combined pan-raf and mek inhibition overcomes multiple resistance...

Small Molecule Therapeutics

Combined Pan-RAF and MEK InhibitionOvercomes Multiple Resistance Mechanismsto Selective RAF InhibitorsSteven R.Whittaker1,2,3, Glenn S. Cowley2, Steve Wagner3, Flora Luo1,2,David E. Root2, and Levi A. Garraway1,2,4,5

Abstract

RAF and MEK inhibitors are effective in BRAF-mutant mela-noma but not in BRAF-mutant colorectal cancer. To gain addi-tional insights into this difference, we performed a genome-scalepooled shRNA enhancer screen in a BRAF-mutant, RAF inhib-itor–resistant colorectal cancer cell line exposed to the selectiveRAF inhibitor PLX4720. We identified multiple genes along thereceptor tyrosine kinase (RTK)/mitogen-activated protein kinase(MAPK) signaling axis that, when suppressed, either geneticallyor pharmacologically, sensitized cells to the selective RAF inhib-itor through sustained inhibition of MAPK signaling. Strikingly,CRAF was a key mediator of resistance that could be overcome bythe use of pan-RAF inhibitors in combination with a MEKinhibitor. Furthermore, the combination of pan-RAF and MEKinhibitors displayed strong synergy in melanoma and colorectal

cancer cell lines with RAS-activating events such as RTK activa-tion, KRAS mutation, or NF1 loss-of-function mutations. Com-binations of selective RAF inhibitors, such as PLX4720 or dab-rafenib, with MEK inhibitors did not incur such profoundsynergy, suggesting that inhibition of CRAF by pan-RAF inhibi-tors plays a key role in determining cellular response. Impor-tantly, in contrast to the modest activity seen with single-agenttreatment, dual pan-RAF and MEK inhibition results in theinduction of apoptosis, greatly enhancing efficacy. Notably,combined pan-RAF and MEK inhibition can overcome intrinsicand acquired resistance to single-agent RAF/MEK inhibition,supporting dual pan-RAF and MEK inhibition as a novel ther-apeutic strategy for BRAF- and KRAS-mutant cancers. Mol CancerTher; 14(12); 2700–11. �2015 AACR.

IntroductionDespite the impressive early successes with BRAF-targeted

therapies in BRAF-mutant melanoma, intrinsic and acquiredresistance presents an enormous challenge and ultimately leadsto progressive disease (1). Furthermore, widespread, intrinsicresistance of BRAF-mutant colorectal cancers to selective RAFinhibitors has severely blunted the use of these agents in thiscontext (2). This observation could be taken to suggest thatcolorectal cancers are indifferent to RAF inhibitors and that aBRAFmutation alone is not sufficient to inform drug selection inthe clinic. However, BRAF has been shown to be a high-rankingdependency that discriminates between BRAF-mutant and BRAFwild-type cell lines, solidifying its status as a key oncogenic driver

(3). These data are therefore consistent with a continued depen-dency on BRAF signaling for cell survival and validates it as a drugtarget in colorectal cancer. Furthermore, initial studies suggestedthat some cancers are able to evade the effects of BRAF inhibitionand reactivate the MAPK pathway. Sustained MAPK pathwayactivity can be brought about by amplification of BRAF, over-expression of CRAF, mutation of NRAS, mutation of NF1,PDGFRb signaling, IGF1R/PI3K activity,MEK1mutation, expres-sion of MAP3K8/COT, or an altered transcriptional state (4–12).Surprisingly, second-site mutations of the BRAF gene have not yetbeen implicated in resistance to BRAF inhibition, despite dem-onstrating the potential to do so in preclinical models (13).

Therefore, to enable an unbiased and global assessment ofpotential RAF inhibitor combination therapies for BRAF-mutantcancers, we performed a pooled shRNA enhancer screen config-ured to identify genes, which when suppressed, sensitized cells tothe effects of the selective RAF inhibitor PLX4720 (14). Candidatehits from the screen would then enable us to identify specificvulnerabilities in BRAF-mutant cancers that might indicate novelopportunities for combination therapies to maximize patientbenefit.

Materials and MethodsCell lines and reagents

Cell lines were obtained from the ATCC or the National CancerInstitute (NCI) and cultured in DMEM (CellGro), 10% FBS(Gemini Bio-Products). Cells were passaged for less than 6months after receipt and authenticated by short tandem repeatprofiling. PLX4720, AZD6244, AZ628, RAF265, GSK1120212,

1Department of Medical Oncology, Dana-Farber Cancer Institute,Boston, Massachusetts. 2The Broad Institute, Cambridge, Massachu-setts. 3Division of Cancer Therapeutics, Institute of Cancer Research,London, United Kingdom. 4Center for Cancer Genome Discovery,Dana-Farber Cancer Institute, Boston, Massachusetts. 5Departmentof Medicine, Brigham andWomen's Hospital, HarvardMedical School,Boston, Massachusetts.

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

Corresponding Author: Levi A. Garraway, Department of Medical Oncology,Dana-Farber Cancer Institute, 450 Brookline Avenue, D1542, Boston, MA 02215.Phone: 617-632-6689; Fax: 617-582-7880; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0136-T

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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and GSK2118436 were purchased from Selleck Chemicals.MLN2480 was purchased from Chemiscene. Expression con-structs for LacZ, KRASG12V, and MEKQ56P have been describedpreviously (5, 11).

Cell proliferation assaysCells were seeded into 96-well plates at a density permitting

logarithmic growth for the duration of the experiment. Thefollowing day, compounds were added to the cells dissolved inDMSO/medium and incubated for 96 hours. CellTiter-Gloreagent (Promega) was then added to the wells, the plateswere shaken for 15 minutes, and then luminescence was readusing an Envision plate reader (Perkin-Elmer). GI50 values or% inhibition were calculated using GraphPad Prism. Wherecell proliferation was determined relative to the starting celltiter, an untreated control plate was frozen at the time oftreatment and inhibitor-treated plates were frozen followingthe 96-hour incubation. Plates were then thawed and CellTiter-Glo reagent was used as described to determine cell prolifer-ation. For colony formation assays, cells were seeded at lowdensity into 12-well plates. The following day, cells weretreated with compounds and the medium/compound wasreplaced every 5 days. Following 7 to 10 days of treatment,cells were washed with PBS, fixed in 4% formaldehyde for 30minutes, and then stained with 0.5% crystal violet solution.Unbound dye was removed by washing with excess water.Wells were photographed and the dye was resuspended in 10%acetic acid and absorbance measured at 595 nm. Relative cellgrowth was determined as a percentage of the DMSO-treatedcontrol. The Cytotox-Glo assay (Promega) was used as recom-mended by the supplier following a 72-hour exposure to theinhibitors.

In-cell WesternCell lines were seeded into clear-bottomed, black-walled

96-well plates. The next day, cells were treated with a 10-pointtitration of PLX4720 for 24 hours. Cells were then fixed andpermeabilized with 4% formaldehyde, 0.1% TX-100 for 30minutes. Wells were blocked with LI-COR blocking buffer andthen incubated with an antibody to phospho-ERK1/2 (Sigma)and to ERK2 (Santa Cruz Biotechnology) for 4 hours. Cells werethen washed with 0.1% Tween-20. Cells were then incubatedwith secondary antibodies conjugated to IR dye for 1 hour.Cells were then washed with 0.1% Tween-20 and PBS wasadded to the wells. Plates were then read using the In-cellWestern protocol on an Odyssey scanner (LI-COR). Phos-pho-ERK1/2 was normalized to total ERK2 and % inhibitiondetermined relative to control wells.

Cell lysis and Western blottingCells were lysed in SDS lysis buffer (1%SDS, 10mmol/L EDTA,

50 mmol/L Tris, pH 8.0). Protein concentration was determinedusing theBCAassay (Sigma). Proteinswere resolvedby SDS-PAGEand transferred to PVDF-Fl membranes (Millipore) using iBlot(Life Technologies). Blots were blocked with LI-COR blockingbuffer and probedwith the indicated antibodies overnight at 4�C.Detection of proteins was via IR dye–conjugated secondary anti-bodies and IR-fluorescence detection via the Odyssey system(LI-COR).

Pooled lentiviral shRNA screenThe conditions and procedure for pooled lentiviral shRNA

screening have been described previously (11). Briefly, RKO cellswere seeded into12-well plates at a density of 3� 106 cells perwell,a total of 7.2 � 107 cells per replicate were infected with a 90,000shRNA, pooled library at an MOI of 0.3 to 0.5. Cells were cen-trifuged at 2,000 rpm for 2 hours and medium changed immedi-ately after. The next day, cells were pooled and selected in thepresence of 1 mg/mL puromycin for 3 days. Cells were thenpassaged in the presence of either DMSO or 3 mmol/L PLX4720for16populationdoublings.Cellswereharvestedby trypsinizationand stored in PBS at�80�C. Extraction of gDNA, amplification ofshRNA sequences, and screen deconvolution by massively parallelsequencing (Illumina) were performed as previously described(11). The depletion of shRNAs in the PLX4720-treated arm relativeto the DMSO-treated arm was determined using the RIGER algo-rithm and this produced a statistically significant ranked list ofgenes according to the "second best shRNA" analysis in Gene-E(http://www.broadinstitute.org/cancer/software/GENE-E/).

shRNA/ORF lentiviral infectionCells were seeded into 6- or 96-well plates as required. The

following day, the medium was supplemented with 4 mg/mLpolybrene (Millipore), and lentiviral particles were added tothe cells to give an MOI of around 1. Plates were centrifuged at2,000 rpm for 30minutes andmediumwas changed immediatelyafter. Seventy-two hours later, cells were treated with inhibitors asrequired. shRNA constructs used are as follows: shLuc TRC-N0000072243; shMET TRCN0000000393, TRCN0000121233;shPTPN11 TRCN0000005002, TRCN0000005003; shRAF1 TRC-N0000001066, TRCN0000197115, shSHOC2TRCN0000151603,TRCN0000154502; shNF1 TRCN0000039717. Further detailsare available via The RNAi Consortium portal http://www.broadinstitute.org/rnai/public/.

ResultsWe profiled a panel of 20 BRAF-mutant melanoma and

colorectal cancer cell lines for sensitivity to PLX4720 (Fig.1A). The majority of colorectal cancer lines showed reducedsensitivity to the inhibitor compared with the melanoma lines.To determine whether this was related to inhibition of theMAPK pathway, we measured ERK1/2 phosphorylation follow-ing a 24-hour incubation with PLX4720 by in-cell Western(Fig. 1B). Compared with the melanoma lines, only one colo-rectal cancer line achieved near-complete suppression of phos-pho-ERK (COLO205) and was highly sensitive to PLX4720. Theother lines tested all failed to reach >75% inhibition of phos-pho-ERK and, as demonstrated in clinical trials of vemurafenib,complete (>90%) suppression of the pathway was required forefficacy (15). Interestingly, when RKO cells were treated with 3mmol/L PLX4720, ERK phosphorylation was inhibited by >90%at 1 to 2 hours, but this recovered during 4 to 24 hours toapproximately 50% of controls (Supplementary Fig. S1A).Furthermore, the cells continued to proliferate in the presenceof the drug (Supplementary Fig. S1B).

To understand what factors might permit reactivation ofMAPKsignaling in the presence of PLX4720, we conducted a pooledshRNA screen targeting >16,500 genes to identify which genes,when silenced, sensitized cells to the inhibitor (Fig. 1C). The RKOcolorectal cancer cell line for used this study, as it was readily

Synergy between Pan-RAF and MEK Inhibition

www.aacrjournals.org Mol Cancer Ther; 14(12) December 2015 2701




transduced with the lentiviral shRNA library, was highly resistantto PLX4720 (GI50, 17 mmol/L) and had a short doubling time(<24 hours). Moreover, these cells have previously been shown topossess a dependency onBRAF for proliferation (3). The cellswereinfected with a pooled library of about 90,000 shRNAs, thencultured in the presence of either DMSO vehicle or 3 mmol/LPLX4720 (Fig. 1D). shRNAs depleted only in the presence ofPLX4720 were identified by Illumina sequencing (11). The Pear-son correlation between replicate samples for each experimentalcondition confirmed reproducibility of the screen result (Fig. 1E).RNAi Gene Enrichment (RIGER; ref. 16) was used to rank genesaccording to the second best scoring shRNA per gene (Fig. 1F;Supplementary Table S1). The top-ranking gene wasMET, whichencodes the receptor tyrosine kinase (RTK) MET/HGFR, followed

by MLL/KMT2A (encoding a lysine methyl-transferase) andPTPN11, which encodes the protein tyrosine phosphatase SHP2.We used the protein interaction tool DAPPLE (17) to identifypossible connections between the top 300 genes in the screen(Supplementary Fig. S2).Notably, a node encompassing genes forthe RTKs MET, ERBB3, and NTRK2, adapter proteins GAB1 andCRKL, the protein tyrosine phosphatasesPTPN6 andPTPN11, andseveral members of the MAPK pathway SHOC2, RAF1, MAP2K2stood out due to the known functional relationships among theproteins encoded by these genes.

We therefore decided to validate key genes in this node asmediators of resistance to BRAF inhibition. Compared with acontrol shRNA targeting luciferase, knockdown of MET/HGFR,PTPN11/SHP2, SHOC2, and RAF1/CRAF all sensitized RKO cells

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Figure 1.A synthetic lethal shRNA screenidentifies enhancers of BRAFinhibition in colorectal cancer.A, BRAF-mutant melanoma andcolorectal cancer cell lines wereprofiled for sensitivity to PLX4720following a 4-day exposure. GI50values were determined usingnonlinear regression analysis inGraphPad Prism. Box and whiskerplots represent the median values,25th and 75th percentiles, andmin/max. B, cell lines weretreated with PLX4720 and ERKphosphorylation was determined byin-cell Western. Results are presentedas a heatmap with PLX4720 GI50values plotted for comparison. C,protocol for a synthetic lethal RNAiscreen in RKO colorectal cancer cellsfor modifiers of sensitivity toPLX4720. D, proliferation of RKO cellsinfected with the shRNA library andcultured in the presence of eitherDMSO or 3 mmol/L PLX4720. E,Pearson correlation was determinedbetween experimental replicates foreach condition in the screento assess reproducibility.F, following 16 population doublings,the abundance of each shRNA wasdetermined and used to generate aranked list of shRNAs on the basis ofthe log fold change between theDMSO- and PLX4720-treatedreplicates. RIGER analysis was used torank synthetic lethal genes accordingto the second best hairpin metric. Thetop 10 ranking genes are indicated.

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Mol Cancer Ther; 14(12) December 2015 Molecular Cancer Therapeutics2702




to PLX4720 (Fig. 2A) and permitted greater sustained suppres-sion of ERK1/2 phosphorylation at 24 hours following treat-ment with 3 mmol/L PLX4720 (Fig. 2B). Suppression of most ofthese genes sensitized cells to BRAF inhibition in other colo-rectal cancer cell lines SW1417, LS411N, and WIDR (Supple-mentary Fig. S3); the exception to this was MET, whose sen-sitization effects were restricted to the RKO cell line. Thesensitization of the RKO line by MET suppression is likely dueto high expression of HGF by these cells, which activates METsignaling, thus creating a dependency on MET in the absence of

signaling by oncogenic BRAF (18). We confirmed that inhibi-tion of MET using crizotinib, SGX523, or foretinib in combi-nation with PLX4720 resulted in near-complete inhibition ofERK1/2 phosphorylation and synergistic antiproliferative activ-ity as determined using the Bliss independence model (Sup-plementary Fig. S4; ref. 19). Interestingly, inhibition of SHP2using the tool compounds NSC87877 (20) or PHPS1 (21) incombination with PLX4720 also yielded greater antiprolifera-tive activity than single-agent treatment and greater suppres-sion of ERK1/2 phosphorylation, resulting in modest synergy

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Figure 2.Validation of candidate synthetic lethal genes. A, RKO cells were infected with individual lentiviral shRNA expression vectors targeting high-ranking genesfrom the primary screen or a control shRNA targeting luciferase. Cells were treated with increasing concentrations of PLX4720 for 4 days. Cell proliferationwas determined using the CellTiter-Glo assay. GI50 values were determined using GraphPad Prism. B, RKO cells were infected as in A, then 72 hours afterinfection were treated with 3 mmol/L PLX4720 for 18 hours, and protein lysates were analyzed by Western blotting for the indicated proteins.






(Supplementary Fig. S5). Thus, it seems likely that other RTKs,such as EGFR that has been demonstrated to confer resistanceto BRAF inhibition in colorectal cancer cell lines (4, 22), alsodepend upon SHP2 to signal to the MAPK pathway and driveresistance. We confirmed that combined inhibition of BRAFand EGFR was synergistic in WiDr and SW1417 cells (data notshown).

Near-complete inhibition of ERK1/2 phosphorylationseemed essential to elicit an antiproliferative response in thePLX4720-resistant cell lines. Hence, we sought to examinedrugs that were more likely to extinguish RAF/MEK/ERK sig-naling to this degree. We selected AZ628 for these studies,which is an inhibitor of BRAFV600E, BRAF, and CRAF (ref. 23;a so-called "pan-RAF" inhibitor). Notably, while PLX4720 is

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Figure 3.PLX4720-resistant cells are exquisitely sensitive to combined pan-RAF and MEK inhibition. A, sensitivity of BRAF-mutant melanoma and colorectal cancer cell linestreated with either AZ628 or AZD6244 was assessed following a 96-hour treatment with a 10-point titration of the drugs. CellTiter-Glo data were used togenerate GI50 values. B, RKO and LOXIMVI cells were treated with a titration of AZD6244 in the presence of either DMSO vehicle, 10 nmol/L AZ628, or100nmol/LAZ628 for 96hours. Cell proliferationwas assessedbyCellTiter-Glo. C, RKOand LOXIMVI cellswere treatedwith 300nmol/LAZD6244, 30nmol/LAZ628alone or in combination. Cells were cultured for 7 to 10 days and then stained with crystal violet. Cell proliferation was quantified by measuring theabsorbance of solubilized crystal violet stain.

Whittaker et al.





technically a pan-RAF inhibitor based on enzymatic assays, incells, the functional outcome is selective for mutant BRAFinhibition (14, 23). Alternatively, we reasoned that profoundMAPK pathway inhibition downstream of RAF (e.g., using aMEK inhibitor) might overcome CRAF-mediated resistance.Consistent with this, both melanoma and colorectal cancercell lines were generally more sensitive to AZ628 or the MEKinhibitor AZD6244 than PLX4720, although some lines stillexhibited resistance (Fig. 3A). Therefore, we explored combi-nation strategies using AZ628 and AZD6244 in PLX4720-resis-tant lines. The RKO cell line has a GI50 of 0.5 � 0.04 mmol/L forAZ628 and 4.7 � 0.9 mmol/L for the MEK inhibitor AZD6244(Fig. 3B). However, when 10 nmol/L AZ628 was added to atitration of AZD6244, the GI50 value for AZD6244 droppedconsiderably to 240 � 131 nmol/L, and if the concentration ofAZ628 was increased to 100 nmol/L, the GI50 value furtherdecreased to 25 � 9 nmol/L. Similarly, LOXIMVI cells, whichhave a GI50 value of 8.3 � 3.6 mmol/L for AZ628, weresensitized to AZD6244 when treated with 10 and 100 nmol/L AZ628, resulting in the GI50 decreasing from 8.2� 1.1 mmol/Lto 384 � 129 nmol/L and 128 � 39 nmol/L, respectively.Furthermore, in longer term colony formation assays, treat-ment of RKO and LOXIMVI cell lines with 300 nmol/LAZD6244 and 30 nmol/L AZ628 potently inhibited cell pro-liferation only when used in combination (Fig. 3C).

Next, we investigated the effects of combining AZD6244 andAZ628 on MAPK pathway activity. PLX4720-resistant RKO, LOX-IMVI, and WIDR cells and PLX4720-sensitive A375 andCOLO205 cells were treated with 1 mmol/L AZD6244 or 100nmol/L AZ628 either alone or in combination for 16 hours(Fig. 4A). In the PLX4720-resistant lines, single-agent treatmentfailed to completely inhibit ERK1/2 phosphorylation and did notsuppress the expression of cyclin D1, a MAPK-regulated protein.However, when the two compounds were combined, ERK1/2phosphorylation was completely inhibited and cyclin D1 expres-sion was markedly reduced. Strikingly, treatment with therelatively high concentration of 10 mmol/L PLX4720 did notinhibit either ERK1/2 phosphorylation or cyclin D1 expressionand even in combination with AZD6244 did not appreciablyinhibit MAPK pathway activity. PLX4720-sensitive cell linesshowed good suppression of the MAPK pathway by single agentsand the combination. Notably, the effects on MAPK signalingcorrelated well with the inhibition of cell proliferation (Fig. 4B)whereby in the PLX4720-resistant cell lines, only the combinationof AZD6244 and AZ628 resulted in robust inhibition. Using theBliss independence model, this combination proved to behighly synergistic across a broad range of concentrations and wasgreatest in lines resistant to single-agent treatment (Fig. 4C). Forcomparison, the combination of AZD6244 and PLX4720 did notpotently inhibit cell proliferation, nor did it produce a strongsynergistic effect in PLX4720-resistant lines (SupplementaryFig. S6).

Given that the combination of a pan-RAF inhibitor and a MEKinhibitor could drive a profound response, we sought to establishwhat factors were responsible for this. Accordingly, we screened apanel of melanoma and colorectal cancer cell lines and deter-mined the sum excess over Bliss score across the range of con-centrations tested. Strikingly, we observed that some of the celllines that exhibited resistance to the single agents but weresynergistically inhibited by the combination also had an activat-ing mutation in either KRAS (SW480, SW837, DLD1, HCT116,

HCT15) or loss-of-function mutations in NF1 (LOXIMVI,HCT116, RKO, MeWo; Fig. 5A; ref. 24). All of these events couldpromote elevated RAS signaling, which may confer resistance tothe aforementioned single agents. We did however observe KRAS-and NRAS-mutant cell lines (SW620 and SKMEL2, respectively)that did not display synergy, likely because they were moresensitive to single-agent treatment alone. RAS activity would beexpected to promote CRAF activation (25) and potentially lead tosustained signaling in the presence of a selective RAF inhibitorsuch as PLX4720 (23, 26, 27). In this case, a greater dependencymay exist on CRAF, which would be more efficiently targeted byAZ628 (7).

Therefore, to test the involvement of RAS in the response topan-RAF/MEK inhibition, we introduced KRASG12V or shRNAtargeting the RAS-GAP NF1 into the A375 melanoma cell line,which confer>200-fold or>30-fold resistance toPLX4720, respec-tively (11). The cells were then treated with AZD6244 and AZ628alone or in combination and cell proliferation was assessed after96 hours (Fig. 5B). In the control LacZ or shLuc cells, AZD6244and AZ628 potently inhibited proliferation alone and in combi-nation. In contrast, expression of KRASG12V or knockdown ofNF1conferred resistance to single-agent treatment but retained sensi-tivity to the combination of both drugs. The cells were also treatedas above for 16 hours andMAPK pathway activity was assessed byWestern blotting (Fig. 5C). In the control cells expressing LacZ orshLuc, both inhibitors reduced ERK1/2 phosphorylation anddecreased the expression of cyclin D1. However, cells expressingKRASG12V or shRNA targetingNF1maintained ERK1/2 phosphor-ylation and the expression of cyclin D1 when treated with thesingle agents but were acutely sensitive to the combination ofboth agents, resulting in loss of ERK1/2 phosphorylation andcyclin D1 expression.

Given that the majority of resistance mechanisms to BRAFinhibitors involve either RAS activation by mutation, amplifica-tion, or upstreamactivation ofRTKs,weposited that cells showingacquired resistance to PLX4720 would be sensitive to the com-bination of MEK and pan-RAF inhibition. We generated aPLX4720-resistant cell line by exposing A375 cells to 1 mmol/LPLX4720 for approximately 1month and isolated resistant clones.This A375R10 clonewas 23-fold less sensitive to PLX4720 than itsparental pretreatment A375 cells (Supplementary Fig. S7A) anddisplayed a greater degree of CRAF S338 phosphorylation, amarker of CRAF activation promoted by RAS (28). Furthermore,the levels of phospho-MEK1/2, phospho-ERK1/2, and cyclin D1were far less affected by PLX4720-treatment than compared withthe sensitive, parental cells (Supplementary Fig. S7B). Whileresistant to treatment with single-agent AZD6244 or AZ628, thecombination of these two compounds markedly inhibited pro-liferation of the cells (Supplementary Fig. S7C) and this correlatedwith greater suppression of MAPK pathway activity in theA375R10 cell line (Supplementary Fig. S7D).

To test the possibility that the antiproliferative effect of the drugcombination was due to off-target effects of either inhibitor, wetested combinations of different pan-RAF and MEK inhibitors.The combination of RAF265 (29) and AZD6244, MLN2480 (30)and AZD6244, and of AZ628 and trametinib (GSK1120212;ref. 31) all resulted in highly synergistic activity in lines that wereresistant to the single agents and this again correlated well withnear-complete MAPK pathway inhibition (Supplementary Figs.S8–S10). We also tested the combination of AZD6244 anddabrafenib (GSK2118436; ref. 32) or trametinib and dabrafenib.






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Figure 4.Combinatorial inhibition of BRAF, CRAF, and MEK is synergistic in cell lines displaying intrinsic resistance to PLX4720. A, RKO, LOXIMVI, WiDr, A375, and COLO205cancer cell lineswere treatedwith 1mmol/L AZD6244, 0.1 mmol/L AZ628, 10 mmol/L PLX4720, or a combination of AZD6244with either AZ628 or PLX4720 for 18 hours. Celllysateswere analyzed byWestern blotting for the indicated proteins. B, cell lineswere treated as in A for 96 hours, and cell proliferationwas assessed by CellTiter-Glo assay.C, cells were treated with a titration of AZD6244 versus a titration of either AZ628 or PLX4720 in combination for 96 hours. Cell proliferation was assessed using CellTiter-Glo and presented in the blue–red heatmaps. The green–red heatmaps present the degree of synergistic interaction between the compounds, as determined using the"Bliss Independence model" where values greater than 0 indicate an effect greater than the combined fractional inhibition of the single agents, indicative of synergy.

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Strikingly, while somemodest synergy was observed between theMEK inhibitors and dabrafenib, the concentrations required toachieve a substantial reduction in proliferation were considerablyhigher than AZ628 and the degree of synergy was lower (Supple-mentary Fig. S11A). Interestingly, when we treated RKO cells withAZ628 and an inhibitor of ERK2, VTX-11e (33), little or nosynergy was observed, despite potent inhibition of proliferation(Supplementary Fig. S11B). This is consistent with ERK2 activitybeing the critical integrator of RAS/RAF/MEK upstream signalingand that single-agent ERK2 inhibition may overcome the resis-tance exhibited to single-agent pan-RAF or MEK inhibition. Thisalso underscores the specificity of the pan-RAF/MEK combinationfor its effect on the MAPK pathway. Importantly, we could rescuecells from the combination of AZD6244 andAZ628by expressionof the drug-resistant mutant MEKQ56P (5), which permittedsustained ERK1/2 phosphorylation and cyclin D1 expression inthe presence of the inhibitors (Supplementary Fig. S12A) andrestored cell proliferation (Supplementary Fig. S12B) consistent

withMAPK pathway inhibition being the key effector of responseto the combination.

To further characterize the strong synergistic response pro-duced by the combination of the pan-RAF and MEK inhibitors,we measured proliferation relative to the number of cellspresent at the commencement of treatment. When treated withsingle agents, cells were still able to proliferate, albeit at areduced rate (Fig. 6A). Strikingly, when used in combination,we observed a net loss of cells relative to the starting numberconsistent with cell death. We therefore assessed cytotoxicityusing the Cytotox-Glo assay, which measures protease activityassociated with cell death. The combination of AZ628 andAZD6244 led to significantly increased cytotoxicity comparedwith the single agents (Fig. 6B; Supplementary Fig. S13). Theinduction of apoptosis by the drug combination was furtherconfirmed by the detection of cleaved PARP following a 72-hour exposure to the inhibitors (Fig. 6C). Therefore, whileexposure to single-agent pan-RAF/MEK inhibition was able to

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Figure 5.RAS activation is associated withsensitivity to combined pan-RAF andMEK inhibition. A, synergy betweenAZD6244 and AZ628 was determinedusing the Bliss independencemodel incolorectal and melanoma cell lineswith genetic lesions in BRAF, NF1,KRAS, or NRAS (red dots, mutation;black, wild-type). The sum of excessover Bliss was calculated across thematrix of concentrations tested.Values in excess of 2, signified by thedotted red line, indicate a synergisticresponse. B, A375 cells were infectedwith lentiviral expression vectorsencoding ORFs for LacZ and KRASG12V

or shRNAs for luciferase andNF1. After72 hours, cells were treated with 1mmol/L AZD6244, 0.1 mmol/L AZ628,or a combination of both compoundsfor 96 hours. Cell proliferation wasassessed using CellTiter-Glo. C, A375cells were infected with lentiviralexpression vectors as in B. After 72hours, cells were treatedwith 1 mmol/LAZD6244, 0.1 mmol/L AZ628, or acombination of both compounds for 18hours. Cell lysates were analyzed forthe indicated proteins.






reduce, but not stop, cell proliferation; the combination of bothagents resulted in significant apoptosis.

DiscussionAlthough BRAF is a clear driver of melanoma and colorectal

cancers, attempts at targeting this oncogene have delivered mixedresponses due to intrinsic or acquired resistance mechanisms(34). Our shRNA screening approach identified multiple poten-tial candidates for RAF inhibitor–based combinatorial therapies.Strikingly, our analysis of genes that promote resistance toPLX4720 indicated enrichment for genes within the RTK/MAPKpathway, including MET, ERBB3, NTRK2, GAB1, CRKL, SHP2,SHOC2, MAP2K2, and CRAF. These combinations have clearmechanistic rationales; for example, MET inhibition overcomessignaling induced by overexpression of the MET ligand HGF, asothers and we have observed (18, 35). Similarly, inhibition ofSHP2 is consistent with blocking RTK-mediated signaling thatmay compensate for inhibition of BRAF, through relief of negativefeedback (36, 37). Furthermore, inhibition of SHP2 may havewider use than inhibition of specific RTKs, as multiple RTKs areknown to depend upon SHP2 tomediatemitogenic signaling andhave been implicated in resistance to BRAF inhibition (e.g., EGFR;WIDR, SW1417, and LOXIMVI cells; refs. 4, 22, 38) and MET(RKO cells; refs. 18, 35, 39). However, more potent and selectiveinhibitors of the SHP2 phosphatase are needed before this strat-egy can be tested clinically. We also identified SHOC2, a proteinthat mediates the activation of CRAF by RAS, as a candidate targetto block RTK- or RAS-driven resistance to BRAF inhibition (40,41). Targeting protein–protein interactions with small moleculesis technically challenging but may be an alternative to targetingspecific RAS mutants directly as recently described (42). Interest-ingly, our data also show that loss of CRAF sensitizes cells toinhibition of BRAF, consistent with CRAF being activated byselective RAF inhibitors either through relief of negative feedbackmechanisms, so-called "paradoxical activation" or relief of auto-inhibition (26, 36, 43). Notably, RKO cells have multiple loss-of-function mutations in NF1 and have activated MET due to highexpression of the MET ligand HGF, which could cooperate toincrease RAS activation and promote activation of CRAF. Takentogether, the data suggest that reactivation of the MAPK pathwayin a CRAF-dependent manner plays a major role in the manifes-tation of resistance to BRAF inhibition.

In light of these observations, we targeted the MAPK pathwaywith the combination of a pan-RAF inhibitor and aMEK inhibitorto fully suppress signaling. Combined treatment with AZD6244and AZ628 resulted in a profound synergistic inhibition of cellproliferation. In comparison, the combination of AZD6244 andPLX4720 or trametinib and dabrafenib failed to show strongsynergy. We hypothesized that the synergistic activity of combi-nationswithAZ628was due to inhibition of both BRAF andCRAF(7). Pan-RAF inhibitors such as AZ628 reportedly retain potencyagainst CRAF in cells whereas inhibitors such as PLX4720 do not(23). This is because kinase assays conducted at low ATP con-centrations in vitro suggest that PLX4720 is equipotent againstBRAFV600E and CRAF. However, in the cellular environment withphysiologically relevant ATP concentrations of 1 mmol/L, thepotency of PLX4720 against CRAF is dramatically reducedbecause the Km(app) for ATP is much higher for BRAFV600E thanfor wild-type CRAF (14, 23). Thus, in cells, the wild-type proteinsare relatively resistant to PLX4720 when compared with the

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Figure 6.Pan-RAF and MEK inhibition is cytotoxic and induces apoptosis. A, RKO,WIDR, and HCT116 cells were treated with 1 mmol/L AZD6244,0.1 mmol/L AZ628, or a combination of the two for 96 hours. Cellproliferation was determined relative to the starting number of cells priorto treatment. Genes known to play a role in resistance to BRAFinhibition are indicated in red for each cell line. B, RKO, WIDR, and HCT116cells were treated as in A and, after 72 hours, cytotoxicity wasdetermined relative to the proportion of viable cells using the Cytotox-Gloassay. C, cells were treated as in B and, after 72 hours, cell lysateswere generated and analyzed by Western blotting for thestated proteins.

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BRAFV600E mutant. Furthermore, both PLX4720 and AZ628 canpromote RAF dimerization, but only PLX4720 causes paradoxicalMAPK pathway activation that is only partially reversed by MEKinhibition, consistent with our observation that PLX4720 incombinationwithAZD6244displays no synergy andhas a limitedantiproliferative effect (23, 26). Other mechanisms have recentlybeen describedwherebyMEK is less susceptible toMEK inhibitorswhen activated by CRAF than when it is activated by BRAFV600E

(44). This may also explain why despite being downstream ofboth BRAF and CRAF, the MEK inhibitor AZD6244 was only ableto reduce ERK1/2 phosphorylation by about 50%when used as asingle agent, perhaps also, in part, due to relief of negativefeedback (36). Consistent with this, we have previously shownthat moderate RAS activation by NF1 loss can confer resistance toAZD6244 in BRAF-mutant melanoma (11). Therefore, AZ628inhibits BRAF and CRAF, prevents MEK activation, andmaintainssensitivity to the MEK inhibitor, resulting in greater pathwayinhibition and synergistic effects on proliferation. It should benoted that AZ628, RAF265andMLN2480are all type II inhibitors,whereas PLX4720, vemurafenib, and dabrafenib are type I inhi-bitors. It is conceivable that the inactive confirmation induced bytype II inhibitor binding is less favorable for transactivation ofRAF dimers, as correct positioning of the DFGmotif is required toform the "regulatory hydrophobic spine" as found in the activeconformation (45, 46). Thus, a reduced ability to transactivateRAF dimers translates into more complete pathway inhibitionwith type II inhibitors versus type I inhibitors. Overall, ourcomplimentary pharmacologic and genetic approaches supportthe concept that more effective targeting of wild-type CRAF drivesthe synergy observed between pan-RAF and MEK inhibitors.

Our data show the strongest synergy in cell lines that wereresistant to the single agents and also harbored RAS activatingevents such as mutation of KRAS (SW480, SW837, DLD1,HCT116, HCT15) or mutation/activation of upstream regulatorssuch as NF1 (RKO, LOXIMVI, HCT116, MEWO), MET (RKO), orEGFR (LOXIMVI, WIDR). We confirmed this in A375 cells, maderesistant to RAF or MEK inhibition by the expression of KRASG12V

or knockdown of NF1, where the combination of AZ628 andAZD6244 potently suppressed proliferation. These data furtherdemonstrate the ability of pan-RAF and MEK inhibition totreat common resistance mechanisms to BRAF inhibition(4, 11, 18, 22). Excitingly, the combination of a pan-RAF inhibitorand aMEK inhibitor appears to drive significant apoptosis. This ispotentially transformative in terms of rapidly reducing tumorburden and thus reducing the opportunity for resistant popula-tions to emerge.

This study set out to discover new approaches to treat BRAF-mutant colorectal cancer, but this strategy may also have use inmelanoma and in tumors driven by oncogenic RAS. Our data is in

agreement with recent reports describing synergistic activity fol-lowing combined genetic or pharmacologic suppression of BRAF,CRAF, and MEK1/2 in RAS-mutant contexts (44, 47–49). This isattributed to the inhibition of BRAF and CRAF activities inducedby relief of negative feedback following MEK inhibition. Overall,these studies underline the continued dependence on MAPKsignaling in RAF inhibitor–resistant cancers and nominate poten-tial therapeutic options for patients exhibiting resistance tovemurafenib- or dabrafenib-based therapies. The synergistic activ-ity observed in models of resistance driven by RTK activationsuggests this strategymaybe analternative todual BRAFandEGFRtargeting (50) or triple BRAF, EGFR, and PI3K inhibition, whichhave shown response rates of up to 32% (50, 51). However, thetolerability of pan-RAF inhibitors has been questioned on thebasis of a phase I study of RAF265 that suggested intermittentdosing might be required, although this is possibly due to theexceptionally prolonged half-life of the drug of 11 days (52). Asthe latest generation of pan-RAF inhibitors progress into clinicaltesting (49, 53, 54), it will be important to do so in combinationwith MEK inhibitors to see if such treatment is well tolerated andwhether it yields the striking responses described herein.

Disclosure of Potential Conflicts of InterestL.A. Garraway reports receiving a commercial research grant from Novartis;

has ownership interest (including patents) in Foundation Medicine; and is aconsultant/advisory Board member of Novartis, Foundation Medicine, andBoehringer Ingelheim. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: S.R. Whittaker, L.A. GarrawayDevelopment of methodology: S.R. Whittaker, G.S. Cowley, D.E. RootAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.R. Whittaker, G.S. Cowley, F. Luo, D.E. RootAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.R. Whittaker, F. LuoWriting, review, and/or revision of the manuscript: S.R. Whittaker, S. Wagner,F. Luo, L.A. GarrawayStudy supervision: L.A. Garraway

Grant SupportThis study was supported by grants to L.A. Garraway from the National

Cancer Institute (5P50CA127003-05) STARR Cancer Consortium (I4-A448),the NIH New Innovator Award (DP2OD002750), and the Melanoma ResearchAlliance.

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 February 12, 2015; revised August 5, 2015; accepted August 30,2015; published OnlineFirst September 8, 2015.

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2015;14:2700-2711. Published OnlineFirst September 8, 2015.Mol Cancer Ther Steven R. Whittaker, Glenn S. Cowley, Steve Wagner, et al. Resistance Mechanisms to Selective RAF InhibitorsCombined Pan-RAF and MEK Inhibition Overcomes Multiple

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