bgb-283, a novel raf kinase and egfr inhibitor, displays ... · raf kinase and egfr inhibitor with...

Small Molecule Therapeutics

BGB-283, a Novel RAF Kinase and EGFRInhibitor, Displays Potent Antitumor Activityin BRAF-Mutated Colorectal CancersZhiyu Tang1, Xi Yuan2, Rong Du2, Shing-Hu Cheung2, Guoliang Zhang3, Jing Wei2,Yuan Zhao2, Yingcai Feng4, Hao Peng4, Yi Zhang4, Yunguang Du4, Xiaoxia Hu1,Wenfeng Gong1,Yong Liu1,Yajuan Gao1,Ye Liu4, Rui Hao4, Shengjian Li4, Shaohui Wang3,Jiafu Ji5, Lianhai Zhang5, Shuangxi Li5, David Sutton1, Min Wei4, Changyou Zhou3,Lai Wang1, and Lusong Luo2

Abstract

Oncogenic BRAF, which drives cell transformation and prolif-eration, has been detected in approximately 50% of humanmalignant melanomas and 5% to 15% of colorectal cancers.Despite the remarkable clinical activities achieved by vemurafeniband dabrafenib in treating BRAFV600E metastatic melanoma, theirclinical efficacy inBRAFV600E colorectal cancer is far less impressive.Prior studies suggested that feedback activationofEGFRandMAPKsignaling upon BRAF inhibition might contribute to the relativeunresponsiveness of colorectal cancer to the first-generation BRAFinhibitors. Here, we report characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical inves-tigation. In vitro, BGB-283 potently inhibits BRAFV600E-activated

ERK phosphorylation and cell proliferation. It demonstrates selec-tive cytotoxicity and preferentially inhibits proliferation of cancercells harboring BRAFV600E and EGFR mutation/amplification. InBRAFV600E colorectal cancer cell lines, BGB-283 effectively inhibitsthe reactivation of EGFR and EGFR-mediated cell proliferation.In vivo, BGB-283 treatment leads to dose-dependent tumor growthinhibitionaccompaniedbypartial and complete tumor regressionsin both cell line-derived and primary human colorectal tumorxenografts bearing BRAFV600E mutation. These findings supportBGB-283 as a potent antitumor drug candidate with clinicalpotential for treating colorectal cancer harboring BRAFV600E muta-tion. Mol Cancer Ther; 14(10); 2187–97. �2015 AACR.

IntroductionThe MAPK pathway plays an essential role in regulating cell

proliferation and survival. Activation of the RAS–RAF–MEK–ERKkinase cascade by external stimuli transduces signals from theplasmamembrane into the cell nucleus to control gene expressionand determine cell fate (1). Aberrant activation of the MAPKsignal transduction pathway is frequently found in different typesof cancers, contributing to increased cell division, suppressedapoptosis, and enhanced cell motility and invasion (2, 3). BRAF,

one of the three members of the RAF kinase family, has beenidentified as a target for cancer therapy (4, 5). A sequencing screenof 923 cancer samples detected mutations in the BRAF gene inapproximately 50%of humanmalignantmelanomas and 15%ofcolorectal cancers (6–11), with the V600E mutation accountingfor at least 90% of oncogenic BRAF mutations (6). This V600Emutation introduces a negative charge in the BRAF kinase domainthat mimics and bypasses the phosphorylation required for BRAFactivation, which is normally achieved through growth factor–activated receptor tyrosine kinases. As a result, this "gain offunction" of BRAF gives rise to a constitutive MAPK signalingthat promotes tumor progression, in which BRAFV600E activatesMEK1/2 in a RAS-independent manner (12–15).

Small molecules that selectively target mutant BRAF exhibitgood efficacy and yield impressive clinical responses in melano-ma patients with BRAFV600E mutation. The RAF inhibitors thatselectively inhibit BRAFV600E tumors, vemurafenib (PLX4032)and dabrafenib (GSK 2118436), have generated objectiveresponse rates from 50% to 70%, respectively, in early clinicaltrials treating metastatic melanoma (5, 16, 17). The clinicalexperience with these BRAF inhibitors in benefiting melanomapatients confirms BRAFV600E as a bona fide oncogenic target andvalidates the utility of cancer therapies that target BRAF andMAPKsignaling (4, 5). However, these first-generation BRAF inhibitorsstill have limitations, including development of cutaneous squa-mous cell carcinomas and treatment-related keratoacanthomas(KA) due to paradoxical activation of MAPK signaling. In addi-tion, they have inadequate clinical activity outside of melanoma

1Department of In Vivo Pharmacology, BeiGene (Beijing) Co., Ltd.,Beijing, P.R. China. 2Department of Discovery Biology, BeiGene(Beijing) Co., Ltd., Beijing, P.R. China. 3Department of Chemistry,BeiGene (Beijing) Co., Ltd., Beijing, P.R. China. 4Department of Molec-ular Sciences, BeiGene (Beijing) Co., Ltd., Beijing, P.R. China. 5KeyLaboratory of Carcinogenesis and Translational Research (Ministry ofEducation),DepartmentofSurgery, PekingUniversityCancerHospitaland Institute, Beijing, P.R. China.

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

Z. Tang and X. Yuan contributed equally to this article.

Corresponding Author: Lusong Luo, BeiGene (Beijing) Co., Ltd., No. 30 SciencePark Road, Zhong-Guan-Cun Life Science Park, Changping District, Beijing102206, P.R. China. Phone: 86-10-5895-8201; Fax: 86-10-5895-8088; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0262

�2015 American Association for Cancer Research.

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with BRAFV600E mutation (16, 18–20). In particular, the clinicalresponse among colorectal cancer patients with BRAFV600E muta-tions ismuch lower than that observed inmelanoma patients. Forexample, vemurafenib was reported to have a mere 5% objectiveresponse rates in colorectal cancer patients with BRAFV600E muta-tion (21). Recent studies suggest that increased EGFR activitythrough a feedback activation mechanism might account for thedrastic difference in objective response rates between BRAFV600E

melanoma and colorectal cancer to the first-generation BRAFinhibitors (22, 23). Combination of BRAF and EGFR inhibitorswas found to significantly enhance the efficacy in preclinicalmodels (22, 23). Therefore, a second-generation BRAF inhibitorthat can inhibit both BRAFV600E and EGFR-driven RAF activationmay have a therapeutic advantage in treating BRAF-mutatedcolorectal cancer.

We describe herein BGB-283, a novel fused tricyclic benzoimi-dazole compound that inhibits both BRAF and EGFR. BGB-283potently inhibits RAF family kinases and EGFR activities inbiochemical assays. It demonstrates selective cytotoxicity to celllines harboring BRAFV600E or EGFRmutations. BGB-283 is highlyefficacious in BRAFV600E colorectal cancer xenograft models,including HT29, Colo205, and two primary tumor xenograftsharboring BRAFV600E mutation. In addition, BGB-283 showscompelling efficacy in a WiDr xenograft model where EGFRreactivation was shown to be induced upon BRAF inhibition(22, 23). Together, these data suggest that BGB-283 is a novelRAF kinase and EGFR inhibitor with therapeutic potential intargeting oncogenic BRAF in human colorectal carcinoma.

Materials and MethodsMaterial

BGB-283 used in this study exceeded a purity of 99% asmeasured by proton nuclear magnetic resonance, liquid chroma-tography–mass spectrometry and high-performance liquid chro-matography. Reference compounds were purchased from thefollowing sources: PLX4032 and dabrafenib, WuXi AppTec; gefi-tinib, MedChem; erlotinib, Beijing Ouhe Technology Co. Ltd.;cetuximab (Merck KGaA), Beijing Cancer Hospital (Beijing, P.R.China). Stock solutions of compounds were prepared in DMSO.

BRAFV600E kinase domain/BGB-283 co-crystallization andstructure determination

BRAFV600E (444–723) was expressed and purified using meth-ods similar to those previously reported (22). To co-crystallizeBRAFV600E with BGB-283, protein solution was incubated withBGB-283 at a ratio of 1:5 for 1 hour, and mixed with reservoirsolution (100 mmol/L Bis Tris at pH 6.5, 23% PEG3350, and 200mmol/L MgCl2) at equal volume. Co-crystals grew by sitting dropvapor diffusion method at 4�C. Diffraction data were collectedfrom the Synchrotron Radiation beam line (Shanghai SynchrotronRadiation Facility). Crystals belonged to the space group P212121(a ¼ 49.395 Å, b ¼ 101.601 Å, c ¼ 109.786 Å) and contain twoBRAFV600E molecules in an asymmetric unit. Diffraction imageswere processed and scaled with HKL2000. Phase was solved usingsoftware MOLREP by molecular replacement method with previ-ously published structure. The resultant model was subsequentlyrefined in PHENIX using rigid-body refinement and maximumlikelihood method (Supplementary Table S1). Complex structureof the BRAFV600E kinase domain with BGB-283 was submitted tothe Protein Data Bank (PDB) with the PDB ID code 4R5Y.

In vitro kinase assayCompounds were tested for inhibition of RAF and WT EGFR

kinase activity in assays based on time-resolved fluorescence-resonance energy transfer (TR-FRET) methodology. MEK1(K97R) was used as a substrate for RAF kinases and a biotinylatedpeptide substrate was used for EGFR (61TK0BLC, CisBioBioassys). The kinase was incubated with a serial dilution ofcompounds for 60 to 120 minutes at room temperature (RT),ATP (final concentration at 100 mmol/L) and kinase substrateswere added to initiate the reaction. The reactionwas stoppedby anequal volume of stop/detection solution according to the man-ufacture's instruction (CisBio Bioassays). Plates were sealed andincubated at RT for 2 hours, and the TR-FRET signals (ratio offluorescence emission at 665 nm over emission at 620 nm withexcitation at 337 nm wavelength) were recorded on a PHERAstarFS plate reader (BMG Labtech).

BGB-283 was screened for activity in a panel of 277 kinases at afixed concentration of 10 mmol/L by Life Technologies using theirstandard assays at Km concentration of ATP for perspectivekinases. The IC50 was then determined for kinases showing>80% inhibition at 10 mmol/L BGB-283.

Cell cultureA375, Sk-Mel-28, HT29, Colo205, WiDr, Ba/F3, A431,

HCC827, SW620, HCT116, and cell lines used in cell panelprofiling were purchased from ATCC. Cell lines were tested andauthenticated at ATCC before purchase using morphology, kar-yotyping, and PCR-based approaches. All the cell lines werecultured in the designated medium supplemented with 10% FBS(Thermo Scientific), 100 U/mL penicillin (Gibco), 0.1 mg/mLstreptomycin (Gibco), and in a humidified 37�C environmentwith 5% CO2. Cell lines were reinstated from frozen stocks laiddown within three passages from the original cells purchased andpassaged no more than 30 times. Culturing condition for cellpanel profiling is listed in Supplementary Table S2. Stimulation ofEGFR phosphorylation in A431 cells was achieved by addition ofEGF to serum-free DMEM with 10-minute incubation. EGF-stim-ulated cell growth was achieved by addition of EGF to DMEM orRPMI supplemented with 10% FBS.

Western blotting analysisFor in vitro studies, cells were harvested after 1 hour treatment at

37�C and lysed immediately as previously described (24). Forin vivo studies, tumors were harvested at the indicated time points,snap-frozen in liquid nitrogen, and stored at�80�C. Tumorswerehomogenized in 500 mL lysis buffer in MP homogenization unit(Fast prep-24; MP bio, Cat. # 6004.2) and lysates were thencentrifuged at 13,000 rpm for 10 minutes at 4�C to removeinsoluble debris. The protein concentration of lysates was deter-mined using the Pierce BCA protein assay kit (Thermo Scientific).Proteins were separated by 10%SDS-PAGE gel orNuPAGENovex4% to 12% Bis-Tris protein gels (Life Technologies) and trans-ferred to nitrocellulose membranes using iBlot Dry BlottingSystem (Life Technologies). Blots were blocked with TBSTM[50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.1% Tween 20,and 5% non-fat milk] at RT for 1 hour and probed with indicatedantibodies diluted in TBSTM. The membranes were probed forphospho-proteins and then stripped to probe for total proteins.For reprobing, the membranes were stripped in stripping buffer(25 mmol/L glycine, pH 2.0, 1% SDS) for 30 to 60 minutes atRT, rinsed twice with TBST for 10 minutes, and probed for other

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proteins. Antigen–antibody complexes were visualized usingthe chemiluminescent substrate (Millipore) and detected withImage Quant LAS4000 mini digital imaging system (GEHealthcare).

Antibodies usedwereobtainedcommercially fromthe followingsources: anti-BRAF (SC-5248), Santa Cruz Biotechnology; anti-C-RAF (610152), BD Biosciences; antibodies to MEK (9122, 9126),phospho-MEK1/2 (Ser217/221) (9154), ERK (4695), phospho-ERK1/2 (Thr202/Tyr204; 4370), EGFR (2646s), phospho-AKT(Ser473; 4060), AKT (4685), c-Myc (3058), DUSP6 (5605), phos-pho-EGFR (Tyr1068; 3777), GAPDH (2118s) and anti-rabbit IgGhorseradish peroxidase (HRP)-linked secondary antibody, CellSignaling Technology; anti-mouse IgGHRP-linked secondary anti-body, Sigma-Aldrich.

Cell-based phospho-ERK and phospho-EGFR detection assayCellular phospho-ERK and phospho-EGFR were measured

using a TR-FRET–based method. Cells were seeded at 3 � 104

per well of a 96-well plate and left to attach for 16 hours.Growth medium was then replaced with 100 mL of DMEMcontaining no serum. Cells were then treated with a 10-pointtitration of compound. After 1 hour of compound treatment,50 mL of lysis buffer (Cisbio) was added to each well. Plateswere then incubated at room temperature with shaking for 30minutes. A total of 16 mL of cell lysate from each well of a 96-well plate was transferred to a 384-well small volume whiteplate. Lysate from each well was incubated with 2 mL of Eu3þ- orTb3þ- cryptate (donor) labeled anti-ERK or anti-EGFR antibody(Cisbio) and 2 mL of D2 (acceptor) labeled anti-phospho-ERKor anti-phospho-EGFR antibody (Cisbio) for 2 hours at roomtemperature. FRET signals were measured using a PHERAstar FSreader (BMG Labtech).

Proliferation assayThe growth-inhibitory activity of compounds in a panel of

melanoma, colon, breast, and lung cancer cells was determinedusing CellTiter-Glo luminescent cell viability assay (Promega).The number of cells seeded per well of a 96-well plate wasoptimized for each cell line to ensure logarithmic growth overthe 3 days treatment period (Supplementary Table S2). Cells wereleft to attach for 16 hours and then treated with a 10-pointdilution series in duplicate. Following a 3-day exposure to thecompound, a volume of CellTiter-Glo reagent equal to the vol-ume of cell culture medium present in each well was added.Mixturewasmixed on an orbital shaker for 2minutes to allow celllysing, followedby10minutes incubation at roomtemperature toallowdevelopment and stabilization of luminescent signal. Lumi-nescent signal was measured using PHERAstar FS reader (BMGLabtech). EC50 values for cell viability were determined withGraphPad Prism software.

Tumor xenografts experimentsFemale NOD/SCID and BALB/c nude mice, ages 4 to 6 weeks,

and weighing approximately 18 g, were purchased from BeijingHFK Bioscience Co., Ltd. All procedures involving animals wereconducted in accordance with the Institutional Animal Care andUse Committee (IACUC) of BeiGene.

ForHCC827,A431,HT29,Colo205, andWiDr xenografts, eachmouse was injected subcutaneously with 2.5 to 5 � 106 cells in200mLPBS in the right frontflank via a 26-gauge needle.When theaverage tumor size reached 110 to 200 mm3, animals were

randomized to treatment groups (7–9 mice per group) andtreated twice per day (b.i.d.) or once daily (qd) by oral gavage(p.o.) with vehicle alone or 2.5 to 30 mg/kg of BGB-283. Ascontrol, mice were treated with erlotinib (100 mg/kg qd) orcetuximab (40 mg/kg twice weekly). BGB-283 and erlotinib wereformulated at the desired concentration as a homogenous sus-pension in 0.5% (w/v) methylcellulose in purified water. Cetux-imab was formulated by diluting the injection solution withsaline before dosing.

For BCCO-002 and BCCO-028 primary human tumor xeno-grafts (PDX), colorectal cancer samples were collected from Beij-ing Cancer Hospital (Beijing, P.R. China) after patient's informedconsent and immediately transferred in DMEM culture mediumcontained 200 U/mL penicillin and 200 mg/mL streptomycin.Within 2 to 4 hours of surgery, small fragments (3mm� 3mm�3 mm) were subcutaneously engrafted into the scapular area orflank of anesthetized NOD/SCID mice. After three successfulpassages on NOD/SCID, tumors were subsequently passaged inBALB/c nude mice. Efficacy studies were conducted within sixpassages of the patient tumors. When the average tumor sizereaches 100 to 200 mm3, animals were randomized to treatmentgroups (8 mice per group) and treated orally with vehicle (0.5%MC) alone, BGB-283 (5–10 mg/kg, b.i.d.) or dabrafenib (50 mg/kg, b.i.d.). A further group received intravenous cetuximab (40mg/kg every 3days). BGB-283was formulated as described above.Dabrafenib was formulated in 10% DMSO þ 90% HP-b-CD(Hydroxypropyl-b-cyclodextrin)/PBS.

In both cell line and primary tumor xenograft studies, individ-ual body weights and tumor volumes were determined twiceweekly, with mice being monitored daily for clinical signs oftoxicity during the study.

Efficacy endpointsTumor volumes were calculated using the formula: V¼ 0.5� (a

� b2), in which a and b are the long and short diameters of thetumor, respectively. Partial regression (PR) was defined as tumorvolume smaller than50%of the starting tumor volumeon thefirstday of dosing for at least three consecutive measurements. Com-plete regression (CR) was defined as tumor volume less than 14mm3 for at least three consecutive measurements. Tumor growthinhibition (TGI) was calculated using the following formula: %growth inhibition ¼ 100 � [1�(treated t� treated t0)/(placebo t�placebo t0)], inwhich treated t represents tumor volume at day tin the treated group, treated t0 represents tumor volume of thesame treated group on the first day of treatment, placebo trepresents placebo tumor volume day t in the control group,placebo t0 represents tumor volume of the same group on the firstday of treatment. Statistical analysis was conducted using theStudent t test. P < 0.05 was considered statistically significant.

ResultsBGB-283 is an inhibitor of RAF family kinases and EGFR

BGB-283, with the chemical formula 5-(((1R,1aS,6bR)-1-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1a,6b-dihy-dro-1H-cyclopropa[b]benzofuran-5-yl)oxy)-3,4-dihydro-1,8-naphthyrdin-2(1H)-one (Fig. 1A), is a synthetic inhibitordesigned to interact with the ATP-binding site of the BRAFV600E

(Fig. 1B). The crystal structure of the BGB-283/BRAFV600E kinasedomain complex revealed that BGB-283binds to the ATP-bindingpocket of BRAFV600E in an inactive conformation (Fig. 1B andSupplementary Table S1). BGB-283 binds to both protomers of

Characterization of a Novel BRAF EGFR Dual Inhibitor

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BRAFV600E with a DFG-out and a-C helix-in conformation. Incomparison, PLX4032binds to only oneof the protomers inDFG-out and a-C helix-in conformation in its co-crystal structure with

BRAFV600E (PDB code: 3OG7; ref. 25). The pyridine lactam groupof BGB-283 interactswith thehinge region,while the fused tricyclegroup binds between the N-terminal lobe domain and activationloop. The enzimidazole and trifluoro groups locate in the pocketformed by a-C helix and activation loop (Fig. 1B). There are twohydrogen bonds formed between BGB-283 and the BRAF protein.One is between hydroxyl group of E501 on the a-C helix and thenitrogen atom from the benzimidazole. The other is between thenitrogen atom on the pyridine lactam group and the carbonyl ofC532 from the hinge region of BRAFV600E.

The biochemical potency of BGB-283 in inhibiting RAF kinaseswas measured under steady-state conditions using GST-MEK1(K97R) as a substrate. BGB-283 was shown to be a pan-RAFinhibitor of the kinase domains of BRAFV600E, WT BRAF, C-RAFY340/341D, and WT A-RAF in vitro (Table 1A). In particular, BGB-283 potently inhibited the activity of the recombinant BRAFV600E

kinasedomainwithan IC50 valueof 23�5nmol/L (Table 1A). Theinhibition by BGB-283 was time dependent and with a slow off-rate. The estimated t1/2 of dissociation was measured to be muchlonger than 240 minutes (Supplementary Fig. S1A). BGB-283 wasfurther shown to be a reversible inhibitor of the BRAF kinase(Supplementary Table S3). The selectivity of BGB-283 was evalu-ated bymeasuring the percentage of inhibition against 279 kinasesat 10 mmol/L, a concentration that is approximately 450 timeshigher than its IC50 value against the purified kinase domain ofBRAFV600E. Of the 279 kinases tested, 43 of them showed >80% ofinhibition by 10 mmol/L BGB-283 (Supplementary Table S4).Further profiling of BGB-283 against these 43 kinases revealed itsinhibition of several other kinases, including EGFR, DDR1, DDR2,EPHA3, FLT3, VEGFR2, and ZAK (Supplementary Table S5). Thebiochemical inhibition of EGFR by BGB-283 was further charac-terized in the TR-FRET assay. BGB-283 was shown to inhibit thekinase activity of EGFR with IC50s of 29 � 18 nmol/L, and EGFRT790M/L858Rmutantwith IC50s of 495� 124nmol/L (Table 1A).

BGB-283 potently inhibits BRAFV600E-activated ERKphosphorylation and cell proliferation

The effect of BGB-283 on oncogenic BRAF-mediated ERK phos-phorylation was investigated in A375, a BRAFV600E-driven mela-noma cell line. Consistent with data from biochemical character-ization, BGB-283 potently inhibited ERK phosphorylation (Fig.2A). The potency of BGB-283 in-inhibiting BRAFV600E-driven

Figure 1.BGB-283, a compound designed for inhibiting oncogenic BRAF. A, chemicalstructure of BGB-283. B, the crystal structure of BGB-283 bound toBRAFV600E. Dashed lines are hydrogen bonds.

Table 1. In vitro kinase and cellular activity of BGB-283

A. Biochemical activity of BGB-283 against RAF family and EGFR kinases.Enzyme IC50 (nmol/L; mean � SD) n

BRAFV600E kinase domain (aa416-766) 23 � 5 7WT BRAF kinase domain (aa416-766) 32 � 8 3C-RAF Y340/341D kinase domain (aa306-648) 7.0 � 2.3 6WT A-RAF kinase domain (aa282-609) 5.6 1EGFR kinase domain (aa669-1210) 29 � 18 10EGFR T790M/L858R (aa669-1210) 495 � 124 2

B. Inhibition of ERK or EGFR phosphorylation by BGB-283 in different cancer cell lines. BGB-283 potently inhibited BRAFV600E-driven ERK phosphorylation inA375, SK-Mel-28, HT29, and Colo205 cells. BGB-283 also inhibited EGFR auto-phosphorylation in A431 and HCC827 cells, where EGFR is activated byoverexpression or E746-A750 deletion.

Phosphorylated kinase Cell lines BGB-283 IC50 (nmol/L; mean � SD) npERK A375 64 � 31 8

Sk-Mel-28 95 � 58 6HT29 50 � 18 6Colo205 92 � 57 7

pEGFR A431 385 � 40 7HCC827 195 � 102 2

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Figure 2.BGB-283 potently inhibited ERK phosphorylation and EGFR activity. A, immunoblot analyses for BRAF, phospho-ERK1/2, ERK1/2, and GAPDH in A375cell lysates prepared 1 hour after treatment with indicated concentrations of PLX4032 or BGB-283. B, immunoblot analyses for BRAF, phospho-ERK1/2, ERK1/2, andGAPDH in HCT116 and SW620 cell lysates prepared 1 hour after being treated with indicated concentrations of PLX4032 and BGB-283. C, immunoblotanalyses for EGFR, phospho-Tyr1068-EGFR, phospho-ERK1/2, ERK1/2, and GAPDH in A431 (EGFR overexpression) cell lysates prepared 2 hours after 20 ng/mL EGFstimulation and treatment with indicated concentrations of PLX4032, BGB-283, or gefitinib. D, immunoblot analyses for phospho-Tyr1068-EGFR, EGFR,phospho-Thr202/Tyr204-ERK1/2, ERK1/2, c-Myc, and GAPDH in WiDr and HT29 cell lysates prepared 3 and 24 hours after treatment with indicated compounddiluted in RMPI-1640 with 5% FBS. HCC827 (EGFR E746-A750 deletion; E) tumor cells (5 � 106) or A431 (EGFR overexpression; F) tumor cells (5 � 106)were implanted subcutaneously in female BALB/c nude mice. When the tumors reached a mean volume of approximately 100 to 180 mm3 in size, mice wererandomly allocated into groups and treated as indicated. Data are presented as average tumor volume � SEM of 7 animals in each group.






ERK phosphorylation was further confirmed by a quantitative TR-FRET assay in four different cell lines harboring BRAFV600E, withIC50 values ranging from 32 to 153 nmol/L (Table 1B). Previously,itwas reported that PLX4032 treatment of SW620 andHCT116 celllines expressing BRAFWT resulted in a paradoxical induction ofERK1/2 phosphorylation (26–28). Unlike PLX4032, BGB-283induced much less ERK activation in both of the two cell lines(Fig. 2B).

BGB-283 inhibits cellular EGFR activity and blocks cellproliferation and tumor growth driven by EGFR

The ability of BGB-283 in-inhibiting EGFR activity in cellswas examined by measuring EGFR phosphorylation in A431and HCC827 cells, where EGFR is activated by overexpressionor EGFR E746-A750 deletion. BGB-283 inhibited the EGF-induced EGFR autophosphorylation on Tyr1068 in A431 cellsin a dose-dependent manner (Fig. 2C and Table 1B). Gefitinib,a known EGFR inhibitor, also potently inhibited the samephosphorylation site. In comparison, no significant inhibitionof EGFR autophosphorylation was observed in A431 andHCC827 cells treated with PLX4032 (Fig. 2C and Supplemen-tary Fig. S1B). It has been reported that BRAF inhibition couldinduce feedback activation of EGFR thus contributing to theinsensitivity of BRAFV600E colon cancer cells to PLX4032 (22,23). To evaluate the feedback activation of EGFR upon BRAFinhibition with BGB-283 or PLX4032 treatment, two BRAFV600E

colon cancer cell lines (HT-29 and WiDr) were treated by thecompounds and measured for EGFR and ERK phosphorylationlevels. PLX4032 treatment resulted in an upregulation of phos-pho-EGFR on Tyr1068, and strongly increased downstreamphospho-ERK in WiDr cells and partially reduced phospho-ERK levels in HT29 cells. Consequently, PLX4032 resulted in aninsufficient inhibition of ERK-downstream signaling as repre-sented by c-Myc expression in both cell lines after 24-hourtreatment. Addition of an EGFR inhibitor, erlotinib, abrogatedthe EGFR reactivation induced by PLX4032 and led to betterinhibition of MAPK signaling after 24 hours. In comparison,BGB-283 alone reduced EGFR phosphorylation, and inhibitedERK phosphorylation and c-Myc expression more effectivelythan PLX4032 (Fig. 2D). These findings suggest that the anti-EGFR activity of BGB-283 could suppress the feedback activa-tion of EGFR signaling upon BRAF inhibition (Fig. 2D).

Ba/F3 cells normally grow in an IL3-dependent manner (29),but their growth can be rendered IL3 independent by overexpres-

sion of EGFR exon 19deletion or EGFR L858Rmutant (30, 31). Inaccordancewith its EGFR inhibition effect, BGB-283was found toblock the proliferation of Ba/F3 cells that express EGFR exon 19deletion or EGFR L858Rmutants, suggesting that BGB-283 couldinhibit EGFR-driven cell proliferation (Table 2A and Supplemen-tary Fig. S1C). Interestingly, BGB-283 also showed moderateinhibitory effect on EGFR Exon19d/T790M mutant that is insen-sitive to gefitinib (Table 2A and Supplementary Fig. S1C). Thisresult is consistent with the observation from biochemical studieswhere BGB-283 showed inhibition to T790M containing EGFRmutant. There were no significant differences in sensitivity toBRAF inhibitors between BRAFV600E melanoma cells (A375) andBRAFV600E colon cancer cells (HT29 andWiDr) in both short-termand long-term proliferation assay (Table 2B). However, in thepresent of the EGFR ligand EGF, both HT29 and WiDr becameinsensitive to PLX4032 at concentration up to 10 mmol/L. Incontrast, BGB-283 retained inhibitory activity against HT29 andWiDr cells albeit with reduced potency (Table 2B). Together, thesedata demonstrate that BGB-283 inhibits EGFR in cells and caninhibit EGF-induced cell proliferation in BRAFV600E colon cancercell lines.

The in vivo activity of BGB-283 in suppressing EGFR-mediatedtumor growth was examined in HCC827 lung carcinoma andA431 epidermoid carcinoma xenografts. Consistent with in vitrostudy results, BGB-283 treatment at 10 and 30 mg/kg b.i.d. or qdeffectively inhibited tumor growth (Fig. 2E and F). In correspon-dence with different anti-EGFR activities between the two celllines (Table 1B), BGB-283 induced tumor regression in HCC827but not in A431 xenograft. As a control, erlotinib, a potent EGFRinhibitor, achieved similar TGI effect at 100 mg/kg qd in theHCC827 xenograft model (Fig. 2E). In summary, these findingssuggested that BGB-283 is a bona fide EGFR inhibitor both in vitroand in vivo.

BGB-283 inhibits proliferation of tumor cells expressingBRAFV600E or harboring an EGFR mutation

In order to test the selectivity of BGB-283 in suppressing cellproliferation, a panel of 107 human tumor cell lines was exposedto BGB-283 in the presence of serum and examined for viabilityafter 3 days. The mutational status of BRAF, H-RAS, K-RAS, andN-RAS was noted for each cell line (Fig. 3 and SupplementaryTable S2). BGB-283 selectively inhibited the growth of cancer celllines expressing BRAFV600E but not BRAFWT, similar to PLX4032(Fig. 3A and B). The EC50 values in themajority of nonresponsive

Table 2. Inhibition of EGFR-driven cell proliferation by BGB-283

A. BGB-283 inhibited EGFR-driven cell growth in Ba/F3 cells expressing different EGFR mutants.BGB-283Mean EC50 � SD (nmol/L) n

Ba/F3 >10,000 1Ba/F3 EGFR Exon19d 188 � 152 2Ba/F3 EGFR L858R 224 � 136 2Ba/F3 EGFR Exon19d/T790M 613 � 40 2

B. BGB-283 inhibited EGF-induced cell proliferation in BRAFV600E colon cancer cell lines.Mean EC50 � SD (nmol/L)

PLX4032 BGB-283 PLX4032 BGB-283

Cell line No additional EGF Plus 2.5 ng/mL EGFA375 191 � 16 (n ¼ 7) 200 � 79 (n ¼ 5) 129 � 61 (n ¼ 3) 150 � 97 (n ¼ 3)HT29 293 � 103 (n ¼ 5) 270 � 66 (n ¼ 5) >10,000 (n ¼ 2) 1,830 � 927 (n ¼ 2)WiDr 367 � 132 (n ¼ 4) 305 � 132 (n ¼ 4) �10,000 (n ¼ 2) 1,890 � 668 (n ¼ 2)

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cell lines were >10 mmol/L (Fig. 3A and Supplementary TableS2). In addition, BGB-283 was found to inhibit proliferation ofthe HCC827 lung cancer cell line with EGFR exon 19 deletion,

the ZR-75-30 breast cancer cell line with HER2 amplification,and the NCI-H322M lung cancer cell line with EGFR over-expression (Fig. 3A).

Figure 3.BGB-283 selectively inhibited proliferation of cancer cells harboring BRAFV600E and EGFR mutations. Antiproliferative effect of BGB-283 (A) and PLX4032 (B)following a 3D exposure across a panel of human cancer cell lines determined by the CellTiter-Glo assay.






BGB-283 exhibits antitumor activity inmouse xenograftmodelsof colorectal cancer

The in vivo efficacy of BGB-283 was assessed in subcutaneousxenograft models derived from HT29 and Colo205 colorectalcancer cell lines harboring the BRAFV600E mutation. It was previ-ously reported that vemurafenib had limited efficacy against HT-29 xenograft and combination with EGFR inhibitor improved itsantitumor activity (22, 32). BGB-283 significantly inhibitedtumor growth of HT29 xenograft (P < 0.001) at 5 mg/kg b.i.d.,which was well tolerated by animals. Addition of cetuximab, anEGFR-targeting monoclonal antibody, did not further enhancethe therapeutic effect of BGB-283 in this xenograft model (P >0.05, BGB-283þ cetuximab vs. BGB-283; Fig. 4A), suggesting thatBGB-283 alone might be sufficient in blocking the feedbackactivation of EGFR. Against Colo205 xenograft, BGB-283 pro-duced dose-dependent tumor inhibition from 3 to 30 mg/kg(Fig. 4B). More significantly, partial regression was observed at10 mg/kg (1/7 mice). At 30 mg/kg BGB-283, regressions wereobserved in 3 of 7mice (2 PR and 1CR; Supplementary Table S6).

The tumor inhibitory activity of BGB-283was further evaluatedin human tumor tissue derived primary colorectal cancer xeno-graft models. A total of 23 patient-derived colorectal cancermodels were established in vivo and two of them, BCCO-002 andBCCO-028, were identified to harbor BRAFV600E mutation. Bothof these patient-derived colorectal cancermodels were sensitive totreatment with BGB-283 (Fig. 4C and D); >100% TGI wasobserved on day 24 following oral treatment with BGB-283(10 mg/kg, b.i.d.; Fig. 4C and D and Supplementary Table S6).For BCCO-002, partial regressions were observed in 2 of 8 (25%)mice treated with BGB-283 (10 mg/kg b.i.d.). Addition of cetux-imab did not further enhance the antitumor activity of BGB-283(P>0.05, BGB-283þ cetuximab vs. BGB-283) against BCCO-002,which is consistent with the results observed in the HT29 xeno-graft model (Fig. 4A and C). BCCO-028 appeared to be moresensitive to treatment with BGB-283; partial regressions wereobserved in 3 of 8 (38%) mice treated with BGB-283 (5 mg/kgb.i.d.). Increasing the BGB-283 to 10 mg/kg, resulted in regres-sions in 7 of 8 (88%)mice (5 partial and 2 complete regressions).In contrast, dabrafenib (50 mg/kg b.i.d.) treatment was lesseffective against BCCO-028 with an observed 86% TGI, and notumor regression (Fig. 4D Supplementary Table S6). It should benoted that for dabrafenib at 50mg/kg b.i.d., its exposure inmouseis already 2- to 3-fold higher than the exposure it has achieved inpatients at 150mgb.i.d. dosing. Treatmentwith BGB-283 at dosesup to 30mg/kg had no significant effect on body weight in any ofthe tumor models tested (Supplementary Fig. S2).

BGB-283 inhibits phosphorylation of both ERK1/2 and EGFRand displays potent antitumor activity in WiDr tumorxenografts

BGB-283 was further evaluated against WiDr tumor xeno-grafts, a BRAFV600E colorectal cancer model where strong feed-back activation of EGFR was reported upon BRAF inhibition intwo independent studies (23). In both reports, it was shownthat BRAFV600E-selective inhibitors vemurafemib and its closeanalogue PLX-4720 were inactive as single agent in WiDrxenografts, and their antitumor activities were markedlyenhanced when combined with erlotinib or cetuximab (22,23). In contrast, BGB-283 induced clear dose-dependent inhi-bition of tumor growth in the WiDr xenograft model as a singleagent. In this study, BGB-283 was orally administrated to

testing mice at 5 and 10 mg/kg twice daily (Fig. 4E). Ninety-five percent TGI was observed at lowest dosage of 5 mg/kg and>100% TGI plus partial regression in 4 of 8 (50%) mice wasachieved at dosage of 10 mg/kg (Supplementary Table S6). Inorder to determine whether the tumor suppression was corre-lated to effective inhibition of EGFR and MAPK signaling,phospho-EGFR (pEGFR), phospho-MEK (pMEK), and phos-pho-ERK (pERK) and its downstream DUSP6 levels in tumorlysate were examined by Western blot analysis at various doselevels of BGB-283. BGB-283 did not induce EGFR feedbackactivation as reported for vemurafenib. In addition, BGB-283potently inhibited pEGFR after either the first or the fifth dose atboth dosages. Correspondingly, BGB-283 potently inhibitedMEK and ERK phosphorylation and DUSP6 expression in vivowhen dosed repeatedly (Fig. 4F). There is no detectable differ-ence on AKT phosphorylation. In sum, these findings showedthat BGB-283, which inhibits both RAF family kinases andEGFR, could have sustained inhibition of the MAPK pathway.Its ability to inhibit EGFR may contribute to its potent antitu-mor activity in this WiDr xenograft model.

DiscussionIn this article, we describe the activity of BGB-283, a second-

generation BRAF inhibitor, with potential for the treatment ofcancers with aberrations in the MAPK pathway. BGB-283 showedpotent and reversible inhibitory activities against RAF familykinases, including wild-type A-RAF, BRAF, C-RAF, andBRAFV600E. In addition, BGB-283 also potently inhibited EGFRat both the biochemical and cellular level. BGB-283demonstratedremarkable selectivity in a panel of 107 cancer cell lines forantiproliferation activity. BGB-283 potently inhibited theserum-induced cell proliferation of BRAFV600E-mutant cancer celllines, with IC50 values ranging from137 nmol/L to 580 nmol/L. Itshowed little or no inhibitory activity in cell lines lackingBRAFV600E mutation, with the exception of the HCC827 lungcancer cell line (EGFR E746-A750 deletion), ZR-75-30 (HER2amplification), and the NCI-H322M lung cancer cell line (EGFRoverexpression). These results suggested that RAF kinase andEGFR-inhibitory activities of BGB-283 contributed the most toits antiproliferative activities in the tested cancer cells. Despite thedifferent kinase selectivity profile between BGB-283 and vemur-afenib, both agents displayed noticeable selectivity toward cancercells harboring BRAFV600E in a cell viability assay (Fig. 3A and B).

Despite the remarkable responses to vemurafenib and dab-rafenib in melanoma, the clinical response of other BRAFV600E

cancers to the first generation of BRAF inhibitors is much lessimpressive (7, 17, 20, 21). The reported response of BRAFV600E

colorectal cancer to vemurafenib is merely 5% (21). Two inde-pendent studies suggested that EGFR feedback activation couldbe one of the main mechanisms of the observed resistance tofirst-generation BRAF inhibitors. This article demonstrates thatBGB-283 is a bona fide EGFR inhibitor and displays good EGFRinhibitory activity in in vitro and in vivo experiments. In WiDrcolorectal cancer cells, BGB-283 was shown to be able to inhibitthe feedback activation of EGFR signaling and achieves sus-tained inhibition of pERK. This sustained inhibition ofpERK translates into remarkable antitumor activity in vivo.Notably, BGB-283 single-agent treatment at 10 mg/kg b.i.d. ledto 50% partial regression in WiDr colorectal adenocarcinomaxenografts. In comparison, both PLX4720þcetuximab and

Tang et al.





vemurafenibþerlotinib combinations seemed to have achievedmostly TGI but not tumor regression in WiDr xenograft models(22, 23).

BRAFV600E mutation is reported to occur in 5% to 15% ofcolorectal cancer patients. Among the 23 colorectal cancer pri-

mary tumor xenograft models established in this study, two ofthem were found to have the BRAFV600E mutation. BGB-283demonstrated good efficacy in both models with the objectiveresponse rate ranging from 25% to 100%. We are carrying outmore comprehensive characterizations of thesemodels and trying

Figure 4.BGB-283 inhibited tumor growth inboth cell line–derived and primaryhuman colorectal cancer xenograftmodels harboring BRAFV600E

mutation. HT29 tumor cells(3 � 106; A), Colo205 tumor cells(2.5 � 106; B), or WiDr tumor cells(5 � 106; E) were implantedsubcutaneously in female BALB/cnude or NOD/SCID mice. When thetumors reached a certain volume insize, mice were randomly allocatedand treated as indicated. Data arepresented as average tumor volume� SEM in each group. Primarycolon cancer BCCO-002 (C) orBCCO-028 tumor fragments(P6; 3 mm� 3mm� 3mm; D) wereimplanted subcutaneously in femaleBALB/c nude mice. When thetumors reached a certain volume insize, mice were randomly allocatedand treated as indicated. Data arepresented as average tumor volume�SEM in eachgroup. F, immunoblotanalyses for EGFR, phospho-EGFR,pAKT, AKT, pMEK, MEK, ERK1/2,phospho-ERK1/2, DUSP6,and GAPDH in WiDr xenografttumor lysates. Tumor lysates wereprepared 4 hours after the first doseor the fifth dose on day 3 (b.i.d.�3)with indicated concentrations ofBGB-283. Lysates from tworepresentative mice each groupwere equally mixed based on totalprotein concentrations and loadedfor SDS-PAGE.






to better understand the MAPK and EGFR pathways in these twoprimary tumor xenograft models. Currently, phase I clinical trialsare in progress to test the safety, tolerability, pharmacokinetics,and pharmacodynamic activity of BGB-283 in human. To ourknowledge, BGB-283 is the only small-molecule inhibitor in theclinic that simultaneously targets RAF kinases and EGFR. Therehave been strong interests from the community to test thehypothesis that EGFR feedback activation leads to lack ofresponses in colorectal cancer for BRAFV600E-selective inhibitors.A number of clinical trials that combines BRAF inhibitors withEGFR small-molecule inhibitors or monoclonal antibodies arecurrently under way (see www.clinicaltrials.gov). The preclinicalresults reported in this study warrant evaluation of BGB-283 as asingle agent in BRAFV600E-mutated colorectal cancer patients.

Disclosure of Potential Conflicts of InterestG. Zhang and D. Sutton have ownership interest in BeiGene (Beijing) Co.,

Ltd. C. Zhou reports receiving commercial research grant from and has own-ership interest in BeiGene (Beijing) Co., Ltd. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: S.-H. Cheung, J. Wei, D. Sutton, M. Wei, C. Zhou,L. Wang, L. LuoDevelopment of methodology: Z. Tang, R. Du, G. Zhang, J. Wei, Y. Zhao,Y. Feng, Y. Zhang, Y. Du, X. Hu, Y. Liu, R. Hao, S. Li, S.Wang, D. Sutton, L.Wang,L. Luo

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Tang, X. Yuan, J. Wei, X. Hu, R. Hao, J.-f. Ji,L.-H. Zhang, S. Li, D. SuttonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):Z. Tang, R.Du, S.-H. Cheung, J.Wei, Y. Zhao,H. Peng,X. Hu, Y. Liu, R. Hao, D. Sutton, L. Wang, L. LuoWriting, review, and/or revision of the manuscript: Z. Tang, X. Yuan, R. Du,S.-H. Cheung, G. Zhang, Y. Liu, D. Sutton, M. Wei, L. Wang, L. LuoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Tang, S.-H. Cheung, J. Wei, X. Hu, W. Gong,Y. Liu, Y. Gao, S. Li, D. Sutton, L. LuoStudy supervision: Z. Tang, X. Yuan, S.-H. Cheung, X. Hu, Y. Liu, D. Sutton,M. Wei, C. Zhou, L. Wang

AcknowledgmentsThe authors thank Drs. Zhan Yao and Neal Rosen for critical review of the

manuscript.

Grant SupportThis work was supported by grants from the Beijing Municipal Science and

Technology Commission and the National Science and Technology MajorProject (to L. Luo, No. 2013ZX09102005).

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 March 31, 2015; revised July 8, 2015; accepted July 15, 2015;published OnlineFirst July 24, 2015.

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2015;14:2187-2197. Published OnlineFirst July 24, 2015.Mol Cancer Ther Zhiyu Tang, Xi Yuan, Rong Du, et al.

-Mutated Colorectal CancersBRAFAntitumor Activity in BGB-283, a Novel RAF Kinase and EGFR Inhibitor, Displays Potent

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