epithelial tissue hyperplasia induced by the raf inhibitor ... · pf-04880594 is attenuated by a...

Preclinical Development

Epithelial Tissue Hyperplasia Induced by the RAF InhibitorPF-04880594 Is Attenuated by a Clinically Well-ToleratedDose of the MEK Inhibitor PD-0325901

Vince R. Torti1, Donald Wojciechowicz2, Wenyue Hu1, Annette John-Baptiste1, Winston Evering1,Gabriel Troche2, Lisa D. Marroquin1, Tod Smeal2, Shinji Yamazaki3, Cynthia L. Palmer4,Leigh Ann Burns-Naas1, and Shubha Bagrodia2

AbstractClinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated

BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment

of advanced melanoma. However, about a third of patients developed resectable skin tumors during the

course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal–

regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical

models. Because these findings raise safety concerns about RAF inhibitor development, we further

investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK

phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia

and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extra-

cellular signal–regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically

well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional

cell culture model of epithelial layering that recapitulated the RAF inhibitor–induced hyperplasia and

reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the

inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing

observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not

seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity

usually associated with such doses. These findings indicated that combination treatment with MEK

inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of

melanoma and other cancers. Mol Cancer Ther; 11(10); 2274–83. �2012 AACR.

IntroductionThe BRAF protein kinase plays a central role in pro-

moting normal cell proliferation and survival as a com-ponent of the RAS/RAF/MEK/ERK signaling pathway.Mutations in the BRAF gene that activate BRAF kinase

activity independently of RAS activation have beenobserved in approximately 50% of malignant melanomas(1–3). BRAF has therefore attracted much attention as apotential target of inhibitor therapies in melanoma andother tumors in which BRAF is mutated. Indeed, theselective RAF inhibitor vemurafenib (PLX4032) wasrecently approved by the U.S. Food and Drug Adminis-tration for treatment of metastatic melanomas in patientswith BRAFV600E mutation. In a pivotal phase III trial of675 patients with previously untreated metastatic mela-noma, 74% had a reduction in risk of progression or deathwith vemurafenib compared with the reference drug,dacarbazine (4). Mean progression-free survival withvemurafenib was 5.3 months, compared with 1.6 monthsfor the dacarbazine cohort, and response rates of 48%withvemurafenib and 5% with dacarbazine were observed.Another selective RAF inhibitor, GSK2118436, has recent-ly entered phase III trials after showing similar effective-ness in melanoma patients, and other compounds are inearly clinical development.

Despite the remarkable clinical effectiveness of vemur-afenib and other RAF inhibitors, there remain 2 key

Authors' Affiliations: 1Drug Safety Research and Development, 2Oncol-ogy Research Unit, 3Pharmacokinetics, Dynamics, and Metabolism, and4Oncology Chemistry Design at Pfizer, San Diego, California and PearlRiver, New York

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

V.R. Torti and D. Wojciechowicz contributed equally to this work.

Current address for L.A. Burns-Naas, Gilead Sciences Inc, 333 LakesideDrive, Foster City, CA 94404

CorrespondingAuthor:ShubhaBagrodia,OncologyResearchUnit, PfizerWorldwide Research and Development, La Jolla Laboratories, 10724Science Center Drive, San Diego, CA 92121. Phone: 858-526-4828; Fax:877-481-3086; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0984

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(10) October 20122274

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Published OnlineFirst July 2, 2012; DOI: 10.1158/1535-7163.MCT-11-0984

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concerns over long-term efficacy and safety. First, clinicaldevelopment of vemurafenib revealed that a large num-ber of patients with BRAFV600Emutation had primary oracquired resistance to the RAF inhibitor. Acquired resis-tance does not seem to develop as a result of mutation of"gatekeeper" residues in BRAF itself, as occurs in resis-tance to BCR-ABL and EGFR inhibitors (5), but fromactivation of other proteins in the same or parallel signal-ing pathways (6). Two of the most common resistancemechanisms involve mutation in the NRAS gene andupregulation of PDGFRb expression (7). Second, a highpercentage of patients in clinical trials of vemurafenib andGSK2118436 developed skin tumors, particularly keratoa-canthoma and squamous cell carcinoma. This is regardedas an acceptable risk for treatment of advancedmelanomabecause in all cases, the tumors were resected and did notprogress to metastases. Nonetheless, the possibility ofRAF inhibitor–induceddevelopment of tumors in internalorgans less amenable to observation remains a majorconcern, andunderstanding the causativemechanismhasbeen a priority.The most likely explanation for development of skin

tumors during RAF inhibitor treatment comes from stud-ies showing that RAF inhibitors cause activation of theextracellular signal–regulated kinase (ERK) pathway incells with wild-type BRAF (8–11). Pathway activationinvolves formation of homo- or heterodimers betweenBRAF and CRAF, in which an inhibitor-bound protomertransactivates its dimeric partner in a RAS-GTP–depen-dent manner (12, 13). In keeping with this model, ectopicexpression of kinase-defective BRAF also causes ERKpathway activation in tumor cells with wild-type BRAF(14). Stimulation of ERK activity, cell proliferation, andtumor growth in vivo was observed in response to RAFinhibitor treatment in preclinical models with bothmutant and wild-type RAS genes (11).It is possible that development of squamous cell carci-

noma in RAF inhibitor–treated patients depends on pre-existing mutations or it is also possible that tumorigenicgenetic changes accumulate as a secondary consequenceof hyperplasia. Indeed, Carnahan and colleagues (8)showed increased cell proliferation and hyperplasia inmouse stomach epithelial tissue after treatment with theRAF inhibitor C-19 that was prevented by cotreatmentwith the mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor PD-0325901. In a followup study, Wisler and colleagues (15) showed hyperplasiain response to a series of RAF inhibitors in multiple ratepithelial tissues. Moreover, severe hyperplasia in thebladder transitional epithelium was found to be associ-atedwith development of a very rare carcinoma in one rat(15). Clearly this will need to be reproduced, but it doesraise the possibility of development of nonskin neoplasiasin melanoma patients receiving RAF inhibitor therapy.PD-0325901, is a highly potent, second-generation

selective MEK1/2 inhibitor that inhibits phosphorylationof ERK1/2 and proliferation in BRAF-mutant tumorcells (16). PD-0325901 showed promising efficacy in early

clinical development, but trials were discontinued afterphase II because of unacceptable musculoskeletal, neuro-logic, and ocular toxicities at doses greater than 15 mgdosed twice daily intermittently (17–19). The potent andselective RAF inhibitors PF-04880594 (20) and WYE-130600 (21), which inhibit both wild-type and mutantBRAF and CRAF, have shown tumor growth inhibitionin BRAF-mutant melanoma xenograft models. However,during the course of preclinical studies, we observedhyperplasia in response to both inhibitors. Because the2 inhibitors are structurally unrelated, this indicated thatthe effect was mechanism specific. Indeed, we hadobserved activation of ERK in cell lines with mutant RASor activated growth factor receptor signaling (unpub-lished observations). On the basis of these observations,we investigated whether RAF inhibitor–induced hyper-plasia was associated with ERK activation and BRAF-CRAF dimerization in hyperplastic tissues and, mostimportantly, whether hyperplasia could be prevented bydoses of MEK inhibitor that would extrapolate to clini-cally safe doses. To facilitate further studies, we alsodeveloped a three-dimensional (3D) cell culture modelthat recapitulated the RAF and MEK inhibitor effects onepithelial hyperplasia in vitro. We were also able to showthat RAF inhibitors induce expression of the inflamma-tory cytokine interleukin 8 (IL-8), providing a possibleexplanation of some of the observed preclinical effects ofRAF inhibitor treatment.

Materials and MethodsModulation of ERK phosphorylation in mice treatedwith the RAF inhibitor (PF-04880594) and the MEKinhibitor (PD-0325901)

Nude mice were treated with either 10 mg/kg ofPF-04880594 alone, 0.5 mg/kg of PD-0325901 alone, or incombination. Chemical structures of the 2 drugs are pro-vided (Fig. 1). Animals were administered 3 doses over 2days: an amandpmdose on day 1 and then an amdose onday 2 approximately 2 hours before the animals werenecropsied and the tissues harvested. A selective panel oftissues, including esophagus, skin, tongue, and urinarybladder, were collected and flash frozen in liquid nitro-gen. Samples were powderized with a mortar and pestleand homogenized in NP40 lysis buffer (25 mmol/L Tris,150mmol/LNaCl, 1mmol/L EDTA, 5% glycerol, and 2%NP-40, pH 7.4), with 10� Halt protease and phosphataseinhibitors (Thermo Scientific). Lysate was centrifuged at12,000 � g for 10 minutes at 4�C and the supernatant wascollected. Protein concentrationwasdeterminedusing thebicinchoninic acid protein assay (Thermo Scientific).

Immunoprecipitation and immunoblottingLysates (500 mg of protein) were incubated with 2 mg of

anti-BRAF antibody (sc-166; Santa Cruz Biotechnology)for 1 hour at 4�Cwhile rocking, followed by an additional30 minutes incubation with 40 mL of resuspended A/GPlus agarose (Santa Cruz Biotechnology). Samples werespundownat 200�g for 1minute andwashedwith 500mL

Improving Tolerance to RAF Inhibitors

www.aacrjournals.org Mol Cancer Ther; 11(10) October 2012 2275




of NP40 buffer 3 times. Then 35 mL of lithium dodecylsulfate sample loading buffer with reducing agent wasadded. Samples were boiled at 95�C to 100�C for5 minutes and then spun at 2,000 � g for 1 minute andsupernatant was collected.

Immunoprecipitates or protein homogenates wereelectrophoretically separated with a NuPAGE 4% to12% Bis-Tris gel (Invitrogen) and transferred to a0.45-mm nitrocellulose membrane. After blocking influorescent blocking buffer (Rockland Immunochem-icals) for 1 hour at room temperature, the membranewas incubated with primary antibodies overnight at4�C. Primary antibodies included anti-phospho-MAPK(ERK1/2; M 8159; Sigma Chemical Co.), anti-MAPK(ERK 1/2; 9102; Cell Signaling), anti-CRAF (610152; BDBiosciences), and anti-BRAF (sc-5284; Santa Cruz Bio-technology). ERK control extracts (9194; Cell Signaling)and Caco-2 whole-cell extract were used as negativeand positive controls for p-ERK and BRAF/CRAF mea-surement. Membranes were washed, probed with sec-ondary infrared antibodies (donkey anti-rabbit IgG orrabbit anti-mouse IgG; Rockland Immunochemicals) for1 hour at room temperature, and washed again. Afterthe final wash, membranes were visualized on theOdyssey infrared scanner (Li-Cor Biosciences).

Microscopic pathology of tissues from PF-04880594and PD-0325901–treated nude mice

Nudemice (6–8weeks old) were treatedwith either PF-04880594 10, 20, or 40mg/kg twice daily or PD-0325901 at1 mg/kg and/or at 0.1, 0.3, 0.5, 1.0, or 2.5 mg/kg twicedaily in combination with PF-04880594 as indicatedin Table 1. Treatment occurred daily for 3 weeks, afterwhich all surviving mice were euthanized and necrop-sied. The esophagus, tongue, footpad, urinary bladder,and dorsal skin were excised and preserved in 10% neu-tral buffered formalin (VWR). Following fixation, all tis-sues were embedded in paraffin blocks, sectioned,mounted on slides, and stained with hematoxylin andeosin (H&E). Slides were evaluated for the incidence andseverity of a hyperplastic response.

All animal studies were conducted in a facility accre-dited by the Association for Assessment and Accredita-tion of LaboratoryAnimal Care International. In addition,all animal experiments were carried out in accordancewith the principles described in the guide for care and useof laboratory animals by theNIH (22) andapprovedby theInstitution Animal Care and Use Committee associatedwith the facility in which the laboratory animals werehoused.

Reconstructed human epidermis 3D cell culture andtreatment

The in vitro reconstructed human epidermis (RHE;Skinethic Laboratories) consists of normal human kerati-nocytes cultured on an inert polycarbonate filter at the air–liquid interface in the proprietary growth culture medi-um. These cells have been tested and authenticated. Thesecells express major differentiation markers that include(i) filaggrin and involucrin in granular layers, (ii) trans-glutaminase I and keratin 10 in suprabasal cell layers, and(iii) loricrin in upper granular cell layers, and (iv) thebasement membrane markers, type IV collagen, integrina6, integrin b4, antigen BP, laminin 1, and laminin V. Thecells were tested for identity via immunohistochemistryby Skinethic Laboratories for each batch before shipping.Cells were treated with dimethyl sulfoxide (DMSO) orRAF inhibitor PF-04880594 at 62.5 nmol/L and MEKinhibitor PD-0325901 at 5 nmol/L separately or in com-bination in the basal chamber for 2 days. Duplicate sam-ples from each treatment were fixed overnight in 10%formalin and subjected to histologic evaluation usingH&E staining. A second set of samples with the sametreatments was flash frozen in liquid nitrogen for proteinextraction. The phosphorylated and total forms of ERKwere evaluated using immunoblotting, as described.

Induction of IL-8 release from HL-60 cells by PF-04880594 and attenuation of release by PD-0325901

HL-60 cells were procured from the American TypeCulture Collection and stored for 5 years. Cells weretested for authenticity by differentiating with phorbolmyristate acetate (PMA), as has been previously reported(23). On an intermittent basis, PMA was added to HL-60

HNN

NN

N

N

NH

N

F

F

PF-04880594

A

B

NH

NH

O

HO

O

OH

F

I

FF

PD-0325901

Figure 1. Chemical structures for PF-04880594 (BRAF inhibitor; A) andPD-0325901 (MEK inhibitor; B).

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Mol Cancer Ther; 11(10) October 2012 Molecular Cancer Therapeutics2276




cells in RPMI-1640 at a concentration of 100 ng/mL, andthe cells were observed for morphologic signs of differ-entiation to authenticate their identity. Secreted IL-8 wasdetermined using a standard ELISA-based assay (SABiosciences/Qiagen), 3 � 106 HL-60 cells were incubatedwith 100 nmol/L PF-04880594 or 100 nmol/LMEK inhib-itor or both for 48 hours in RPMI media containing 10%FBS. Control cells were treated with DMSO (0.25% final).After 48 hours, the culture media was separated from thecells by centrifugation at 200� g for 5minutes. The culturesupernatant was stored at 4�C and assayed the followingday. The cellswerewashed once in cold PBS and lysed in abuffer as described above. Protein determination wascarried out using the BCA protein assay (Pierce).

ResultsThe RAF inhibitor PF-04880594 induces ERKphosphorylation and BRAF-CRAF dimerization inmultiple epithelial tissuesHatzivasiliou and colleagues (13) showed elevation of

p-ERK, by immunohistochemistry, in skin sections frommice treated with RAF inhibitor. To extend this observa-tion, we examined p-ERK levels in target tissues frommice treated with PF-04880594 alone or in combinationwith PD-0325901. As shown in Fig. 2A, tissue (urinarybladder, tongue, skin, and esophagus) homogenates frommice dosed with PF-04880594 at 10 mg/kg for 2 daysshowed higher levels of p-ERK compared with tissuehomogenates from animals treated with vehicle. Theinduction of ERK phosphorylation by PF-04880594 treat-ment was attenuated by cotreatment with 0.5 mg/kg PD-0325901. A similar induction of p-ERKwas seen in tissues

taken from dogs dosed with the RAF inhibitor WYE-130600 (data not shown).

It has been shown that the activation of ERK in tumorcells with wild-type BRAF involves the formation ofhomo- and heterodimers between BRAF and CRAF iso-forms (12, 13). To determine whether dimerization mightbe responsible for the observed ERK in normal mousetissues, we immunoprecipitated BRAF from tissueshomogenate lysates and immunoblots were probed withanti-CRAF antibodies. As shown in Fig. 2B, CRAF wasdetectable in BRAF immunprecipitates from skin andesophagal lysates from animals that were treated withPF-04880594 alone or in combination withMEK inhibitor,but not in animals treated with vehicle or MEK inhibitoralone. These results are entirely consistent with the pro-posed model of RAF inhibitor transactivation of RAFisoforms via inhibitor-induced dimerization. The MEKinhibitor, although blocking ERK activation, would not beexpected to inhibit dimer formation.

Induction of epithelial hyperplasia by PF-04880594is prevented by low doses of the MEK inhibitor PD-0325901

Wisler and colleagues showed hyperplasia in multiplestratified squamous and transitional epithelial tissuesafter treatment of rats with a series of RAF inhibitorsfor 7 days (15). In agreement, we observed a similar levelof hyperplasia in squamous epithelial layers from esoph-agus, tongue, urinary bladder, and footpad in micetreated for 21 days with the RAF inhibitor PF-04880594at 10, 20, and 40 mg/kg (Fig. 3 and Table 1). Carnahanand colleagues (8) have previously shown that RAF

Table 1. Severity and incidence of hyperplastic lesions in mice treated with BRAF inhibitor (PF-04880594)and MEK inhibitor (PD-0325901)

Compound Tissuesa incidence/total mice per group (severity)

Group PD-0325901 PF-04880594 Esophagus Tongue Feet

1 0 0 – – –

2 0 10 4/6 (1,2) 4/6 (1) 2/6 (1)3 1 0 – – –

4 0.5 10 – – –

5 0.3 10 – – –

6 0.1 10 4/6 (1–2) – –

7 0 20 4/6 (2–3) 4/6 (2–3) 4/6 (2–3)8 2.5 20 – – –

9 1.0 20 – – –

10 0.3 20 – – –

11 0 40 5/6 (2–3) 6/6 (2–3) 6/6 (2–3)12 2.5 40 – – –

13 1 40 – – –

14 0.3 40 – – –

aTissues positive for hyperplasia are denoted as a/b (c), in which a¼ the number of hyperplasia positive mice, b¼ the total numberof mice examined per group, and c ¼ the severity. Severity of lesions is described as 1, minimal, 2, mild, 3, moderate, and 4,severe.






inhibitor–induced hyperplasia in nonglandular stomachepitheliumwas blocked by theMEK inhibitor PD-0325901at 3 mg/kg. To test the effectiveness of PD-0325901 inpreventing RAF inhibitor–induced hyperplasia, we useda dose range of PD-0325901 (0.1–1 mg/kg) that, on thebasis of pharmacokinetic and toxicologic considerations,extrapolated to a dose range that was well toleratedin clinical trials (Supplementary Fig. S1). At 1 mg/kg,PD-0325901 blocked hyperplasia in response to 10mg/kgPF-04880594 (Fig. 3). Titration of the PD-0325901 doserevealed that it was effective at ratios up to at least 1:33at all doses of PF-04880594 (Table 1).

PF-04880594 at 10 mg/kg was well tolerated in thesestudies; however, mice treated with 20 and 40 mg/kg PF-04880594 were euthanized at week 1 and 2, respectively,because ofmoribundity associatedwith toxicity. Postdoseobservations included up to a 9% body weight loss over6 days (Supplementary Fig. S2) and noticeable hypoactiv-ity. Mice in the 10 mg/kg RAF inhibitor dose group werebeginning to show signs of RAF inhibitor–induced toxic-ity by the end of the study just before scheduled eutha-nasia after 3 weeks of dosing. However, mice in RAFinhibitor–treated groups with up to 40 mg/kg PF-04880594 treatment that also received PD-0325901 treat-ment survived the treatment period and showed lesserweight loss to no weight loss (Supplementary Fig. S2).

A 3D human epidermal model recapitulates PF-04880594–induced hyperplasia in vitro

To further evaluate the clinical relevance of the hyper-proliferative response to RAF inhibitors, we used a 3Dculture model of RHE cells (Skinethic), which bears his-tologic similarity to human epidermal layers. Weobserved growth and development of all squamous epi-thelial cell layers (stratum geminativum, stratum spino-sum, stratum granulosum, and stratum corneum) acrossall groups (Fig. 4A). There was a thick layer of viable cellsthat was 4 to 6 cells deep and covered by a cornified layer(stratum corneum) that was approximately half the totalthickness of the sample (Fig. 4A).After theRHEcellmodelwas treated with PF-04880594 at 62.5 nmol/L for 2 days,marked necrosis was evident, characterized by the pres-ence of ghost cells in a layer superficial to the viable cells,with cell debris scattered at the ghost cell–viable cellinterface and within the viable cell layer (Fig. 4B). Theghost cell layer was considerably thicker than the viablecell layer below it, approximately 6 to 7 cells deep andconsisted approximately 50% to 60% of the thickness ofthe culture, whichwere approximately 10 to 14 cells deep,compared with 4 to 6 cells deep in untreated cells(compare Fig. 4A with B). Cells treated with PF-04880594 and PD-0325901 in combination closely resem-bled untreated cells, with a viable cell layer 4 to 6 cells

Figure 2. RAF inhibitor (PF-04880594) induces ERK phosphorylation and BRAF/CRAF dimerization in epithelial tissues. Tissue homogenates from urinarybladder, tongue, skin, and esophagusweremade frommice treatedwith PF-04880594 at 10mg/kg or PD-0325901 at 0.5mg/kg separately or in combinationfor 2 days. A, levels of phosphorylated and total ERK were evaluated using immunoblotting and the band intensity was digitally quantified (Odyssey 2.1software). The levels of phosphorylated ERK were normalized against total ERK and expressed as a fold change over vehicle control. Jurkat wholecells extract treated with U0126 at 10 mmol/L for 1 hour were used as negative control, and Jurkat cells treated with TPA at 200 nmol/L for 10 minutesserved as positive control for ERK phosphorylation. B, the dimerization of BRAF/CRAF was evaluated in the esophagus and skin samples viaimmunoprecipitation using BRAF-specific antibody, followed by immunoblotting using CRAF-specific antibody. The total level of CRAF was also assessedin the same membrane as the loading control. Caco-2 whole-cell extract was used as positive control.

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deep covered by a cornified layer that was approximately40% to 50% of the total thickness of the culture withminimal single cell necrosis and no detectable ghost cells.The level of p-ERK and total ERK were evaluated in RHEcells treated with PF-04880594 or PD-0325901 separatelyor in combination (Fig. 4D). Consistent with observationsin mouse epithelial tissues, the level of p-ERK was sig-nificantly induced by RAF inhibitor treatment in RHEcells. This increase in p-ERK was attenuated by cotreat-ment with PD-0325901, at doses comparable with the invivo exposure level in mice.

RAF inhibitors induce IL-8 release in vitroAfter relatively short periods of exposure (1–3 days),

mice treated with PF-04880594 and dogs treated withWYE-130600 were seen to experience flushing of the skin(Supplementary Fig. S3). We speculated that this effectmight be because of production and release of proinflam-matory factors in response to RAF inhibitor–induced ERKpathway activation. To address this possibility, HL-60cells were treated with the RAF inhibitor WYE-130600(300 nmol/L for 48 hours) and analyzed the tissue culturesupernatant for the presence of inflammatory cytokines.The only cytokine detected at elevated levels after

inhibitor treatment was IL-8 (data not shown). Interest-ingly, exposure of HL-60 cells to WYE-130600 did notincrease proliferation, butmarkers of differentiationweredetected, consistent with previous reports of constitutiveERK phosphorylation inducing differentiation of HL-60cells (ref. 18; data not shown). To confirm these findingsand establish the role of ERK activation, we treatedHL-60cells with PF-04880594 (100 nmol/L), with or without PD-0325901 (100 nmol/L for 48 hours) and measured IL-8levels in tissue culture media. As shown in Fig. 5, PF-04880594 caused ERK activation and stimulation of IL-8release by more than 5-fold, both of which were blockedby PD-0325901 treatment.

DiscussionPrevious studies have shown that different RAF inhi-

bitors activate the ERK pathway in cells harboring wild-type BRAF via a mechanism involving BRAF/CRAFdimerization and transactivation of the inhibitor-free pro-tomer in a RAS-GTP–dependent manner (12, 13). It hasalso been shown that a series of RAF inhibitors inducehyperplasia in multiple epithelial tissues and that hyper-plasia in the nonglandular stomach epithelium can beprevented by coadministration of a MEK inhibitor,

Figure 3. Combination dosing witha MEK inhibitor (PD-0325901)prevents RAF inhibitor(PF-04880594)-induced hyperplasia.Microscopic pathology is shownafter 21 days of treatment with RAFinhibitors. Urinary bladder,esophagus, and tongue tissue invehicle-treated mice are shown forcomparison and display normaltissue morphology. Tissues frommice treated with PF-04880594alone display the microscopicpathology typical of unopposedBRAF inhibition. Thickened layers ofsquamous cell epithelia, bracketedby arrows, mark the extent of cellularhyperplasia after 21 days of PF-04880594 alone. In contrast, thesame tissues from mice treated with1:10 ratio of MEK:BRAF inhibitor(1 mg/kg PD-0325901 and 10 mg/kgPF-04880594) display none of thehyperplastic response, instead beingcomparable with the untreatedtissues.






suggesting ERK activation as a contributing factor (8). Wehave nowextended these sets of studies and characterizedthe underlying biology by showing that treatment ofmicewith the RAF inhibitor PF-04880594 results in CRAF-BRAF dimerization, ERK activation, and hyperplasia inthe same epithelial tissues. Most importantly, we haveshown that hyperplasia induced by the RAF inhibitor PF-04880594 can be prevented by relatively low, clinicallyrelevant doses of the MEK inhibitor PD-0325901 and thatthis translates to human epithelial tissue model.

The RAF inhibitors vemurafenib and GSK2118436showed excellent effectiveness in clinical trials ofadvanced melanoma in patients with BRAFV600E muta-tions (4, 24, 25). For instance, vemurafenib had a 48%response rate, with 5.3 months progression-free survival(1.8 months for the dacarbazine comparator). However,15% to 30% of patients treated with these RAF inhibitorsdeveloped skin tumors diagnosed as keratoacanthomasand squamous cell carcinomas, as did patients treatedwith less selective RAF inhibitors such as sorafenib(24, 26, 27). These tumors were successfully resected anddid not metastasize; however, there remain concerns

about the possibility of patients developing undetectedskin tumors or tumors at sites less amenable to detectionand resection. The hyperplasia associated with ERK acti-vation in epithelial tissues, including skin, reported hereand in previous studies (8, 13, 15) lend strong support tothe idea that skin tumor formation in patients is linked toRAF inhibitor–induced ERK activation. Tumor formationmay be secondary to ERK-mediated hyperproliferation,by promoting accumulation of oncogenic mutations, or itmay require the presence of preexisting mutations, suchas sunlight-induced RAS or p53mutations. In either case,it will be important to minimize this unwanted effect ofRAF inhibitors; although keratoacanthomas generallyspontaneously regress and do not metastasize, squamouscell carcinomasdohavemetastatic potential if undetected.Moreover, the ERK pathway is ubiquitous and, as shownhere and elsewhere (15), hyperplasia occurs in multipleepithelial tissues in response to RAF inhibitors, raising thepossibility of tumor formation at extracutaneous sites.

Our finding that treatment of HL-60 cells with PF-04880594 increases IL-8 release raises further concerns,as IL-8 promotes angiogenesis, stimulates proliferation

Figure 4. MEK inhibition eliminates RAF inhibitor–induced epithelial cell hyperplasia in 3D reconstructed human epithelium (RHE). Treatment of RHE for 2 dayswith vehicle (DMSO), RAF inhibitor (PF-04880594), or a combination of RAF and MEK (PD-0325901) inhibitors. A, DMSO treatment. Indicated are a viablesquamous epithelial cell layer 4 to 6 cells deep covered with a thick cornified layer (1) that is superficial to the granular cell (2), prickle cell (3), andbasal cell layers (4). Occasional necrotic cells (thin arrows) are present. B, PF-04880594 (62.5 nmol/L) treatment. The culture is thickened as a result ofhyperplasia of epithelial cells that are now necrotic and appear as cell ghosts (between parentheses). Single-cell necrosis (thin arrows) is also present. C,combined PF-04880594 (62.5 nmol/L) and PD-0325901 (5 nmol/L) treatment. The cell culture is comparable with control with viable squamous epithelial cellsevident subjacent to a thick cornified layer. An occasional necrotic cell (thin arrow) is evident. Photomicrographs tissues stained with H&E. D, the level ofp-ERK was significantly induced by RAF inhibitor treatment in RHE cells, which was attenuated by cotreatment with PD-0325901, at dose comparablewith the in vivo exposure level in mice.

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and survival, and facilitates cancer cell migration (28). It isalso possible that IL-8 may mediate the acute redness, orflushing, in the skin observed in dogs after RAF inhibitortreatment on areaswhere the furwas thin and the skinwasvisible (areas surrounding themuzzle, nose, and eyelids).This observation was noted in dogs administered with4 structurally distinct RAF inhibitors, suggesting theeffect is target mediated. Interestingly, production ofIL-8, which plays a role in the extravasation of melanomacells by mediating ICAM-1 interaction with b2 integrin,was shown to be decreased following inhibition ofBRAFV600E (29). Moreover, elevated plasma levels of

IL-8 in mice bearing BRAFV600E melanoma xenograftswere decreased by RAF inhibitor treatment (30). Ourobservation is consistent with these findings and, takentogether, these findings suggest that reduction of IL-8productionmaybe important for prevention ofmetastasisin melanoma by RAF inhibitors and minimizing tumor-igenic risk in nontarget tissues by MEK inhibitors.

TheMEK inhibitor PD-0325901was found to effectivelyblock PF-04880594–induced hyperplasia and ERK activa-tion in epithelial tissues and IL-8 release from cells in vitro.These results suggest a means of reducing the risk oftumorigenesis in nontarget tumor tissues with wild-typeBRAF in patients treated with RAF inhibitors. Moreover,we found that the coadministration of PD-0325901allowed elevation of the PF-04880594 dose by 2-fold with-out the toxicity usually observed at such doses. Thisindicates that combinationwith aMEK inhibitor will bothincrease RAF inhibitor safety and potentially increaseefficacy by allowing dose escalation. A key point to beraised here is that the effective systemic exposures of PD-0325901 (MEK inhibitor) used in these studies extrapolateto exposures thatwere shown to bewell tolerated in phaseI and II clinical trials (17, 18). Specifically, the doses of PD-0325901 used in this study (0.1–1 mg/kg) have beencalculated to correlate with clinical exposures resultingfrom oral twice-daily doses in the 4 to 10 mg range. Inthose trials, higher exposures of PD-0325901 were foundto have unacceptable toxicity, particularly neurotoxicityand retinal vein occlusion. Unfortunately, these higherexposures were needed to achieve efficacy and thusled to the discontinuation of the clinical trials (17–19).When used at the lower levels shown here, the MEKinhibitor (PD-0325901) is highly effective at inhibitingRAF inhibitor-induced ERK activation and would beexpected to be well-tolerated clinically.

The reconstructed human epidermis 3D cell modeldescribed here will be useful for future mechanistic andscreening studies. First, rodent and human skin havesignificant differences in architecture and reconstructedhuman skin models can be used to study melanomaprogression and invasion (31) and may be useful as apredictor of drug efficacy. Second, as shown here, themodel can be used as a measure of adverse effects, in thiscase skin hyperplasia. Finally, the model may prove mostuseful in screening future drug candidates, particularlythose impacting the ERK pathway, for their potential tocause epidermal hyperplasia.

Amajor challenge facing the clinical application of RAFinhibitors as antitumor agents (aside from the squamouscell carcinoma side effect) lies in the development of drugresistance. Most patients treated with vemurafenibacquired resistance within 8 to 12 months after initiationof treatment. In the case of other kinase-targeted thera-pies, such as those targeting BCR-ABL, KIT, or EGFR,resistance arises as a result of acquisition of "gatekeeper"mutations in the target kinase (5). However, so far anal-ogousmutations in BRAFV600Ehave not beendetected intumors from patients receiving RAF inhibitors. Nazarian

Figure 5. RAF inhibitor PF-04880594 induces ERK phosphorylation andinduces IL-8 production in HL-60 cells. HL-60 cells were incubated withthe designated compounds for 2 days followed by harvesting the cells foranalysis of ERK phosphorylation and IL-8 secretion. A, lysates of HL-60cells treated with the designated compounds were analyzed byimmunoblotting to determine levels of phosphorylated ERK. B, equalvolumes of cell culture supernatants from control and treated cells weretested for the presence of IL-8 using an ELISA-based immunoassay (SABioproducts). Data are presented in terms of the amount of colorimetricproduct produced by catalysis of the secondary detection antibodymeasured at OD 450 nm. Each sample was tested in duplicate andduplicates were within 2% of the calculated mean.






and colleagues (7) analyzed 12 matched pairs of drug-sensitive and drug-resistant tumors and discovered acti-vatingmutations inNRASor activation of PDGFRb in halfof the samples, suggesting that acquisition of mutationsthat bypass BRAF to activate ERK (NRAS/PDGFRb) oractivate parallel pathways (PDGFRb) may cause RAFinhibitor resistance. Combination treatment with a MEKinhibitor would most likely prevent tumor cells with theformer category of mutation, including NRAS mutation,from gaining a proliferative advantage and thereforeprotect against resistance development. Early clinicalstudies in which both RAF and MEK inhibitors wereadministered seem to validate the predicted advantagesof this combination and has recently been published (32).It is not clear whether a MEK inhibitor would protectagainst PDGFR-mediated resistance, as there is the poten-tial for signaling through multiple pathways, includingERK. Indeed, Shi and colleagues (33) have shown that acombination of RAF inhibitor with PI3K/mTOR inhibitorconfers sensitivity to PDGFRb-positive, vemurafenib-resistant melanoma cell lines. Also a recent reportdescribes the presence of an activating mutation inMEK1in a resistant tumor (34). The extent to which MEK inhi-bitors protect against RAF inhibitor–acquired resistancewill no doubt become clearer with more extensive geno-typing of drug-resistant tumors.

Disclosure of Potential conflicts of InterestAll authors are either former or current employees of Pfizer Inc.

Authors' ContributionsConception and design: V.R. Torti, D. Wojciechowicz, W. Hu, A. John-Baptiste, S. Yamazaki, C.L. Palmer, L.A. Burns-Naas, S. BagrodiaDevelopment of methodology: V.R. Torti, D. Wojciechowicz, W. HuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V.R. Torti, D. Wojciechowicz, W. Hu, G. Troche,L.D. MarroquinAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): V.R. Torti, D. Wojciechowicz, W. Hu, W.Evering, L.D. Marroquin, S. Yamazaki, L.A. Burns-Naas, S. BagrodiaWriting, review, and/or revision of the manuscript: V.R. Torti, D. Woj-ciechowicz,W.Hu,A. John-Baptiste,W. Evering, T. Smeal, S. Yamazaki, L.A. Burns-Naas, S. BagrodiaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V.R. Torti, D. Wojciechowicz, W.Evering, G. TrocheStudy supervision: V.R. Torti, D. Wojciechowicz, T. Smeal, S. Bagrodia

AcknowledgmentsTheauthors thank JosephPiraino for his technical contributions to the in

vivo studies in Table 1 and in the Supplementary Data; Anthony Wong,Pamela Plumlee, April Marr, Orlando Esparza, Beatrice Debrosse-Serra,Jeri Eades, and Brittany Snider for their histology support; Su Khoh-Reiterfor support with the RHE cells cultures; Constance Benedict for assistancewith the tables and figures; Sylvia Vekich and Zhongzhou Shen for thebioanalytical analysis; and Steve Bender for his scientific guidance andsupport.

Grant SupportThe work was funded by Pfizer Inc.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 6, 2011; revised June 11, 2012; accepted June 26, 2012;published OnlineFirst July 2, 2012.

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2012;11:2274-2283. Published OnlineFirst July 2, 2012.Mol Cancer Ther Vince R. Torti, Donald Wojciechowicz, Wenyue Hu, et al. the MEK Inhibitor PD-0325901PF-04880594 Is Attenuated by a Clinically Well-Tolerated Dose of Epithelial Tissue Hyperplasia Induced by the RAF Inhibitor

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