Molecular and Cellular Pathobiology

Mechanisms of Acquired Resistance to BRAFV600E Inhibition in Colon Cancers Converge onRAF Dimerization and Are Sensitive to ItsInhibitionRona Yaeger1, Zhan Yao2, David M. Hyman1, Jaclyn F. Hechtman3, Efsevia Vakiani3,HuiYong Zhao2,Wenjing Su2, LuWang3, Andrew Joelson2, Andrea Cercek1, Jose Baselga1,Elisa de Stanchina2, Leonard Saltz1, Michael F. Berger3, David B. Solit1,4, and Neal Rosen1,2

Abstract

BRAF V600E colorectal cancers are insensitive to RAF inhibitormonotherapy due to feedback reactivation of receptor tyrosinekinase signaling. Combined RAF and EGFR inhibition exerts atherapeutic effect, but resistance invariably develops throughundefined mechanisms. In this study, we determined that colo-rectal cancer progression specimens invariably harbored lesionsin elements of the RAS–RAF–MEK–ERK pathway. Genetic ampli-fication of wild-type RAS was a recurrent mechanism of resistancein colorectal cancer patients thatwas not seen in similarly resistantmelanomas. We show that wild-type RAS amplification increases

receptor tyrosine kinase-dependent activation of RAS morepotently in colorectal cancer than in melanoma and causesresistance only in the former. Currently approved RAF inhibitorsinhibit RAF monomers but not dimers. All the drug-resistantlesions we identified activate BRAF V600E dimerization directlyor by elevating RAS-GTP. Overall, our results show that mechan-isms of resistance converge on formation of RAF dimers andthat inhibiting EGFR and RAF dimers can effectively suppressERK-driven growth of resistant colorectal cancer. Cancer Res; 77(23);6513–23. �2017 AACR.

IntroductionThe RAS–RAF–MEK–ERK pathway is physiologically regulated

by upstream signals generated by receptors and activates multiplecellular processes including proliferation. RAS activation pro-motes the formation of RAF homo- and hetero-dimers, which inturn induce downstream signaling (1, 2). ERK activation alsoinduces feedback inhibition of multiple components of thepathway, which limits the duration and output of the signal.Thus, ERK inhibits activation of RAS by receptor tyrosine kinases(RTK) by phosphorylating the SOS exchange factor and a varietyof receptors and by inducing the expression of members of theSprouty family of proteins. Active ERK also directly phosphor-ylates and inhibits CRAF and BRAF and induces the expression ofmultiple MAPK phosphatases (3, 4).

BRAF V600E is the most common mutant BRAF allele. BRAFV600 mutants are constitutively activated and, uniquely amongRAF mutants, can signal as RAS-independent monomersor dimers, depending on levels of RAS activation in the tumor(5, 6). BRAF V600 mutants are therefore unaffected by upstreamfeedback and drive high levels of ERK signaling output, whichprofoundly inhibit intracellular RAS activity. Thus, in thesetumors, BRAF V600E predominantly exists as a drug-sensitivemonomer. Current RAF inhibitors selectively inhibit BRAFmono-mers and are much less potent inhibitors of RAF dimers. Accord-ingly, RAF inhibitors rapidly inhibit ERK signaling in BRAFV600Etumors. This relieves feedback inhibition of RAS and results ininduction of both BRAF V600E and wild-type RAF dimers. Thesedimers are resistant to RAF inhibitors, so a rebound in ERKsignaling ensues and attenuates the antitumor effects of thesedrugs (7). InBRAFV600Emelanomas, ERK reboundsonly slightlyand remains significantly lower than pretreatment levels. RAFinhibitors have significant therapeutic activity in BRAF V600Emelanoma, but combined inhibition of BRAF and MEK reducesthe rebound and is more effective than BRAF inhibition alone(8, 9). In colorectal and thyroid carcinoma, ERK rebound afterinhibition by RAF inhibitors ismuch greater than that observed inmelanoma and can rise to pretreatment levels. RAF inhibitorshave only marginal therapeutic effects in these tumors (10).

EGFR is the dominant RTK in colon and the marked reboundin ERK signaling is thought to be due predominantly to relief offeedback inhibition of this receptor (11, 12). Consistent withthis idea, the rebound in ERK signaling in colorectal canceris sensitive to EGFR inhibition and combined administrationof RAF and EGFR inhibitors induces tumor regression in mostpatients (13–17). However, acquired resistance invariably

1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York,New York. 2Program in Molecular Pharmacology and Chemistry, Memorial SloanKettering Cancer Center, New York, New York. 3Department of Pathology,Memorial Sloan Kettering Cancer Center, New York, New York. 4Human Oncol-ogy and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, NewYork, New York.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

R. Yaeger and Z. Yao contributed equally to this article.

Corresponding Author: Neal Rosen, 1275 York Avenue, Box 271, New York, NY10065. Phone: 646-888-2075; Fax: 646-422-0246; E-mail: rosenn@mskcc.org

doi: 10.1158/0008-5472.CAN-17-0768

�2017 American Association for Cancer Research.

CancerResearch

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develops, typically within 6 months (13–16). In the recentrandomized trial of cetuximab and irinotecan with or withoutvemurafenib in BRAF V600E colorectal cancer patients (SWOG1406), there was an improved response rate in the triplet arm,but progression-free survival in the triplet arm was only 4.4months (17).

Materials and MethodsGenetic analysis

DNA from pretreatment and disease progression specimenswere analyzed using our custom next-generation sequencingplatform, MSK-IMPACT (integrated mutation profiling of action-able cancer targets). The pretreatment samples were collectedbefore the administration of any chemotherapy and consisted ofthe primary colon tumor in six cases and liver metastasis in twopatients. MSK-IMPACT is a targeted exome capture assay withdeep sequencing coverage. Target-specific probes for hybrid selec-tion were designed as previously described (18, 19) to capture allprotein-coding exons of 341 oncogenes, tumor suppressor genes,and components of pathways deemed actionable by targetedtherapies (for full list, see ref. 20).

All patients were treated on BRAF inhibitor clinical trialsapproved by MSKCC Institutional Review Board/Privacy Board(protocols 12-131, 12-221, 14-019). Progression biopsies andcollection of patient samples were conducted under appropriateInstitutional Review Board/Privacy Board protocols and waivers(protocols 06-107, 12-245, 14-019). Participating patients signedwritten informed consent for these clinical trials and biospecimenprotocols. This study was conducted in accordance with ethicalguidelines in the Declaration of Helsinki.

V600E BRAF immunohistochemistryIHCwith an antibody specific to BRAFV600Ewas performed as

previously described (21).

FISHFISH analysis was performed on formalin-fixed paraffin embed-

ded (FFPE) sections. BRAF FISH was performed using a 2-colorBRAF break apart probe (developed at MSKCC): bacterial artificialchromosomes (BAC) clones mapping to 50BRAF (RP11-715H9,RP11-133N19) labeledwith red dUTP and 30BRAF (RP11-759K14,RP11-788O6) with green dUTP. KRAS FISH analysis was per-formed using a two-color KRAS/Cen12 probe mix (developed atMSKCC). The probe mix consisted of BAC clones containing thefull length KRAS gene (clones RP11-29515 and RP11-707F18;labeled with red dUTP) and a centromeric repeat plasmid forchromosome 12 served as the control (clone pa12H8; labeledwithgreen dUTP). Probe labeling, hybridization, washing, and fluores-cence detection were performed according to standard procedures.Slides were scanned using a Zeiss Axioplan 2i epifluorescencemicroscope equipped with a megapixel CCD camera (CV-M4þCL,JAI) controlled by Isis 5.5.9 imaging software (MetaSystemsGroupInc.). Theentire sectionwas scanned through63�or100� to assesssignal pattern and select representative regions for imaging. Ampli-fication was defined as >10 copies of each locus.

DrugsVemurafenib (PLX4032) and PLX4720 were obtained from

Plexxikon. Cetuximab was obtained from the MSKCC hospitalpharmacy. BGB659 was provided by BeiGene.

Cell cultureAll cells were obtained either from theMSKCC cell collection or

the ATCC. HT-29 and VaCo432 cells weremaintained inMcCoy'smedium with antibiotics and 10% FBS. A375 and SKMel28 cellswere grown in DMEM medium with antibiotics and 10% FBS.Cells with inducible expression constructs were maintained inthis medium with 100 mg/mL hygromycin (Invitrogen) and0.2 mg/mL puromycin (Invitrogen).

HT-29 is a mismatch repair proficient colon cancer cell line(22). Vaco432 is a mismatch repair deficient (dMMR) coloncancer cell line due toMLH1 promoter methylation and silencing(23). The four cell lines used (HT-29, Vaco432, A375, and SKMel-28) do not have any mutations in the KRAS, NRAS, or HRASgenes. All cell lines tested negative for mycoplasma. Gene altera-tions in cell lines from theMSKCCcell collection (SKMel-28)wereconfirmed by MSK-IMPACT sequencing.

Inducible expression systemRetrovirus encoding the tet regulated NRAS or KRAS gene was

packaged in Phoenix-AMPHO cells obtained from ATCC. Themedium containing virus was filtered with 0.45 mm PVDF filtersfollowed by incubation with the target cells for 6 hours. After thisincubation, cells were cultured in virus-free medium for 2 days.Then the cells were selected with puromycin (2 mg/mL) or hygro-mycin (250 mg/mL) for 3 days. The positive infected cells werefurther sortedwithGFPmarker after overnight exposure to 1mg/mLdoxycycline (Sigma-Aldrich). The NRAS gene construct includedthree consecutive FLAG tags in the N-terminus, and the KRAS geneconstruct included one FLAG tag in the N-terminus.

AntibodiesImmunoassays were performed as previously described (24).

Antibodies against phospho-ERK (T202/Y204), total ERK1/2,phospho-MEK (S217/221), phospho-EGFR (Y1068), and totalEGFR were obtained from Cell Signaling Technology; antibodiesagainst NRAS and BRAF from Santa Cruz Biotechnology; anti-RASfrom ThermoScientific; and anti-CRAF from BD Biosciences.

RAS-GTP assayGTP-bound RAS was measured with the RAF1 RAS-binding

domain (RBD) pull-down and detection kit (Thermo Scientific)following the manufacturer's instructions.

Animal model studiesPatient-derived tumor models were generated by mincing

about 1 g of tumor tissue, mixing with matrigel (50%) andimplanting orthotopically into NSG (NOD scid g) mice (Institu-tional Review Board protocols 06-107, 14-091). The growingtumor was then implanted as subcutaneous xenografts for growthexperiments, and tumor measurements were performed asdescribed (24). The patient-derived xenograft (PDX) generatedwas sequenced to confirm the same genomic alterations present inthe progressing biopsy specimen were maintained in the PDX. Allstudies were performed in compliance with institutional guide-lines under an Institutional Animal Care and Use Committee–approved protocol.

ResultsTo define the molecular basis for resistance to combined RAF/

EGFR inhibition, we analyzed nine tumor samples collected fromeight patients at the time of disease progression and compared

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the results with those obtained from matched pretreatmenttumors. The tumors of all eight patients regressed with treatment(10–100%), and all subsequently developed resistance (Table 1).DNA derived from tumor and normal DNA from blood weresubjected to targeted deep sequencing of all exons and selectedintrons of 341 key cancer-associated genes (mean tumor coveragewas 649�; ref. 20). New alterations in genes that encode compo-nents of the RAS/RAF pathway were identified in all nine of thesamples obtained at progression. These included activatingmuta-tions of KRAS orNRAS, amplification of wild-typeNRAS or KRASormutant BRAF V600E, and an intragenic deletion of exons 2 to 8in BRAF V600E (Table 1). Two separate progression samples(collected from the liver and the peritoneum) from patient 3revealed distinct new alterations of genes in the pathway—a BRAFintragenic deletion in the former and an NRAS mutation in thelatter. Amplification in the resistant cells was validated directly(Supplementary Fig. S1A, data not shown), by increased proteinexpression (Supplementary Fig. S1B andS1C), andbydetectionofthe amplified gene in double minute chromosomes or homo-genously staining regions by fluorescence in situ hybridization(Supplementary Fig. S1D and S1E). Amplification or gain of wild-type RAS occurred in three of nine tumors after acquisition ofresistance.

Our previous work showed that RAS mutation, BRAF V600Eamplification, and a splice variant of BRAF V600E all causeresistance to RAF inhibitors by inducing BRAF V600E dimeriza-tion (6, 25). One of the tumor specimens frompatient 3 harboredan intragenic deletion of exons 2 to 8 of BRAF, a region includingthe RAS-binding domain. This is also the domain that is deleted inthe alternatively spliced isoform and in most BRAF fusion pro-teins in cancer (Supplementary Fig. S1F; ref. 26). Its deletioncauses RAS-independent constitutive dimerization and activationof the truncated RAF (26, 27). As demonstrated in the tumor frompatient 3, differentmechanismsof resistance canoccur indifferentlesions, but, in this case, each results in RAF dimerization, sug-gesting that this is a common convergent mechanism underlyingthe progression of tumors treated with this regimen.

In melanoma, RAS mutation, BRAF V600E aberrant spliceisoforms, and BRAF V600E amplification are the most commonmechanisms for acquisition of resistance to RAF inhibitors or toRAF plus MEK inhibitors (Table 2). Amplification of wild-typeRAS has not been reported as a resistance mechanism inmelanoma and the biologic consequences of wild-type RAS over-

expression are not clear. To investigate these issues, colorectalcancer BRAF V600E cell lines sensitive to vemurafenib/cetuximabtreatment (HT-29 and Vaco432) were infected with a virus thatencodes a doxycycline-inducible wild-typeNRAS.Overexpressionofwild-typeNRAS in these cells resulted in an increase inRAS-GTPlevels. In HT-29 and Vaco432 cells, an approximate doubling ofwild-typeNRAS expression led to justmore than double the levelsof the activated, GTP-bound form of RAS (Fig. 1A and Supple-mentary Fig. S2A). RAS overexpression resulted in an increase ofco-immunoprecipitated CRAF with BRAF (Fig. 1B and Supple-mentary Fig. S2B). At baseline levels of RAS, BRAF/CRAF dimerscould not be detected by immunoprecipitation (SupplementaryFig. S2B and Fig. 1B), but a less than doubling in RAS expressionresulted in the detection of RAF hetero-dimers (Fig. 1B). Weobserved a dose-dependent relationship between levels of RASexpression and levels of BRAF/CRAFdimers (Fig. 1B). Thus, inHT-29 cells, an approximate doubling of baseline RAS expressionresulted in RAS-GTP increasing approximately 2.5 times and thisincrease resulted in an induction of RAFhetero-dimers. These datasuggest that modest increases in RAS expression, comparable towhat is seen in clinical progression samples (4 to >20 copies), areenough to increase RAF dimerization.

Increased expression of wild-type RAS led to increased RASactivation and induction of BRAF/CRAF dimers in a model ofKRAS amplification, as well. Increasing doxycycline exposurefrom 100 to 1000 ng/mL led to an approximate doubling of RASexpression and a large (10-fold) increase in RAS-GTP levels(Supplementary Fig. S2C). Doubling of RAS expression was againsufficient to lead to a detectable increase in CRAF/BRAF dimers(Supplementary Fig. S2D). These data suggest that moderateamplification and overexpression of either wild-type NRAS orKRAS inBRAFV600E colorectal cancer is sufficient to increase RASactivation enough to cause resistance to RAF/EGFR therapy bygenerating of RAF-inhibitor resistant RAF dimers.

This turned out to be the case. Overexpression ofNRAS in BRAFV600E HT-29 colorectal cancer cells was sufficient to confervemurafenib/cetuximab resistance (Supplementary Fig. S3A andS3B). A 1.7-fold amplification of RAS was found to be associatedwith a detectable decrease in the inhibition of phosphorylatedERKwith 1-hour vemurafenib/cetuximab treatment (Supplemen-tary Fig. S3C). RAS overexpression led to resistance in HT-29xenografts as well (Fig. 2A). NRAS expression approximatelydoubled in the vehicle treated mouse and increased 3.6-fold in

Table 1. Acquired alterations in BRAF V600E colorectal cancers treated with RAF/EGFR inhibitor combinations

Patient TreatmentBest response(RECIST read)

Duration ofresponse Acquired ERK pathway alteration

1 Vemurafenib þ Panitumumab PR (�100%) 40 weeks NRAS Q61K2 Vemurafenib þ Panitumumab PR (�64%) 24 weeks BRAF V600E amplification (predominantly double minute

chromosomes with 10–100 copies of BRAF)3 Encorafenib þ Cetuximab þ Alpelisib PR (�62%) 24 weeks Peritoneal metastasis: BRAF del exons 2–8

Liver metastasis: NRAS G13R4 Vemurafenib þ Cetuximab PR (�50%) 16 weeks BRAF V600E amplification (predominantly clusters of 6–14 copies

of BRAF)5 Encorafenib þ Cetuximab þ Alpelisib PR (�43%) 18 weeks KRAS G12A6 Vemurafenib þ Panitumumab SD (�24%) 32 weeks KRAS gain (4 copies), MET gain (5 copies)7 Vemurafenib þ Panitumumab SD (�20%) 16 weeks NRAS amplification (>10 copies)8 Vemurafenib þ Cetuximab SD (�10%) 24 weeks KRAS amplification (predominantly homologous staining region

type with 10 to >20 copies of KRAS)

NOTE: Vemurafenibþpanitumumab treatmentwas given in a pilot trial (16), encorafenibþ cetuximabþ alpelisibwas given in a phase Ib/II trial (15), and vemurafenibþ cetuximab treatment was given in the basket trial of vemurafenib (14).Abbreviations: PR, partial response; SD, stable disease.

RAF Dimers Underlie Resistance to RAF/EGFR Inhibitors in Colorectal Cancer

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the mouse treated with RAF and EGFR inhibitors. These dataconfirm that even low level of RAS expression in colorectal canceris sufficient to cause resistance to RAF/EGFR inhibitors in vivo andthe higher level of RAS amplification in the drug treated mousesuggests a selection for RAS amplification in the tumors treatedwith the drug combination.

Inhibition of EGFR phosphorylation by vemurafenib/cetuxi-mab treatment was unaffected by overexpression of wild-typeRAS, but inhibition of ERK phosphorylation was abrogated in theHT-29_NRAS cells (Fig. 2B and Supplementary Fig. S3D). Thesedata suggest that the sensitivity of RAS activation to EGFR inhi-

bition is reduced in cells in which wild-type NRAS is amplifiedand this turned out to be the case. In the HT-29 cells, inhibitionof EGFR with cetuximab alone or together with vemurafenib,inhibited endogenous levels of activated RAS (SupplementaryFig. S3D). Levels of RAS-GTP in HT-29 cells with NRAS over-expression were not sensitive to EGFR inhibition (Fig. 2C andSupplementary Fig. S3D), suggesting that amplified wild-typeRAS may be activated by other RTKs. The baseline levels ofRAS-GTP and the increased RAS-GTP in cells in which NRAS wasoverexpressed remained sensitive to serum starvation (Fig. 2C),suggesting that wild-type RAS amplification leads to increasedRAS-GTP in colorectal cancer cells by amplifying upstream sig-naling by growth factors andmay signal throughmultiple RTKs inaddition to EGFR.

To determine if RAS amplification conferred resistance byreducing sensitivity of the pathway to the RAF inhibitor, weexamined the ability of vemurafenib to inhibit ERK signalingin HT-29 cells in which different levels of NRAS expressionwere induced. The sensitivity of ERK signaling in these cells toone hour exposure to vemurafenib was inversely related toNRAS expression (Fig. 2D). In cells with 3.5-fold RAS over-expression, one hour exposure to vemurafenib caused a 32%decrease in phosphorylated MEK and a 23% decrease inphosphorylated ERK compared to an 88% decrease in bothphosphorylated MEK and ERK levels in the absence of NRASoverexpression.

These findings suggest that wild-type RAS gene amplificationcauses resistance to RAF/EGFR inhibition by increasing cellularRAS-GTP levels to levels sufficient to drive BRAF V600E dimer-ization. To our knowledge, wild-type RAS amplification has notbeen described as a mechanism of resistance to RAF inhibitors inmelanomas (Table 2). To evaluate if wild-type RAS amplificationcan cause resistance in melanoma, we inducibly expressed wild-type NRAS, Q61K mutant NRAS, or control GFP protein in A375or SKMel-28 BRAF V600E melanoma cells. Overexpression ofwild-typeNRAS did not affect phospho-ERK inhibition by vemur-afenib, whereas expression of Q61K NRAS led to insensitivity ofphospho-ERK to even high concentrations of drug (Fig. 3A andSupplementary Fig. S4A). Overexpression of NRAS in A375 cellsled to a small increase in RAS-GTP levels, much lower than thelevels of RAS-GTP that result fromQ61KmutantNRAS expression(Fig. 3B), and did not alter the growth inhibitory effect ofvemurafenib treatment (Supplementary Fig. S4B). Compared tothe colorectal cancer cell lines, we observed that overexpression ofNRAS in A375 and SKMel-28melanoma cells, even to high levels,resulted in a barely perceptible induction of BRAF/CRAF dimers(Fig. 3C).

We compared the effect of increasing levels of NRAS expressionon relative inhibition of phospho-ERK and phospho-MEK inA375 and HT-29 cells (Fig. 3D and Supplementary Fig. S4C). Anapproximately six-fold increase in NRAS expression led to min-imal change in the sensitivity of phospho-ERK and phospho-MEKto vemurafenib in A375 cells, but led to a greater than 50%decrease in the inhibition of phospho-ERK and phospho-MEKby vemurafenib combined with cetuximab in HT-29 cells. Thesedata suggest that overexpression of wild-type NRAS amplifies RASactivation to a much greater degree in colorectal cancers than inmelanomas when BRAF V600E is expressed. This may have to dowith the low levels of endogenous RTK activation in melanomas(28). In this context, amplification of theweakRTK signal bywild-type RASmay be insufficient to cause resistance.We speculate that

Table 2. Clinically validated mechanisms of resistance to RAF inhibitors inmelanoma patients

Reference Treatment Resistance mechanisms

Johannessen andcolleagues (34)

Vemurafenib Increased COT expression

Nazarian and Vemurafenib PDGFb upregulationcolleagues (35) NRAS mutation

Poulikakos andcolleagues (25)

Vemurafenib Aberrantly spliced BRAF

Wagle and colleagues(36)

Vemurafenib MEK1 mutation

Villaneuva andcolleagues (37)

Vemurafenib Increased IGF1R expression

Shi and colleagues Vemurafenib BRAF amplification(38) BRAF truncation

NRAS mutationIncreased RTK expression

Straussman andcolleagues (39)

Vemurafenib ordabrafenib þtrametinib

Stromal HGF secretion

Whittaker andcolleagues (40)

Vemurafenib NF1 loss

Trunzer and Vemurafenib NRAS mutationcolleagues (41) MEK1 mutation

Van Allen and Vemurafenib or NRAS mutationcolleagues (42) dabrafenib BRAF amplification

MEK1 mutationMEK2 mutationMITF amplification

Shi and colleagues Vemurafenib or NRAS mutation(43) dabrafenib BRAF amplification

Aberrantly spliced BRAFMEK1 mutationKRAS mutation

Rizos and colleagues Vemurafenib or NRAS mutationand Johnson and dabrafenib Aberrantly spliced BRAFcolleagues (44, 45) BRAF amplification

MEK1 mutationMEK2 mutationKRAS mutationIncreased IGF1R expressionAKT1 mutationPIK3CA mutationPTEN lossDUSP4 deletionAKT3 mutationMITF amplificationPDGFR upregulation

Sun and colleagues(46)

Vemurafenib ordabrafenib ortrametinib

EGFR expression

Wagle and colleagues Dabrafenib þ MEK2 mutation(47) trametinib BRAF amplification

Aberrantly spliced BRAFVillanueva andcolleagues (48)

Trametinib followedby dabrafenib

MEK2 mutation plus BRAFamplification

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this is the reason that RAS amplification has not been identified asa common cause of RAF inhibitor resistance in melanomas.

Recently, we have identified drugs that inhibit ERK signalingdriven by both mutant RAF monomers and dimers (6). Wehypothesized that such compounds should effectively inhibitBRAF V600E colorectal cancers with dimer-dependent acquiredresistance to RAF/EGFR inhibition. We tested the effects of one ofthese, BGB659, in vemurafenib/cetuximab-resistant colorectaltumors. This drug inhibited ERK signaling in HT-29 and HT-29_NRAS cells at similar concentrations (Fig. 4A). At later timepoints, BGB659 treatment was associated with a rebound inphospho-ERK levels in HT-29 cells that was largely suppressed

by combination treatment with the EGFR inhibitor cetuximab(Supplementary Fig. S5A), suggesting that reactivation of RTKsignaling can also reduce sensitivity to the RAF dimer inhibitor inBRAF V600E colorectal cancers. Feedback reactivation of RTKsignaling likely attenuates sensitivity to RAF dimer inhibitors dueto increased RAS activation and to formation of wild-type RAFdimers. We have shown that, compared to mutant BRAF dimers,RAS driven wild-type RAF dimers are less sensitive to these drugs(6). Thus, RAFdimer inhibitorswill still have to be combinedwithinhibitors of the dominant RTK EGFR in order to maximallyinhibit bothmutant and wild-type RAF. As shown in Supplemen-tary Fig. S5B, the combination of BGB659 with cetuximab led to

Figure 1.

RAS amplification leads to increased RAS-GTP and dimerization of RAF. A, HT-29 cells and HT-29 FLAG-NRAS wild-type (WT; HT-29_NRAS) cells, exposedto increasing doses of doxycycline as indicated, were harvested after 24 hours. Expression of the indicated proteins was assayed by immunoblotting. Thecellular RAS-GTP was determined by the active RAS pull-down assay. Densitometric analysis of the bands was used to calculate the relative amplification ofRAS and RAS-GTP. B, HT-29_NRAS and Vaco432_NRAS cells were treated with the indicated doses of doxycycline for 16 hours. Then the cells were lysed in 0.1%NP-40 Tris-NaCl buffer. The soluble fractions were isolated and incubated with anti-BRAF antibody–coupled IgG beads for 2 hours at 4�C. The immunoprecipitatedprotein complex and 2% input were assayed by immunoblotting with indicated antibodies. Densitometric analysis of the bands was used to calculate relative RASamplification and CRAF/BRAF dimers. Relative levels of CRAF/BRAF dimers were normalized to levels of immunoprecipitated BRAF.

RAF Dimers Underlie Resistance to RAF/EGFR Inhibitors in Colorectal Cancer

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Figure 2.

Increase in NRAS expression is sufficient to cause resistance to RAF/EGFR inhibition in colorectal cancer. A, Growth curves for HT-29 and HT-29_NRAS xenograftstreatedwith vehicle or vemurafenib (PLX472050mg/kgorally twice daily) plus cetuximab (50mg/kg i.p. injection twice perweek). (Continuedon the followingpage.)

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significantly better growth inhibition in vitro than BGB659 alonein both HT-29_NRAS and Vaco432_NRAS cells. In agreementwith our model, although both the BGB659/cetuximab andvemurafenib/cetuximab combinations were able to suppress thegrowth of colorectal cancer cells with BRAF V600Emutation, onlythe former combination could effectively inhibit the growth ofBRAF V600E colorectal cancer cells, which also express high levelsof wild-type NRAS (Fig. 4B).

BGB659/cetuximab treatment was not only effective in the RASamplification-driven vemurafenib resistant colorectal cancer cells,but also in other models of resistance. In a patient-derivedxenograft derived from the progressing liver metastasis obtainedfrom patient 3 (NRAS G13R mutation, Table 1), treatment withPLX4720 plus cetuximab resulted in only amodest delay in tumorgrowth whereas BGB659/cetuximab treatment led to tumorregression in all five mice treated (Fig. 4C). In tumors treatedwith PLX4720/cetuximab, phospho-MEK and phospho-ERKlevels increased by day 20. This is likely due to transactivationof wild-type RAF dimers with PLX4720. By contrast, BGB659/cetuximab continued to profoundly inhibit phospho-MEK andphospho-ERK on day 20. These data suggest that new RAF dimerinhibitors that equipotently inhibit both RAF mutant monomersand dimers represent a new, potentially much more effectivestrategy for treating BRAF V600E colorectal cancer.

DiscussionAcquired resistance to RAF/EGFR inhibitor combination ther-

apy is mediated bymultiple genetic lesions (29–31). In our work,we analyzed nine progression samples from patients and foundthat resistance was mediated by five different genetic aberrations.Allfive lesions prevent effective inhibitionofRAF activationby thecombination. Tumors in which resistance is mediated by parallelor downstream lesions in which RAF activity remains sensitive tothe RAF inhibitors were not found. Four of the lesions we haveidentified have been previously shown to also cause resistance inmelanoma. The exception is amplification of wild-type RAS,which causes resistance in colorectal cancer, but has not beenidentified as a resistance mechanism in melanoma. We have nowdetermined that each of these lesions causes resistance by thesame unifying mechanism—induction of RAF dimers, which areinsensitive to current RAF inhibitors. Basedon this understanding,we show that combined administration of a RAF inhibitor thatequipotently inhibits RAF mutant monomers and dimers and anEGFR inhibitor is able to suppress growth of resistant colorectaltumors, thus proving that inductionofRAFdimers is a key event inresistance.

Amplification of wild-type RAS is often observed in carcino-mas, but functional consequences are unknown. Our data suggestthe possibility that high levels of wild-type RAS can amplify

induction of RAS/RAF/ERK signaling by upstream inputs such asRTKs. Future studies are needed to elucidate the detailed mech-anism by which amplification of RAS facilitates RAS activation byRTKs, but likely mechanisms include a simple linear increase inRAS-GTP as a function of increase in RAS expression, as seen in thecolorectal cancer cell lines, or saturation of inhibitory processesinvolvingRAS-GTP loading andhydrolysis ormembranebinding.

We find that amplification of wild-type RAS in colorectalcancer, but not melanoma, is sufficient to elevate RAS-GTP tolevels that induce enough dimers to cause resistance to RAFinhibitors. This result is reminiscent of the differing response ofmelanoma and colorectal cancer to single-agent RAF inhibitorsand suggests that higher endogenous levels of RTK signaling incolorectal cancer lead to the formation of drug-resistant RAFdimers, causing adaptive resistance to RAF inhibitors and, in thepresence of high RAS expression, rapid acquired resistance. This isthe first example of a lineage dependent mechanism of acquiredresistance and is due either to the higher level of RTK signaling incolorectal cancer than inmelanoma or to the increased sensitivityof RTK signaling to ERK-dependent feedback in melanoma com-pared to colorectal cancer. We have recently shown that hypoac-tive BRAFmutants serve a similar function (32, 33). They amplifyERK signaling in a RAS-dependentmanner and, in lung and coloncarcinomas, cooperate with upstream RTKs to drive transforma-tion. Interestingly, levels of RAS-GTP in melanomas are also notsufficient to cooperate with low activity RAF mutations (33).Tumors with these mutations are invariably found to coexist withNF1 inactivation or RASmutations. Thus, both hypoactive BRAFmutants and amplified wild-type RAS significantly activate ERKsignaling in carcinomas, but not melanoma. It is likely that bothof these lineage dependent phenomena have the same cause—higher levels of steady state RAS-activation in lung and colonepithelial cells than inmelanocytes. Further work will be requiredto prove this hypothesis.

We observed that RAS amplification does not significantlyincrease the level of phosphorylated ERK in BRAF V600E cells.The presence of high levels of RAS expression, however, affectsresponse to vemurafenib treatment differently in melanomaand colon tumors. In BRAF V600E cells, RAS activity is almostcompletely feedback inhibited and ERK phosphorylation is driv-enbyBRAFV600Emonomers. In colorectal cancer cells, but not inmelanomas, RTK activity is high enough to elevate RAS-GTP levelsin tumor cells with high expression of wild-type RAS. This causesRAS-dependent dimerization of BRAF V600E protein, and thusreduces the sensitivity of BRAF V600E colorectal cancer to vemur-afenib, since the drug does not potently inhibit these dimers. ERKphosphorylation does not go up because the activity of BRAFV600E is not regulated by RAS binding or its dimerization.

We recently reported why RAF inhibitors selectively inhibitBRAF V600E monomers at low concentrations, but require much

(Continued.) Five mice were treated in each group, and tumor volumes (and SD) are shown as a function of time on treatment. Right panel shows immunoblots fromrepresentative mice fed doxycycline (dox) and treated with vehicle control (left) or vemurafenib/cetuximab (vem/cx; right). Tumors were collected for immunoblotanalysis at theendof thegrowthexperiment.B,HT-29andHT-29_NRAS, treatedwithdoxycycline2mg/mLfor 24hoursbeforedrugexposure,were treatedwith eithervehicle (DMSO) or vemurafenib/cetuximab (Vem/cx) for 24 hours. Expression of the indicated proteinswas assayedby immunoblotting.C,HT-29_NRAS cells, treatedwith doxycycline 2 mg/mL, were plated for 12 hours to adhere and then serum was removed as indicated. Twelve hours later, cells were subjected to treatment withvehicle control or cetuximab (Cx) for 24 hours. Cells were then collected and expression of the indicated proteins was assayed by immunoblotting. The cellular RAS-GTP was determined by the active RAS pull-down assay. D, HT-29_NRAS cells were treated with doxycycline for 24 hours and then subjected to sorting of the cellpopulations by GFP expression. HT-29 cells, unsorted HT-29_NRAS cells, and HT-29 cells sorted for low, medium (med), or high GFP expression were treated witheither vehicle (DMSO)orvemurafenib 1mmol/L for 1 hour. Expressionof the indicatedproteinswasassayedby immunoblotting.Densitometric analysis of thebandswasused to calculate phosphorylated ERK and phosphorylated MEK levels with vemurafenib treatment at each level of RAS expression.

RAF Dimers Underlie Resistance to RAF/EGFR Inhibitors in Colorectal Cancer

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Figure 3.

Increase in NRAS expression does not cause resistance to RAF inhibition in melanoma. A and B, A375 cells expressing inducible GFP (control), wild-typeNRAS, orNRASQ61Kwere treatedwith doxycycline (2mg/mL for 24 hours), followedby treatmentwith vemurafenib for one hour at the indicated concentrations (A)or collected for RAS-GTP analysis with the active RAS pull-down assay (B). Expression of the indicated proteins was assayed by immunoblotting. C, Increasingexpression of wild-type NRAS was induced into BRAF V600E–mutant colorectal cancer cell lines HT-29 and VACO432 andmelanoma cell lines A375 and SKMEL-28with FLAG-NRAS by doxycycline treatment with the indicated doses for 24 hours. The cells were then collected and lysed. BRAF/CRAF heterodimers were pulleddown with anti-BRAF antibody–conjugated beads. The cell lysate (input) for the binding assay and the immunoprecipitated complexes were assayed byimmunoblottingwith the indicated antibodies.D,A375 andHT-29 cells expressing induciblewild-typeNRASwere treatedwith increasingdoses of doxycycline for 24hours and then treated with vemurafenib 1 mmol/L or vemurafenib 1 mmol/L plus cetuximab 50 nmol/L, respectively, for 24 hours. Expression of phospho-ERK andphospho-MEK was assayed by immunoblotting. Densitometric analysis of the bands was used to calculate the relative change in phosphorylated ERK andMEK levels with drug treatment at each level of ectopic RAS expression, where the change in phospho-MEK or phospho-ERK levels in the absence of doxycycline ineach cell line was defined as 100% inhibition.

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Figure 4.

Combined administration of RAF dimer and EGFR inhibitors overcome resistance. A, HT-29 and HT-29_NRAS cells (treated with 2 mg/mL doxycycline for24 hours) were treated with a range of BGB659 doses as indicated for one hour. Expression of the indicated proteins was assayed by immunoblotting.B, Growth curves for treatment of HT-29 or Vaco432 cells with vehicle (DMSO), vemurafenib 2 mmol/L plus cetuximab 100 nmol/L (Vem/Cx), or BGB659 1 mmol/Lplus cetuximab 100 nmol/L (BGB659/Cx) for 5 days. Relative cell counts were assayed by alamarBlue. For this experiment, HT-29 and Vaco432 NRAS–overexpressing cells were treated with doxycycline 2 mg/mL for 24 hours before drug exposure. Experiments were done in 8 replicates. C, PDX made from theprogression specimen of patient 3 was expanded into mice that were treated with vehicle, vemurafenib (PLX4720 50 mg/kg orally twice daily) plus cetuximab (50mg/kg i.p. injection twice perweek), or BGB659 (100mg/kgorally daily) plus cetuximab (50mg/kg i.p. injection twice perweek). Tumor volumes (and SD) are shownas a function of time on treatment. Tumors were collected at day 20 and two samples from each group were lysed for immunoblotting with the indicated antibodies.

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higher concentrations to inhibit any of the RAF dimers (5). Thebinding of drug to one protomer of the RAF dimer reduces theaffinity of the drug for the other protomer (6). This negativecooperativity leads to reduced sensitivity of RAF dimers, com-pared to monomers, to inhibitors and also underlies adaptiveresistance to these agents in colorectal cancer.

Our work identifies a potential strategy to overcome resistancein BRAF V600E colorectal cancer, using new RAF inhibitors thatare not affected by the negative cooperativity. Unlike MEK andERK inhibitors, these drugs selectively inhibit ERK signaling that isdriven bymutant BRAF (6). This provides a basis for a therapeuticindex for these RAF inhibitors in the treatment of resistant tumors.Our data suggest that combining these newRAF inhibitorswith anEGFR inhibitor may be useful in treating resistance and as initialtargeted therapy for BRAF V600E colorectal cancer and should beexplored in future clinical trials.

Disclosure of Potential Conflicts of InterestR. Yaeger is a consultant/advisory boardmember for GlaxoSmithKline. D.M.

Hyman reports receiving a commercial research grant from PUMA, AstraZeneca,LOXO, and is a consultant/advisory board member for Atara, AstraZeneca,Chugai, CytomX, and Boehringer Ingelheim. L. Saltz reports receiving a com-mercial research support from Taiho Pharmaceuticals. N. Rosen reports receiv-ing a commercial research grant from Chugai; and is a consultant/advisoryboard member for Beigene, AstraZeneca, Chug, Daiitchi, and Novartis.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R. Yaeger, Z. Yao, J. Baselga, L. Saltz, D.B. Solit,N. Rosen

Development of methodology: R. Yaeger, Z. Yao, J.F. Hechtman, H.Y. Zhao,L. Wang, N. RosenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Yaeger, Z. Yao, D.M. Hyman, H.Y. Zhao, W. Su,L. Wang, A. Joelson, E. de Stanchina, D.B. SolitAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Yaeger, Z. Yao, D.M. Hyman, J.F. Hechtman,E. Vakiani, J. Baselga, M.F. Berger, D.B. Solit, N. RosenWriting, review, and/or revision of the manuscript: R. Yaeger, Z. Yao,J.F. Hechtman, L. Wang, A. Cercek, L. Saltz, M.F. Berger, D.B. Solit, N. RosenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Yao, D.M. Hyman, H.Y. Zhao, D.B. SolitStudy supervision: R. Yaeger, N. Rosen

AcknowledgmentsThe authors are grateful to Yijun Gao for helpful discussions. We would like

to thank Ahmet Zehir for help with figure preparation.

Grant SupportThis research was supported by grants to N. Rosen from the NIH (R01

CA169351 and P01 CA129243), fromMr. William H. Goodwin and Mrs. AliceGoodwin and the Commonwealth Foundation for Cancer Research, The Centerfor Experimental Therapeutics at Memorial Sloan Kettering Cancer Center, andsupport from Mr. and Mrs. Robert A. Kramer. This work is also supported by aCareer Development Award from the Conquer Cancer Foundation of theAmerican Society of Clinical Oncology (to R. Yaeger) and the NIH/NCI CancerCenter Support Grant P30 CA008748.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 15, 2017; revised August 8, 2017; accepted September 22,2017; published OnlineFirst September 26, 2017.

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