix, PhoreMost Ltd, Cambridge, UnitedKingdom; current address for M. Hale, Ra Pharmaceuticals, Cambridge, Massa-chusetts; current address for G. Martinez-Botella, Praxis Precision MedicinesIncorporated, Cambridge, Massachusetts; current address for R. Samadani,MedImmune, Gaithersburg, Maryland; current address for M. Namchuk,Alkermes, Waltham, Massachusetts; and current address for S. Saha, AtlasVenture, Cambridge, Massachusetts.

Corresponding Author: Dean J. Welsch, BioMed Valley Discoveries, 4435 MainStreet, Suite 550, Kansas City, MO 64111. Phone: 816-389-8831; Fax: 816-960-6976; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0456

�2017 American Association for Cancer Research.

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combination regimen with vemurafenib; furthermore, thecombination of dabrafenib and trametinib is also approvedin this indication (9, 10). Approved BRAF inhibitors providethe advantage of selectivity for BRAFV600-mutant proteins, byvirtue of ability to signal as monomers, unlike wild-type RAFproteins which require dimerization to activate signaling,therefore providing a greater therapeutic index.

Importantly, the concomitant use of BRAF- plus MEK-targetedtherapies has demonstrated simultaneous targeting of differentnodes in the MAPK pathway can enhance the magnitude andduration of response (11–13).

Despite improvements in clinical outcomes seen with BRAF-/MEK-inhibitor combination therapies, durable benefit is lim-ited by the eventual development of acquired resistance andsubsequent disease progression, with median progression-freesurvival (PFS) ranging from approximately 9 to 11 months(11, 13–15). Genetic mechanisms of acquired resistance tosingle-agent BRAF inhibition have been intensely studied;some examples of identified resistance mechanisms includesplice variants of BRAF (16), BRAFV600E amplification (17),MEK mutations (18), NRAS mutations, and RTK activation(19, 20). Resistance mechanisms in the setting of BRAF/MEKinhibitor combination therapy are beginning to emerge andmirror that of BRAF single-agent resistance (21, 22). Thesegenetic events all share the ability to reactivate ERK signaling.Indeed, reactivated MAPK pathway signaling as measured byERK transcriptional targets is common in tumor biopsies fromBRAF inhibitor–resistant patients (23). Moreover, ERK1/2 reac-tivation has been observed in the absence of a genetic mech-anism of resistance (24, 25). The quest to achieve durableclinical benefit has led researchers to focus on evaluatingadditional agents that target the downstream MAPK compo-nents ERK1/2. Inhibiting ERK may provide important clinicalbenefit to patients with acquired resistance to BRAF/MEKinhibition. ERK family kinases have shown promise as thera-peutic targets in preclinical cancer models, including thoseresistant to BRAF or MEK inhibitors (26–28); however, thepotential for ERK inhibitors expands beyond acquired resis-tance in melanoma.

Targeting ERK1/2 is a rational cornerstone therapy in anytumor type harboring known drivers of MAPK, not only BRAF/MEK therapy–relapsed patients. As ERK1/2 reside downstreamin the pathway, they represent a particularly attractive treatmentstrategy within the MAPK cascade that may avoid upstreamresistance mechanisms. Here, we report the preclinical charac-terization of BVD-523 (ulixertinib) in models of MAPK path-way–dependent cancers, including drug-na€�ve and BRAF/MEKtherapy acquired–resistance models. Phase I clinical studies ofBVD-523 are ongoing (NCT01781429, NCT02296242, andNCT02608229).

Materials and MethodsCompounds

Compounds were sourced from the following vendors;SCH772984 (SelleckChem), pyrazolylpyrrole (Santa Cruz Bio-technology), Temozolomide (Temodar, Schering Corporation),dabrafenib (Chemietek), trametinib (LC Laboratories), vemura-fenib (Focus Synthesis LLC), and selumetinib (Chemietek).BVD-523 was synthesized as per method described in US patentnumber 7,354,939 B2.

Ki determination of ERK2The inhibitory activity of BVD-523 against ERK2 was deter-

mined using a radiometric assay, with final concentration of thecomponents being 100 mmol/L HEPES (pH 7.5), 10 mmol/LMgCl2, 1 mmol/L dithiothreitol (DTT), 0.12 nmol/L ERK2,10 mmol/L myelin basic protein (MBP), and 50 mmol/L33P-g-ATP. All reaction components, with the exception of ATPand MBP, were premixed and aliquoted (33 mL) into a 96-wellplate. A stock solution of compound in DMSO was used tomake up to 500-fold dilutions; a 1.5-mL aliquot of DMSO orinhibitor in DMSO was added to each well. The reaction wasinitiated by adding the substrates 33P-g-ATP and MBP (33 mL).After 20 minutes, the reaction was quenched with 20% (w/v)tricholoracetic acid (TCA; 55 mL) containing 4 mmol/L ATP,transferred to the GF/B filter plates, and washed 3 times with5% (w/v) TCA. Following the addition of Ultimate Goldscintillant (50 mL), the samples were counted in a PackardTopCount. From the activity versus concentration titrationcurve, the Ki value was determined by fitting the data to anequation for competitive tight binding inhibition kinetics usingPrism software, version 3.0.

IC50 determination of ERK2ERK2 activity was assayed by a standard coupled-enzyme assay.

The final concentrations were as follows: 0.1 mol/L HEPES (pH7.5), 10 mmol/L MgCl2, 1 mmol/L DTT, 2.5 mmol/L phospho-enolpyruvate, 200 mmol/L NADH, 50 mg/mL pyruvate kinase, 10mg/mL lactate dehydrogenase, 65 mmol/L ATP, and 800 mmol/Lpeptide (ATGPLSPGPFGRR). All of the reaction componentsexcept ATP were premixed with ERK and aliquoted into assay-plate wells. BVD-523 in DMSO was introduced into each well,keeping the concentration of DMSO per well constant. BVD-523concentrations spanned a 500-fold range for each titration. Theassay plate was incubated at 30�C for 10 minutes in the platereader compartment of the spectrophotometer (MolecularDevices) before initiating the reaction by adding ATP. The absor-bance change at 340 nmwas monitored as a function of time; theinitial slope corresponds to the rate of the reaction. The rate versusconcentration of the BVD-523 titration curve was fitted either toan equation for competitive tight-binding inhibition kinetics todetermine a value for Ki or to a 3-parameter fit to determine theIC50 using Prism software, version 3.0.

Cell linesThe cell lines RKO, SW48, A375, MIAPaCa-2, HCT116,

Colo205,HT-29, ZR-75-1, andAN3Cawere obtained fromATCC,confirmed mycoplasma negative, and authenticated by STR gen-otyping. Cell line G-361 was obtained from ECACC and con-firmed mycoplasma negative and authenticated by STR genotyp-ing. For each cell line, upon thawing, experiments were performedwhen required cell number was achieved to avoid long-termpassaging of cells.

Antitumor activity in A375 xenograftsXenografts were initiated with A375 cells maintained by serial

subcutaneous transplantation in female athymic nudemice. Eachtest mouse received an A375 tumor fragment (1mm3) implantedsubcutaneously in the right flank. Once tumors reached target size(80–120 mm3), animals were randomized into treatment andcontrol groups, and drug treatment was initiated. BVD-523 in 1%

Germann et al.

Mol Cancer Ther; 16(11) November 2017 Molecular Cancer Therapeutics2352




(w/v) carboxymethylcellulose (CMC) was administered orallytwice daily at doses of 5, 25, 50, 100, or 150 mg/kg.

The efficacy of BVD-523 in combination with dabrafenibwas evaluated in mice randomized into 9 groups of 15 and1 group (Group 10) of 10. Dabrafenib was administered orallyat 50 or 100 mg/kg once daily and BVD-523 was administeredorally at 50 or 100 mg/kg twice daily, alone and in combina-tion, until study end; vehicle-treated control groups were alsoincluded. Combination dosing was stopped on Day 20 tomonitor for tumor regrowth. Animals were monitored individ-ually and euthanized when each tumor reached an endpointvolume of 2,000 mm3 or on the final day (day 45), whichevercame first, and median time to endpoint (TTE) calculated. Thecombination was also evaluated in an upstaged A375 modelwhere larger tumors in the range 228–1,008 mm3 were evalu-ated. Here, mice were randomized into 1 group (Group 1) of14 and 4 groups (Groups 2–5) of 20. Dosing was initiated onday 1 with dabrafenib plus BVD-523 (25 mg/kg dabrafenib þ50 mg/kg BVD-523 or 50 mg/kg dabrafenib þ 100 mg/kg BVD-523), with each agent given orally twice daily until study end.The study included 50 mg/kg dabrafenib and 100 mg/kg BVD-523 monotherapy groups as well as a vehicle-treated controlgroup. Tumors were measured twice weekly. Combinationdosing was stopped on day 42 to monitor for tumor regrowththrough study end (day 60). Treatment outcome was deter-mined from % tumor growth delay (TGD), defined as thepercent increase in median TTE for treated versus control mice,with differences between groups analyzed via log-rank survivalanalysis. For tumor growth inhibition (TGI) analysis, % TGIvalues were calculated and reported for each treatment (T)group versus the control (C). Mice were also monitored forcomplete regression (CR) and partial response (PR). Animalswith a CR at the end of the study were additionally classified astumor free survivors (TFS). All in vivo studies were conducted inaccordance with generally recognized good laboratory practices,all procedures were in compliance with the Animal Welfare ActRegulations (9 CFR 3). All in vivo studies were reviewed andapproved by the appropriate Institutional Animal Care and UseCommittees (IACUC).

BVD-523 activity in Colo205 xenograftsHuman Colo205 cells were cultured in RPMI1640 supplemen-

ted with 10% (v/v) FBS, 100 U/mL penicillin, 100 mg/mL strep-tomycin (Invitrogen), and 2 mmol/L L-glutamine. Cells werecultured for <4 passages prior to implantation. Female athymicnude mice (19–23 g) were injected subcutaneously with 2 � 106

Colo205 cells into the right dorsal axillary region on day 0.Mice with an approximate tumor volume of 200 mm3 were

randomized unblinded into 6 experimental groups. Vehicle con-trol, 1% CMC (w/v), was prepared weekly. BVD-523 was sus-pended in 1% (w/v) CMC at the desired concentration andhomogenized on ice at 6,500 rpm for 50 minutes. BVD-523suspensions were prepared weekly and administered orally twicedaily at total daily doses of 50, 100, 150, and 200 mg/kg (n ¼ 12/group)onan8- or 16-hour dosing schedule for 13days. The vehiclecontrol (n¼ 12) was administered using the same dosing regimen.

Efficacy testing in a patient-derived tumor xenograftBVD-523 was evaluated in a patient-derived xenograft

(ST052C) representing melanoma from a BRAFV600E patient whohad become clinically refractory to vemurafenib. Immunocom-

promised mice between 5–8 weeks of age were housed onirradiated corncob bedding (Teklad) in individual HEPA venti-lated cages (Sealsafe Plus, Techniplast USA) on a 12-hour light–dark cycle at 70–74�F (21–23�C) and 40–60% humidity. Animalswere fed water ad libitum (reverse osmosis, 2 ppm Cl2) and anirradiated standard rodent diet (Teklad 2919) consisting of 19%protein, 9% fat, and 4% fiber. Tumor fragments were harvestedfrom host animals and implanted into immunodeficient mice.The study was initiated at amean tumor volume of approximately170 mm3, whereby the animals were randomized unblindedinto 4 groups, including a control [1% (v/v) CMC orally, twicedaily � 31] and 3 treatment groups [BVD-523 (100 mg/kg),dabrafenib (50 mg/kg), or BVD-523/dabrafenib (100/50 mg/kg),n ¼ 10/group]; All treatment drugs were administered orally on atwice daily � 31 schedule.

ResultsDiscovery and initial characterization of the novel ERK1/2inhibitor, BVD-523 (ulixertinib)

Following extensive optimization of leads originally identifiedusing a high-throughput, small-molecule screen (29), a noveladenosine triphosphate (ATP)-competitive ERK1/2 inhibitor,BVD-523 (ulixertinib) was identified (Fig. 1A). BVD-523 is apotent ERK inhibitor with a Ki of 0.04 � 0.02 nmol/L againstERK2. It was shown to be a reversible, competitive inhibitor ofATP, as the IC50 values for ERK2 inhibition increased linearly withincreasingATP concentration,with dependence of enzyme rate onBVD-523 concentration (Fig. 1B andC). The IC50 remained nearlyconstant for incubation times �10 minutes, suggesting rapidequilibrium and binding of BVD-523 with ERK2 (Fig. 1D).BVD-523 is also a tight-binding inhibitor of recombinant ERK1(30), exhibiting a Ki of <0.3 nmol/L.

Binding of BVD-523 to ERK2 was demonstrated using calori-metric studies and compared with data generated using the ERKinhibitors SCH772984 (26) and pyrazolylpyrrole (31). All com-pounds bound and stabilized inactive ERK2 with increasingconcentration, as indicated by positive DTm values (Fig. 1E). The10- to 15-degree change in DTm observed with BVD-523 andSCH772984 is consistent with compounds that have low nano-molar binding affinities (32). BVD-523 demonstrated a strongbinding affinity to both phosphorylated active ERK2 (pERK2) andinactive ERK2 (Fig. 1F), with a stronger affinity to pERK2 com-pared with inactive ERK2. BVD-523 did not interact with thenegative control protein p38a MAP kinase (Fig. 1F).

BVD-523 demonstrated excellent ERK1/2 kinase selectivitybased on biochemical counter-screens against 75 kinases, inaddition to ERK1 andERK2. The ATP concentrationswere approx-imately equal to the Km in all assays. Kinases inhibited to greaterthan 50% by 2 mmol/L BVD-523 were retested to generateKi values (or apparent Ki; Fig. 1G). Twelve of the 14 kinases hada Ki of <1 mmol/L. The selectivity of BVD-523 for ERK2 was>7,000-fold for all kinases tested except ERK1, which was inhib-ited with a Ki of <0.3 nmol/L (10-fold). Therefore, BVD-523 is ahighly potent and selective inhibitor of ERK1/2.

BVD-523 inhibits cellular proliferation and enhancescaspase-3/7 activity in vitro while demonstrating substrateinhibition despite increased ERK1/2 phorphorylation

The antiproliferative impact of BVD-523 treatment on sen-sitive cancer cell lines was characterized by FACS analysis on

ERK Inhibitor BVD-523 in Preclinical Cancer Models

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BRAFV600E-mutant melanoma cell line, UACC-62, following24-hour treatment with BVD-523. BVD-523–treated cells werearrested in the G1 phase of the cell cycle in a concentration-dependent manner (Fig. 2A; Supplementary Fig. S1A). The per-

centage of cells arrested in G1 was also demonstrated to be timedependent, as shown by increasing proportion of KRASG12C-mutant pancreatic cell line MiaPaca-2 in G1 following 24 and48 hours treatment with BVD-523 (Supplementary Fig. S1B).

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Figure 1.

Discovery and characterization ofnovel ERK1/2 inhibitor BVD-523(ulixertinib). A, Structure of BVD-523.B, Inhibitory potency of BVD-523 forERK2 as a function of ATPconcentration, C, Demonstration ofenzyme inhibition as a function ofinhibitor concentration in thepresence of 50 mmol/L ATP, and D,Measurement of inhibitory potency asa function of incubation time. Bindingof the ERK inhibitors BVD-523,SCH772984, and pyrazolylpyrrole toERK2 (E) and phosphorylated ERK2as measured by changes in enzymemelting temperatures (p38a includedas negative control; F). G, Potency ofBVD-523 against a panel of kinases.For B–G, representative data areshown.

Germann et al.





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Figure 2.

BVD-523 inhibits cellular proliferation and enhances caspase-3/7 activity in vitro. A, Percentage of UACC-62 cell population in G2, S, or G1 phase of thecell cycle following incubation for 24 hours with increasing concentrations of BVD-523 (representative data shown). B, Cell line–dependent changes incaspase activity after 48 hours of exposure to BVD-523 (0, 0.082, 0.247, 0.741, 2.222, 6.667, 20 mmol/L) compared with the IC50 for cell viability(maximum change in caspase shown, <2-fold: �; 2-fold: þ/�; 2- 3- fold increase: þ; 3- 6- fold increase: þþþ; >6-fold increase þþþþþ; n ¼ 3). C, MAPKpathway and effector proteins are modulated by acute (4-hour) and prolonged (24-hour) BVD-523 treatment in BRAFV600E-mutant A375 cells(representative data shown). D, Levels of pERK1/2, pRSK, and Cyclin D1 measured in cell lines (A375, AN3Ca, Colo205, HCT116, HT29, and MIAPaca2) following24-hour treatment with ERK1/2 inhibitors BVD-523 (BVD), Vx11e (Vx), GDC-0994 (GDC), or SCH772984 (SCH; n ¼ 4 for each treatment group percell line). Data are average � SEM relative to DMSO control.






In addition, caspase-3/7 activity was analyzed as a measureof apoptosis in multiple human cancer cell lines (Fig. 2B). Acell-line–dependent increase in caspase-3/7 was observed fol-lowing treatment with BVD-523 for 48 hours. BVD-523 treat-ment resulted in pronounced caspase-3/7 induction (greaterthan 3-fold) in a subset of cell lines with sensitivity to BVD-523(<2 mmol/L IC50).

To further characterize the mechanism of action and effectson signaling elicited by BVD-523, the levels of various effectorand MAPK-related proteins were assessed in BVD-523–treatedBRAFV600E-mutant A375 melanoma cells (Fig. 2C). Phospho-ERK1/2 levels increased in a concentration-dependent mannerafter 4 and 24 hours of BVD-523 treatment. Despite prominentconcentration-dependent increases in pERK1/2 observed with2 mmol/L BVD-523 treatment, phosphorylation of the ERK1/2target RSK1/2 was reduced at both 4 and 24 hours, which isconsistent with sustained inhibition. Total protein levels ofDUSP6, transcription of which is a target of ERK1/2, were alsoattenuated at 4 and 24 hours. Following 24 hours of treatmentwith BVD-523, the apoptotic marker BIM-EL increased in adose-dependent manner, while cyclin D1 and pRB were atten-uated at 2 mmol/L. All effects are consistent with on-targetERK1/2 inhibition.

To examine the effects of BVD-523 on signaling relativeto other known ERK1/2 inhibitors (SCH772984, GDC-0994,and Vx-11e), a large-scale reverse-phase protein array (RPPA) of39 proteins was employed in a variety of cell lines withsensitivity to ERK inhibition (Supplementary Fig. S2). Celllines with common alterations in BRAF and RAS were assayed:BRAFV600E-mutant lines A375, Colo205, and HT29; KRASG12C-mutant cell line MIAPACa-2; KRASG13D-mutant cell lineHCT116; and AN3Ca with atypical HRASF82L mutation.Changes in protein levels are shown as a percentage changefrom dimethyl sulfoxide (DMSO)-treated control (Supplemen-tary Fig. S2). All ERK inhibitors elicited qualitatively simi-lar protein effects, with the exception of phosphorylation ofERK1/2 [pERK1/2 (ERK1/2 -T202, -Y204)]; SCH772984 inhib-ited pERK1/2 in all cell lines, while BVD-523, GDC-0994, andVx-11e markedly increased pERK1/2 (Fig. 2D). Phospho-p90RSK (pRSK1) and cyclin D1, which are proximal and distaltargets of pERK1/2, respectively, were similarly inhibited by allinhibitors tested regardless of the degree of ERK1/2 phosphor-ylation (Fig. 2D). These independent findings for BVD-523 areconsistent with studies showing that phosphorylation ofERK1/2 substrates RSK1/2 remained inhibited despite dramat-ically elevated pERK1/2 by Western blot analysis in A375 cells(Fig. 2C), in addition to protein-binding studies demonstratingBVD-523binding and stabilizationofpERK1/2 and inactive ERK1/2(Fig. 1E and F). Therefore, measuring increased pERK1/2 levelscould be considered as a pharmacodynamic biomarker for BVD-523, while quantifying inhibition of ERK1/2 targets such as pRSK orDUSP6 could serve this purpose more directly.

Additional protein changes are of note in this RPPA dataset(Supplementary Fig. S2). Decreased pS6-ribosomal protein isindicated to be another pharmacodynamic marker of ERK1/2inhibition, as evidenced in all cell lines with all inhibitors (Sup-plementary Fig. S3A). Furthermore, prominent induction of pAKTappears to be a cell line–dependent observation, where eachERK1/2 inhibitor induced pAKT in cell lines A375 and AN3CAcells (Supplementary Fig. S3A). Interestingly, the degree of inhi-bition of survival marker pBAD appears to differ between com-

pounds, with only modest inhibition of pBAD by GDC-0994compared with the other ERK1/2 inhibitors tested (Supplemen-tary Fig. S3B).

Next, we investigated how BVD-523 affects cellular localiza-tion of ERK1/2 and downstream target pRSK in a BRAFV600E-mutant RKO colorectal cell line (Supplementary Fig. S3C). Inresting cells, ERK1/2 localizes to the cytoplasm, and once stim-ulated, pERK1/2 migrates to target organelles, particularly thenucleus where transcriptional targets are activated (33). InDMSO-treated control cells, pERK1/2 is evident in both nuclearand cytoplasmic fractions, which is likely reflective of MAPKpathway activity due to the presence of BRAFV600E in this cell line.Treatment with BVD-523 resulted in elevated pERK1/2 in thenucleus and cytoplasm, as well as a modest increase in nucleartotal ERK1/2 compared with DMSO-treated cells, suggesting thatcompound-induced stabilization of pERK1/2 stimulates somenuclear translocation. Despite increased pERK1/2 in both com-partments, pRSK levels are lower in the cytoplasmic and nuclearcompartments compared with DMSO control. ComparatorMAPK signaling inhibitors (i.e., trametinib, SCH772984, dabra-fenib) inhibited phosphorylation of ERK1/2 and RSK, as reflectedby lower levels in the nuclear and cytoplasmic compartments.These data again suggest that BVD-523–associated increases inpERK1/2 are evident in both the cytoplasm and nucleus; how-ever, this does not translate to activation of target substrates. Thisis consistent with data presented in Fig. 2C and D.

BVD-523 demonstrates in vivo antitumor activity inBRAFV600E-mutant cancer cell line xenograft models

On the basis of our in vitro findings that BVD-523 reducedproliferation and induced apoptosis in a concentration-depen-dent manner, BVD-523 was administered by oral gavage todemonstrate its in vivo antitumor activity in models withMAPK/ERK pathway dependency. Xenograft models of melano-ma (cell line A375), and colorectal cancer (cell line Colo205),were utilized, both of which harbor a BRAFV600E mutation.

In A375 cell line xenografts, 5, 25, 50, and 100 mg/kg twicedaily doses of BVD-523 were compared following 18 days oftreatment. For all doses, no significant body weight changes wereobserved (Supplementary Fig. S4A). BVD-523 demonstrated sig-nificant dose-dependent antitumor activity starting at 50 mg/kgtwice daily (Fig. 3A). Doses of 50 and 100 mg/kg twice dailysignificantly attenuated tumor growth, with tumor growth inhi-bition (TGI) of 71% (P ¼ 0.004) and 99% (P < 0.001), respec-tively. Seven partial regressions (PR) were noted in the 100mg/kgtwice daily group; no regression responseswere noted in any othergroup.

BVD-523 demonstrated antitumor efficacy in a Colo205human colorectal cancer cell line xenograft model (Fig. 3B). Nosignificant body weight changes were observed (SupplementaryFig. S4B). BVD-523 again showed significant dose-dependenttumor volume regressions at doses of 50, 75, and 100 mg/kgtwice daily, yielding mean tumor regressions T/Ti (T ¼ end oftreatment, Ti ¼ treatment initiation) of �48.2%, �77.2%, and�92.3%, respectively (all P < 0.0001). Regression was notobserved at the lowest dose of BVD-523 (25 mg/kg twice daily);however, significant tumor growth inhibition, with a T/C (T ¼Treatment, C ¼ Control) of 25.2% (P < 0.0001), was observed.

In addition, BVD-523 resulted in dose-dependent antitumoractivity in KRASG12C-mutant pancreatic cell line xenograft model,MIAPaCa2, (Supplementary Fig. S4C). Here, animals were dosed

Germann et al.





with 10, 25, 50, 75, and 100 mg/kg twice daily BVD-523 for 15days. No significant body weight loss was observed in any treat-ment group (Supplementary Fig. S4D). At the highest doseadministered for BVD-523 (100 mg/kg twice daily), significanttumor growth inhibition compared with vehicle-treated controlswas observed, with a T/C of 5.3% on the last day of treatment (P <0.0001). As a negative control, xenografts ofmammary tumor cellline ZR-75-1were also tested for BVD-523 antitumor activity. Thismodel was not anticipated to respond to ERK inhibition, as it isnot known to harbor alterations in the MAPK/ERK pathway.Indeed, at each dose of BVD-523 tested (10, 30, 60, 100 mg/kgtwice daily), no antitumor activity was observed (SupplementaryFig. S4E), and no significant body weight loss was incurred(Supplementary Fig. S4F).

To establish the relationship between pharmacokineticsand pharmacodynamics, BVD-523 tumor concentrations werecompared with DUSP6 mRNA and pERK1/2 protein levelsover a 24-hour period following a single 100 mg/kg oraldose of BVD-523 (Fig. 3C and D). Phosphorylation ofERK1/2 was low in untreated tumors. Following treatmentwith BVD-523, ERK1/2 phosphorylation steadily increasedfrom 1 hour postdose to maximal levels at 8 hours postdose,then returned to predose levels by 24 hours. This increase inpERK1/2 correlated with BVD-523 drug tumor concentrations.The in vivo observation of increased pERK1/2 with BVD-523

treatment is consistent with earlier in vitro findings (Fig. 2Cand D). Conversely, DUSP6 expression decreased withincreasing BVD-523 tumor concentration (Fig. 3D). MaximalDUSP6 inhibition was observed 3 hours post-BVD-523 dose(�96% change from baseline), and similar to pERK, returnedto baseline by 24 hours.

Combination therapy with BVD-523 and a BRAF inhibitorprovides promising antitumor activity

Patients with BRAF-mutant cancer may acquire resistance tocombined BRAF/MEK therapy (21), warranting considerationof other combination approaches within the MAPK pathway.We assessed the antiproliferative effects of combining BVD-523with the BRAF inhibitors dabrafenib or vemurafenib in theBRAFV600E-mutant melanoma cell lines G-361 and A375. Asanticipated, single agents BVD-523, dabrafenib, and vemura-fenib were each active, and modest synergy was observed with aBVD-523 plus BRAF inhibitor combination (SupplementaryFig. S5A and S5B). This indicated that BVD-523 combinedwith BRAF inhibitors were at least additive and potentiallysynergistic in melanoma cell lines carrying a BRAFV600E muta-tion. Furthermore, generating acquired resistance in vitro fol-lowing continuous culturing of BRAFV600E-mutant cell line(A375) in BRAF inhibitor plus BVD-523 was challenging. Incontrast, generating resistance to dabrafenib alone occurred

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In vivo BVD-523 antitumor activity.Effects of orally dosed BVD-523monotherapy on tumor volume inA375 (BVD-523 groups n ¼ 15,vehicle group n ¼ 10; A) andColo205 (n ¼ 12 per group; B) cellline xenografts. Mean tumor volume� SEM is plotted for each group. InA375, 50 mg/kg and 100 mg/kgBVD-523 twice daily are significantcompared with vehicle control (P ¼0.004 andP <0.001 respectively). InColo205, 50, 75, and 100 mg/kgBVD-523 twice-daily groups aresignificant compared with vehiclecontrol (all P < 0.0001). C, IHCimages for pERK in Colo205xenograft tumors at 0, 1, 3, 8, 16, and24 hours post-single 100 mg/kgdose of BVD-523.D,BVD-523 tumorconcentrations andpercent changesfrom baseline for pERK and DUSP6mRNA are plotted at 0, 1, 3, 8, 16,and 24 hours post-single dose100 mg/kg BVD-523(representative data shown).






relatively rapidly (Supplementary Fig. S5C). Even resistanceto combined dabrafenib and trametinib emerged before dab-rafenib plus BVD-523.

The benefit of combined BRAF and ERK inhibition may notbe fully realized in in vitro combination studies where con-centrations are not limited by tolerability. To understand thebenefit of the combination, efficacy was assessed in vivo uti-lizing xenografts of the BRAFV600E-mutant human melanomacell line A375. Because of the response of combined dabrafe-nib and BVD-523 treatment, dosing in the combination groupswas stopped on day 20 to monitor for tumor regrowth, andwas reinitiated on day 42 (Fig. 4A). Tumors were measuredtwice weekly until the study was terminated on day 45. Themedian time to endpoint (TTE) for controls was 9.2 days, andthe maximum possible tumor growth delay (TGD) of 35.8

days (end of study) was defined as 100%. Temozolomidetreatment resulted in a TGD of 1.3 days (4%) and no regres-sions. The 50- and 100-mg/kg dabrafenib monotherapiesproduced TGDs of 6.9 days (19%) and 19.3 days (54%),respectively, a significant survival benefit (P < 0.001), and 1PR in the 100 mg/kg group. The 100-mg/kg BVD-523 mono-therapy resulted in a TGD of 9.3 days (26%), a significantsurvival benefit (P < 0.001), and 2 durable complete responses.The combinations of dabrafenib with BVD-523 each producedthe maximum possible 100% TGD with statistically superioroverall survival compared with their corresponding mono-therapies (P < 0.001). The lowest dose combination produceda noteworthy 7/15 tumor-free survivors (TFS), and the 3 higherdosage combinations produced a total of 43/44 TFS, consistentwith curative or near-curative activity (Fig. 4B). In summary,

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Effect of combined BVD-523 and BRAF inhibition. A375 BRAFV600E-mutant melanoma cell line xenograft model with a tumor start volume of 75–144mm3 (A andB),and in the same model with larger tumor volume (700–800 mm3) at the initiation of dosing (C and D). Mean � SEM tumor volumes (top) and Kaplan–Meier survival (bottom) are presented for each study (n ¼ 10 for each treatment group). In each experiment, combinations of dabrafenib with BVD-523 resultsin statistically superior overall survival compared with their corresponding controls (P < 0.001).

Germann et al.





the combination of dabrafenib with BVD-523 produced agreater number of TFS and superior efficacy to either singleagent.

On the basis of the activity of BVD-523 plus dabrafenib inA375 xenograft models with a starting tumor volume ofapproximately 75–144 mm3, a follow-up experiment was con-ducted to determine the efficacy of combination therapy in"upstaged" A375 xenografts (average tumor start volume, 700–800 mm3; Fig. 4C). The median TTE for controls was 6.2 days,establishing a maximum possible TGD of 53.8 days, which wasdefined as 100% TGD for the 60-day study. BVD-523 100 mg/kg monotherapy produced a negligible TGD (0.7 day, 1%) andno significant survival difference from controls (P > 0.05). Thedistribution of TTEs and 2 PRs suggested there might have beena subset of responders to treatment with BVD-523 alone.Dabrafenib 50-mg/kg monotherapy was efficacious, yieldinga TGD of 46.2 days (86%) and a significant survival benefitcompared with controls (P < 0.001). This group had 5 PRs and5 CRs, including 3 TFS, among the 11 evaluable mice (Fig. 4D).Both combinations of dabrafenib with BVD-523 produced themaximum 100% TGD and a significant survival benefit com-pared with controls (P < 0.001). Each combination produced100% regression responses among evaluable mice, thoughthere were distinctions in regression activity. The 25 mg/kgdabrafenib and 50 mg/kg BVD-523 combination had 2 PRs and8 CRs, with 6/10 TFS, whereas the 50-mg/kg dabrafenib and100-mg/kg BVD-523 combination had 11/11 TFS on day 60(Fig. 4D). Overall, these data support the rationale for frontlinecombination of BVD-523 with BRAF-targeted therapy inBRAFV600-mutant melanoma, and this is likely to extend toother tumor types harboring this alteration.

BVD-523 exhibits activity in in vitro models of BRAF andMEK inhibitor resistance

Emergence of resistance to BRAF and MEK inhibitors limitstheir clinical efficacy. Here, we sought to model and compare thedevelopment of resistance to BRAF (dabrafenib), MEK (trameti-nib), and ERK1/2 (BVD-523) inhibition in vitro. Over severalmonths, BRAFV600E-mutant A375 cells were cultured in progres-sively increasing concentrations of each inhibitor. Drug-resistantA375 cell lines were readily obtained following growth in highconcentrations of trametinib or dabrafenib, while developing celllines with resistance to BVD-523 proved challenging (Fig. 5A).Overall, these in vitro data suggest that at concentrations yieldingsimilar target inhibition, resistance to BVD-523 is delayed com-paredwith dabrafenib or trametinib, andmay translate to durableresponses in the clinic.

Reactivation and dependence on ERK1/2 signaling is a com-mon feature of acquired resistance to BRAF/MEK inhib-ition (26, 27); therefore, we evaluated the activity of BVD-523 in in vitro models of acquired resistance. First, a dabra-fenib and trametinib combination-resistant A375 populationwas obtained using the increased concentration method (asdescribed in detail in Supplementary Materials and Methodssection of this manuscript). The IC50-fold change from parentalA375 for dabrafenib, trametinib, and BVD-523 in the BRAF/MEK combination-resistant population is shown in Fig. 5B.BVD-523 IC50 was modestly shifted (2.5-fold), while dabrafe-nib and trametinib were more significantly shifted (8.5-foldand 13.5-fold, respectively; Fig. 5B). The cytotoxic agent pac-litaxel was tested as a control with only a modest shift in

potency observed. These data support the investigation ofBVD-523 in the setting of BRAF/MEK therapy resistance,although the mechanism of resistance in this cell populationremains to be characterized.

To further investigate the tractability of ERK1/2 inhibitionin a model with a known mechanism of BRAF inhibitorresistance, we used AAV-mediated gene targeting to generatea pair of RKO BRAFV600E-mutant cell lines isogenic for thepresence or absence of an engineered heterozygous knock-in ofMEK1Q56P-activating mutation. MEK1/2 mutations, includingMEK1Q56P, have been implicated in both single-agent BRAFand combination BRAF/MEK targeting therapy-acquired resis-tance in patients (18, 21, 34–36). Single-agent assays demon-strated that relative to the parental BRAFV600E::MEK1wt cells,the double-mutant BRAFV600E::MEK1Q56P cells displayed amarkedly reduced sensitivity to the BRAF inhibitors vemur-afenib and dabrafenib and the MEK inhibitor trametinib(Fig. 5C). In contrast, response to BVD-523 was essentiallyidentical in both the parental and MEKQ56P-mutant cells,indicating that BVD-523 is not susceptible to this mechanismof acquired resistance. These results were confirmed in 2independently derived double-mutant BRAFV600E::MEK1Q56P

cell line clones, thus validating that results were specificallyrelated to the presence of the MEK1Q56P mutation rather thanan unrelated clonal artifact. Similar results were also observedwith a second mechanistically distinct ERK1/2 inhibitor(SCH772984), supporting the expectation that these observa-tions are specifically related to mechanistic inhibition ofERK1/2 and not due to an off-target compound effect.

To further characterize the mechanistic effects of BVD-523 onMAPK pathway signaling in BRAFV600E::MEK1Q56P cell lines,protein levels were assessed by Western blot analysis (Fig. 5D).In the parental BRAFV600E RKO cells, a reduced level of pRSK1/2was observed following 4-hour treatment with BRAF (vemurafe-nib), MEK (trametinib), or ERK1/2 (BVD-523) inhibitors atpharmacologically active concentrations. In contrast, isogenicdouble-mutant BRAFV600E::MEK1Q56P cells did not exhibitreduced RSK phosphorylation following BRAF or MEK inhibitortreatment, while BVD-523 remained effective in inhibitingpRSK1/2 to a level comparable with parental RKO. Similarly,pRB is reduced, indicating G0–G1 arrest by 24 hours of BVD-523treatment in both parental RKOandBRAFV600E::MEK1Q56P.Nota-bly, as introduction of the MEK-Q56P mutation alone increasedthe level of pERK compared with the RKO parental cell line, BVD-523 treatment did not appear to significantly further boost pERKlevels in the engineered cells.

Acquired KRAS mutations are also known drivers of resis-tance to MAPK pathway inhibitors. To understand the sus-ceptibility of BVD-523 to this mechanism of resistance, anisogenic panel of clinically relevant KRAS mutations incolorectal cell line SW48 was utilized. Sensitivity to BVD-523 was compared with MEK inhibitors selumetinib (37)and trametinib (Fig. 5E and F). Sensitivity to paclitaxel wasunaltered (Supplementary Fig. S6). While several mutantKRAS alleles conferred robust to intermediate levels ofresistance to MEK inhibition, sensitivity to BVD-523 wasunaltered by the majority of alleles, and where a shift insensitivity was observed, it was not to the extent observedwith trametinib or selumetinib. Overall, these data suggestthat BVD-523 is more efficacious in this context than MEKinhibitors.






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BVD-523 demonstrates activity in models of resistance to BRAF/MEK inhibition. A, The emergence of resistance to BVD-523, dabrafenib, or trametinib inBRAFV600E A375 cells following exposure to increasing concentrations of drug is indicated. A strict set of "criteria" was applied (see Materials andMethods section) as to when the concentration of drug was increased. Each point on the plotted line represents a change of medium or cell split. B, In A375cells cultured to be resistant to combined BRAF (dabrafenib) þ MEK (trametinib), the fold change in IC50 compared with parental A375 of BVD-523,dabrafenib, trametinib, and paclitaxel was measured. C, Parental and BRAFV600E RKO cells engineered with endogenous heterozygous knock-in ofMEK1Q56P were incubated with BRAF (vemurafenib or dabrafenib), MEK (trametinib), or ERK (BVD-523) inhibitor and cell viability assessed as a functionof inhibitor concentration. Data presented are average � SEM of three independent titrations of each inhibitor and cell line. D, Western blot analyses ofpERK, pRSK, and pRB from cell lines treated in C. E and F, Knock-in of KRAS-mutant alleles into SW48 cell line significantly diminishes sensitivity to theMEK inhibitors trametinib and selumetinib, while comparative sensitivity to BVD-523 is retained (n ¼ 3 per titration; data are average � SEM).

Germann et al.





BVD-523 demonstrates in vivo activity in a BRAF inhibitor–resistant patient-derived melanoma xenograft model

To confirm and extend the antitumor effects of BVD-523observed in in vitro models of BRAF-/MEK-acquired resistance,we utilized a BRAF-resistant xenograft model derived from apatient with resistance to vemurafenib. BVD-523 was dosed byoral gavage at 100 mg/kg twice daily for 28 days, both aloneand in combination with dabrafenib at 50 mg/kg twice daily(Fig. 6). With all regimens, no significant change in bodyweight was observed (Supplementary Fig. S7). As expected,minimal antitumor activity was demonstrated for single-agentdabrafenib (22% TGI). BVD-523 activity was significant com-pared with vehicle control (P� 0.05), with a TGI of 78%. In thismodel, combining BVD-523 with dabrafenib resulted in a TGIof 76% (P � 0.05); therefore, further benefit was not gained forthe combination compared with single-agent BVD-523 in thisparticular model of BRAF-acquired resistance. Whole-exomesequencing was performed on both the patient biopsy and thePDX model. A number of alterations with unknown signifi-cance were identified in addition to the known driver mutationPIK3CA-H1047L. We speculate that the presence of this muta-tion may actually attenuate response to BVD-523, as stasisrather than regression was observed.

DiscussionBVD-523 is a potent, highly selective, reversible, small-mole-

cule ATP-competitive inhibitor of ERK1/2 with activity in in vitroand in vivo cancer models. In vitro, BVD-523 demonstrated potentinhibition against several human tumor cell lines, particularlythose harboring activating mutations in the MAPK signalingpathway, consistent with its mechanism of action. BVD-523elicited changes in downstream target and effector proteins,including inhibition of direct substrate of ERK1/2, pRSK, andtotal DUSP6 protein levels (transcriptional target of ERK1/2).These findings are in line with those of previous studies of otherERK1/2 inhibitors, which demonstrated effective suppression of

pRSK with ERK1/2 inhibition (26, 27). Interestingly, BVD-523treatment resulted in a marked increase in ERK1/2 phosphoryla-tion in vitro and in vivo. Similar to our findings, an increase inpERK1/2 has been reported with the ERK1/2 inhibitor Vx11e;conversely, pERK1/2 increases are not observed with SCH772984(26). Although differences in pERK1/2 levels were observedamong the various ERK1/2 inhibitors tested, downstream effec-tors (i.e., pRSK1 and total DUSP6) were similarly inhibited. Thesefindings suggest quantifying ERK1/2 target substrates, such aspRSK1, may serve as reliable pharmacodynamic biomarkers forBVD-523–mediated inhibitionof ERK1/2 activity. Thedifferentialeffect of BVD-523 and SCH772984 upon the levels of pERK isstriking. The significance of this to efficacy and tolerability in theclinic remains to be determined.

While BRAF (dabrafenib, vemurafenib) and MEK (trameti-nib, cobimetinib) inhibitors validate the MAPK pathway as atherapeutic target, particularly in patients with BRAFV600 muta-tions, the antitumor response is limited by the emergence ofacquired resistance and subsequent disease progression (38).Reactivation of the ERK1/2 pathway is one common conse-quence of acquired resistance mechanism. When introducedinto the BRAFV600E-mutant colorectal cell line RKO, MEKQ56P

conferred resistance to MEK and BRAF inhibition. In contrast,BVD-523 retained its potent inhibitory activity in the engi-neered MEKQ56P cell line, indicating that ERK1/2 inhibition iseffective in the setting of upstream activating alterations, whichcan arise in response to BRAF/MEK treatment. As further evi-dence of a role for BVD-523 in the context of acquired resis-tance, efficacy of BVD-523 was evident in a xenograft modelderived from a patient whose disease progressed on vemurafe-nib; the BRAF inhibitor dabrafenib was not effective in thismodel. These data support a role for targeting ERK1/2 in thesetting of BRAF/MEK resistance, and complement previouslypublished findings (26, 27).

To further characterize resistance to inhibitors of the MAPKpathway, the emergence of resistance to BVD-523 itself wasinvestigated. We found that single-agent treatment of cancer cellswith BVD-523 was durable, and it was more challenging todevelop resistance against BVD-523 than other agents targetingupstream MAPK signaling components (i.e., dabrafenib, trame-tinib). This may suggest that acquiring resistance to ERK1/2-targeting agents is harder to achieve than acquiring resistance toBRAForMEK therapy.However, in vitro studieswith other ERK1/2inhibitors have identified specific mutants in ERK1/2 that driveresistance (39, 40); these specific mutations have yet to beidentified in clinical samples from ERK1/2 inhibitor–relapsedpatients.

The potential clinical benefit of ERK1/2 inhibition with BVD-523 extends beyond the setting of BRAF/MEK therapy–resistantpatients. As ERK1/2 is a downstream master node within thisMAPK pathway, its inhibition is attractive in numerous cancersettings where tumor growth depends on MAPK signaling.Approximately 30% of all cancers harbor RAS mutations;therefore, targeting downstream ERK1/2 with BVD-523 is arational treatment approach for these cancers. Furthermore,results from a study by Hayes and colleagues indicate thatprolonged ERK1/2 inhibition in KRAS-mutant pancreatic can-cer is associated with senescent-like growth suppression (41).However, a combination approach may be required for max-imal and durable attenuation of MAPK signaling in thesetting of RAS mutations. For example, MEK inhibition in

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Figure 6.

BVD-523 in vivo activity in xenograft derived from a vemurafenib-relapsedpatient. Mean tumor volume (�SEM) is shown for BVD-523 100 mg/kgtwice daily (BID) alone, dabrafenib 50 mg/kg twice daily alone, andBVD-523 100 mg/kg twice daily plus dabrafenib 50 mg/kg twice daily.






KRAS-mutant colorectal cancer cell results in an adaptiveresponse of ErbB family activation, which dampens theresponse to MEK inhibition (42). Similar context-specific adap-tive responses may occur following ERK1/2 inhibition withBVD-523. The optimal treatment combinations for variousgenetic profiles and cancer histologies are the subject of ongo-ing research. In addition to BRAF and RAS mutations, otheralterations which drive MAPK are emerging. For example, novelRAF fusions and atypical (non-V600) BRAF mutations whichpromote RAF dimerization activate the MAPK pathway (43).BRAF inhibitors such as vemurafenib and dabrafenib whichinhibit BRAFV600-mutant monomer proteins have been shownto be inactive in atypical RAF alterations which drive MAPKsignaling in a dimerization-dependent manner (43). However,treatment with BVD-523 to target downstream ERK1/2 in thesetumors may be a novel approach to addressing this unmetmedical need; indeed patients harboring such atypical BRAFalterations are included in an ongoing clinical trial of BVD-523(NCT01781429).

In the setting of BRAFV600-mutant melanoma tumors, com-bined BRAF andMEK inhibition exemplifies how agents targetingdifferent nodes of the same pathway can improve treatmentresponse and duration. Our combination studies in BRAFV600E-mutant xenografts of human melanoma cell line A375 providesupport for combination therapy with BVD-523 and BRAF inhi-bitors. The combination demonstrated superior benefit relative tosingle-agent treatments, including results consistent with curativeresponses. The clinical efficacy and tolerability of combinedBRAF/BVD-523 therapy remains to be determined. It would notbeunreasonable to expect that aBRAF/BVD-523 combinationwillat least be comparable in efficacy to a targeted BRAF/MEK com-bination. Furthermore, the in vitro observation that acquiredresistance to BVD-523 is more challenging to achieve comparedwith other MAPK pathway inhibitors suggests that the BRAF/BVD-523 combination has the potential to provide a moredurable response.

It would be remiss to not highlight that significant progress hasalso been made using immunotherapy for the treatment ofmelanoma. The FDA has approved various immune checkpointinhibitors for the treatment of advancedmelanoma, including thecytotoxic T-lymphocyte antigen-4 targeted agent ipilimumab, andthe programmed cell death protein-1 (PD-1) inhibitors pembro-lizumab and nivolumab. Emerging data suggest MEK inhibitorsmay have benefit in combination with anti-PD-L1 therapy ate-zolizumab (44). Combining BVD-523 with such immunothera-pies is an attractive therapeutic option; further investigation iswarranted to explore dosing schedules and to assess whethersynergistic response can be achieved.

On the basis of the preclinical data, BVD-523 may holdpromise for treatment of patients with malignancies dependent

on MAPK signaling, including those whose tumors haveacquired resistance to other treatments. Phase I clinical studiesof BVD-523 are ongoing (NCT01781429, NCT02296242, andNCT02608229).

Disclosure of Potential Conflicts of InterestU.A. Germann has ownership interest (including patents) in Vertex

Pharmaceuticals Incorporated. J.J. Roix is an associate at BioMed ValleyDiscoveries. D.A. Sorrell has ownership interest (including patents) inHorizon Discovery. M. Fitzgibbon is a senior research scientist and hasownership interest (including patents) in Vertex Pharmaceuticals. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: U.A. Germann, W. Markland, R.R. Hoover,A.M. Aronov, J.J. Roix, M. Hale, R. Samadani, G. DeCrescenzo, S. Saha,D.J. WelschDevelopment of methodology: B.F. Furey, W. Markland, R.R. Hoover,J.J. Roix, D.A. Sorrell, R. Samadani, G. DeCrescenzo, M. Namchuk, S. Saha,D.J. WelschAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.F. Furey, W. Markland, R.R. Hoover, D.M. Boucher,D.A. Sorrell, M. Fitzgibbon, P. Shapiro, M.J. Wick, R. Samadani, K. Meshaw,C.M. Emery, S. Saha, D.J. WelschAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): U.A. Germann, B.F. Furey, W. Markland,R.R. Hoover, J.J. Roix, D.M. Boucher, D.A. Sorrell, P. Shapiro, R. Sama-dani, A. Groover, G. DeCrescenzo, M. Namchuk, C.M. Emery, S. Saha,D.J. WelschWriting, review, and/or revision of themanuscript:U.A. Germann, B.F. Furey,W. Markland, A.M. Aronov, J.J. Roix, M. Hale, D.M. Boucher, D.A. Sorrell,G. Martinez-Botella, M.J. Wick, R. Samadani, A. Groover, G. DeCrescenzo,M. Namchuk, C.M. Emery, S. Saha, D.J. WelschAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.J. Roix,M. Fitzgibbon, A. Groover, C.M. Emery,S. Saha, D.J. WelschStudy supervision: U.A. Germann, J.J. Roix, D.A. Sorrell, M. Namchuk,C.M. Emery, S. Saha, D.J. WelschOther (his group while at Vertex designed and made the compound in thestudy and holds patents on the chemical matter): M. Hale

AcknowledgmentsThe authors thank Corinne Ramos and Nicholas Hoke for performing the

large-scale reverse phase protein array. They also thank SharonMaguire andPaulA. Russell for assay support and Amy Smith and Mark Stockdale for theconstruction of the MEK1Q56P cell line.

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 to indicatethis fact.

Received May 19, 2017; revised August 2, 2017; accepted August 23, 2017;published OnlineFirst September 22, 2017.

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2017;16:2351-2363. Published OnlineFirst September 22, 2017.Mol Cancer Ther Ursula A. Germann, Brinley F. Furey, William Markland, et al. BVD-523 (Ulixertinib)Preclinical Activity with the Novel Selective ERK1/2 Inhibitor Targeting the MAPK Signaling Pathway in Cancer: Promising

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