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Translational Science

Antitumor Properties of RAF709, a HighlySelective and Potent Inhibitor of RAF KinaseDimers, in Tumors Driven by Mutant RAS or BRAFWenlin Shao1, Yuji M. Mishina1, Yun Feng1, Giordano Caponigro1, Vesselina G. Cooke1,Stacy Rivera1, Yingyun Wang1, Fang Shen1, Joshua M. Korn1, Lesley A. Mathews Griner1,Gisele Nishiguchi2, Alice Rico3, John Tellew4, Jacob R. Haling4, Robert Aversa2,Valery Polyakov3, Richard Zang3, Mohammad Hekmat-Nejad5, Payman Amiri6,Mallika Singh6, Nicholas Keen1, Michael P. Dillon3, Emma Lees1, Savithri Ramurthy3,William R. Sellers1, and Darrin D. Stuart1

Abstract

Resistance to the RAF inhibitor vemurafenib arises commonly inmelanomas driven by the activated BRAF oncogene. Here, we reportantitumor properties of RAF709, a novel ATP-competitive kinaseinhibitor with high potency and selectivity against RAF kinases.RAF709 exhibited a mode of RAF inhibition distinct from RAFmonomer inhibitors such as vemurafenib, showing equal activityagainst both RAF monomers and dimers. As a result, RAF709inhibitedMAPK signaling activity in tumormodels harboring eitherBRAFV600 alterations or mutant N- and KRAS-driven signaling, withminimal paradoxical activation of wild-type RAF. In cell lines andmurine xenograft models, RAF709 demonstrated selective antitu-mor activity in tumor cells harboring BRAF or RAS mutationscompared with cells with wild-type BRAF and RAS genes. RAF709demonstrated a direct pharmacokinetic/pharmacodynamic rela-

tionship in in vivo tumor models harboring KRAS mutation. Fur-thermore, RAF709 elicited regression of primary human tumor–derived xenograft models with BRAF, NRAS, or KRAS mutationswith excellent tolerability. Our results support further developmentof inhibitors like RAF709, which represents a next-generation RAFinhibitor with unique biochemical and cellular properties thatenables antitumor activities in RAS-mutant tumors.

Significance: In an effort to develop RAF inhibitors withthe appropriate pharmacological properties to treat RAS mutanttumors, RAF709, a compoundwithpotency, selectivity, and in vivoproperties, was developed that will allow preclinical therapeutichypothesis testing, but also provide an excellent probe to furtherunravel the complexities of RAF kinase signaling. Cancer Res; 78(6);1537–48. �2018 AACR.

IntroductionThe MAPK signaling pathway, comprised of H/K/N-RAS,

A/B/C-RAF, MEK1/2, and ERK1/2, plays a major role in the

regulation of cellular functions such as cell-cycle regulation,proliferation, survival, and migration. The pathway is activatedby extracellular signals that in turn induces the small G proteinRAS to exchange GDP for GTP, and results in activation of theRAF/MEK/ERK cascade. The MAPK pathway is activated in manyhuman cancers such as those harboring mutations in RAS or RAF.The RAS genes are the most frequently mutated oncogenes in allcancers (reviewed in ref. 1); however, therapeutic targeting of RASproteins has been challenging. Recent studies described smallmolecules that specifically target the KRASG12C mutation throughcovalent inhibition and result in cellular active inhibitors in thelow micromolar range, thus offering a potential to developtherapeutics for KRASG12C-driven tumors (2–4). Inhibitors devel-oped that target downstream effectors of RAS, such as RAF, MEK,and ERK kinases, have not demonstrated significant clinicalactivity in RAS-driven tumors. RAF inhibitors such as vemurafeniband dabrafenib, which are efficacious in melanomas withBRAFV600 mutations, are ineffective in RAS-mutant cancers andinstead induce paradoxical activation in BRAF wild-type cellsthrough transactivation of stabilized RAF dimers (5–7). It hasbeen recently demonstrated that these inhibitors have decreasedaffinity for the secondRAFprotomer due to negative cooperativity(8). A wide array of inhibitors targeting MEK has also beendeveloped with several under clinical evaluation (reviewed inref. 9). Although these inhibitors have demonstrated efficacy inpreclinical RAS-mutant tumor models, they have not shown clear

1Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachu-setts. 2Global Discovery Chemistry, Novartis Institutes for BioMedical Research,Cambridge, Massachusetts. 3Global Discovery Chemistry, Novartis Institutes forBioMedical Research, Emeryville, California. 4Genomics Institute of the NovartisResearch Foundation, San Diego, California. 5Infectious Diseases, NovartisInstitutes for BioMedical Research, Emeryville, California. 6Oncology, NovartisInstitutes for BioMedical Research, Emeryville, California.

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

Current address for W. Shao: iMED Oncology, AstraZeneca, Waltham, Massa-chusetts; current address for Y. Wang and R. Zang: Genentech Inc., South SanFrancisco, California; current address for A. Rico and M.P. Dillon: IDEAYABiosciences, South San Francisco, California; current address for M. Singh:Revolution Medicines, Redwood City, California; current address for N. Keen:Bicycle Therapeutics, Lexington, Massachusetts; current address for E. Lees:Jounce Therapeutics, Cambridge, Massachusetts; and current address for W.R.Sellers: Broad Institute, Cambridge, Massachusetts.

Corresponding Author: Darrin D. Stuart, Novartis Institutes for BioMedicalResearch, 250 Massachusetts Avenue, Cambridge, MA 02139. Phone: 617-871-5311; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2033

�2018 American Association for Cancer Research.

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clinical benefits in patients with tumors harboring RASmutationsas monotherapy, likely due to a narrow therapeutic index andfeedback-mediated pathway reactivation (10).

Emerging biology illustrating how RAS proteins activate theirdownstream targets has provided new opportunities for therapeu-tic development. CRAF (RAF1) was recently demonstrated as thecritical mediator of mutant KRAS-driven cell proliferation andtumor development in both cellular and genetically engineeredmouse tumor models (11, 12). CRAF was also shown to be themediator of feedback-mediated pathway reactivation followingMEK inhibitor treatment in KRAS-mutant cancers (10, 13). Inaddition, CRAF plays an essential role in mediating paradoxicalactivation following BRAF inhibitor treatment (5–7). Thus selec-tive inhibitors that potently inhibit the activity of CRAF could beboth effective in blocking mutant RAS-driven tumorigenesis andalleviating feedback activation. This notion is supported by therecent report of LY3009120, a pan-RAF inhibitor that effectivelyinhibits active RAF homo-and heterodimers and exhibits activitiesin tumors with RAS mutations (14). However, in addition to RAFisoforms, LY3009120 potently inhibits several other kinases,including those that have important biological functions such asEphrin receptors, JNK, p38, and SRC family members. Althoughthe clinical results from LY3009120 have not been published, it isclear from studies with other RAF inhibitors that off-target kinaseinhibition can limit dose escalation. For example, RAF265 andBGB-283 inhibit multiple kinases at concentrations similar to theRAFs and their toxicities are suggestive of receptor tyrosine kinaseinhibition (e.g., thrombocytopenia, hypertension; refs. 15–17).Wehypothesized that a highly selectiveRAFdimer inhibitorwouldbe valuable to assess on-target antitumor activities of this new classof molecules. In this report, we describe the pharmacologic char-acterizationofRAF709, a type2ATP-competitive inhibitorwehavedeveloped that potently inhibits RAF kinases with high selectivity.RAF709 demonstrates equal potency in inhibiting RAFmonomersand dimers, and exhibits inhibition of MAPK signaling in tumormodels harboring BRAF or N/KRAS mutations with minimalparadoxical activation. Correspondingly, RAF709 exhibits greaterantitumor activity in cell line and tumor xenograft models har-boring BRAF or RAS mutations as compared with those that arewild-type. Furthermore, RAF709 in combination with a MEKinhibitor leads to increased antitumor activity in RAS-mutantmodels that are insensitive to RAF709 single agent.

Materials and MethodsRAF in vitro enzyme assays

Enzymatic activities of purified B/CRAF proteins were measuredusing inactive MEK1 protein as a substrate. Substrate phosphoryla-tion was detected using the anti-pMEK1/2 (S217/S221) antibody,AlphaScreen Protein A coated acceptor beads and streptavidincoated donor beads, and read in an EnVision reader. To measurethe inhibitory activity of RAF709, compound was added to theenzyme assay plates with the final concentration from 25 to1.74E-6 mmol/L. Kinase selectivity of RAF709was determined usingtheKINOMEscan screeningplatform (DiscoverX) that quantitativelymeasures interactions between RAF709 and 456 human kinases.

ImmunoprecipitationCells were seeded in 15-cm dishes and incubated with the

indicated concentrations of compound for 1 hour. Cell lysateswere prepared in immunoprecipitation buffer supplementedwith1� protease and 1� phosphatase inhibitor cocktails. Cleared

lysates were normalized for protein concentration and incubatedwith specific antibody overnight at 4�C. Protein AUltra Link Resin(Thermo Scientific)was then added to each sample and incubatedfor 2 hours at 4�C. Resin was washed with immunoprecipitationlysis buffer before bound proteins were eluted in SDS samplebuffer.

Cell proliferationAll cell lines were purchased from commercial sources and

maintained and as described previously (18). Cell lines wereconfirmed to be mycoplasma-free by PCR detection and authen-ticated by SNP genotyping. Cells were seeded in 384-well platesand incubatedwith a 1:3 serial dilutionof compound starting from30 mmol/L for 5 days. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) and readon an Envision (Perkin Elmer) plate reader. Percent growth inhi-bition was calculated by normalizing treatment to DMSO control.IC50 values, where cell growth was inhibited by 50%, were calcu-lated from dose–response curves generated in GraphPad PRISMusing a nonlinear regression 4-parameter curve fitting model.

Combination activityCombination testing was performed using a large-scale com-

pound screening platformasdescribedpreviously (19). Cellswereplated in triplicate into 384-well plates and treated with com-pounds serially diluted 1:3 across a dose range. Following 5 daysof compound treatment, cell viability was determined asdescribed above. Compound combination effects were assessedby weighted "Synergy Scores" calculated using the Loewe doseadditivity model. In addition, isobologram and combinationindex values were generated using the same model to betterinterpret the doses at which the most profound synergy effectswere seen.

In vivo pharmacodynamics and efficacyMice were maintained and handled in accordance with the

Novartis Institutes for BioMedical Research (NIBR) InstitutionalAnimal Care and Use Committee (IACUC) and all studies wereapproved by the NIBR IACUC. Calu-6 and HPAFII tumor xeno-grafts were generated by implanting cells in 50% Matrigel sub-cutaneously into the right flank of female nude mice (6–8 weeksold). For tumor pharmacodynamics measurements, tumor-bear-ingmice were randomized into treatment groups and treatedwithindicated inhibitor. Tumor samples were collected at differenttime points after single dose (n¼ 3/time point) and analyzed forlevels of phospho- and total MEK1/2 using the MesoScale Dis-covery (MSD) platform or DUSP6mRNA by qPCR. For the in vivoefficacy study, mice were randomized into treatment groups.Tumor volume and body weights were collected at the time ofrandomization and twice per week for the study duration. Tumorvolume was determined by measurement with calipers and cal-culated using a modified ellipsoid formula, where tumor volume(TV) (mm3) ¼ [((l � w2) � 3.14159))/6], where l is the longestaxis of the tumor andw is perpendicular to l. The general health ofmice was monitored daily and behavior and well-being weremonitored twice weekly.

Efficacy in primary tumor modelsPatient-derived tumor xenograft models (PDX) models were

established and characterized as described previously (20).Tumor response was determined by comparing tumor volume

Shao et al.

Cancer Res; 78(6) March 15, 2018 Cancer Research1538




change at time measured (t) to its baseline: % tumor volumechange ¼ DVolt ¼ 100% � ((Vt – Vinitial)/Vinitial). The BestRe-sponsewas theminimumvalue ofDVolt for t�10d. For each timet, the average of DVolt from t ¼ 0 to t was also calculated. Wedefined the BestAvgResponse as the minimum value of thisaverage for t� 10 d. This metric captures a combination of speed,depth, and durability of response into a single value.

ResultsDiscovery of RAF709 as a highly selective RAF kinase inhibitor

By leveraging the existing knowledge of targeting RAF andstructure-based rational design, we have developed RAF709, N-(2-methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide, a highlyactive and selective inhibitor of RAF isoforms. In in vitro biochem-ical assays, RAF709 exhibited potent inhibitory activity targetingBRAF, BRAFV600E, and CRAFwith IC50 values ranging between 0.3to 1.5 nmol/L (Fig. 1). Data obtained from the cocrystal structureof RAF709 in complexwith the BRAF kinase domain revealed thatthe protein adopts an inactive conformation with the DFG outand the aC-helix in, characteristic of a type II inhibitor bindingmode (21). Kinase selectivity of RAF709 was evaluated using theKINOMEscan screening platform, which quantitatively measuredthe binding of RAF709 against 456 human kinases and theirmutated derivatives (22). Of the 456 kinases tested, RAF709showed a high degree of selectivity for RAF kinases with onlyBRAF, BRAFV600E, CRAF, and DDR1 demonstrating greater than99% binding at 1 mmol/L (Fig. 1; Supplementary Tables S1 andS2). Three additional kinases, PDGFRB, FRK, and DDR2, showedgreater than 85% binding by RAF709. The remaining 449 kinasesall showed less than 65% binding by RAF709 (SupplementaryTables S1 and S2). While we have not confirmed the potencyagainst these off-target kinases in biochemical or cellular assay,the kinase selectivity of RAF709 was further assessed using the

KiNativ platform, in which the cellular selectivity is determinedby competition with an ATP-competitive covalent probe andread-out by mass spectrometry (23). In HCT116 cells treated for2 hours at 10 mmol/L RAF709, the only kinases inhibited � 80%were BRAF and CRAF, the next most potently inhibited kinaseswere ARAF (47%) and EPHA2 (58%) and of the remaining 253kinases detected, including FRK, none were inhibited >50%(Supplementary Table S3). The apparent inferior binding to ARAFcompared with BRAF and CRAF was difficult to confirm inbiochemical kinase assays due to the low enzymatic activity ofrecombinant purified ARAF; however, the potency was in-linewith the potency against BRAF andCRAF. The KiNativ data furtherconfirm the exquisite selectivity of RAF709 and provide a com-parative dataset to LY3009120, which was evaluated using thesame platform in A375 cell lysates and inhibited 17 kinases >50%at concentrations less than 1 mmol/L (14). The cellular selectivityof RAF709was also evaluated in a Ba/F3 cell panel usingwild-typeor Ba/F3 cells rendered IL3 independent by stably expressing 38different kinase oncogenes (24). Consistentwith on-target activityagainst RAF kinases, RAF709 was active in Ba/F3 cells expressingthe BRAFV600E oncogene with an IC50 of 0.52 mmol/L with littleactivity observed in cells expressing 36 additional kinases (Sup-plementary Table S4). Collectively, these data demonstrated thatRAF709 is an active and highly selective inhibitor targeting theRAF kinases.

RAF709 is an effective inhibitor of BRAF monomers and RAFdimers in BRAFmut and KRASmut tumor cells

The pharmacologic activity of RAF inhibitors in cells expressingdifferent BRAF mutations and mutant RAS have highlighted therole of RAF dimers in signaling and inhibitor sensitivity. BRAFV600

mutants are activated monomers and sensitive to vemurafeniband other RAF inhibitors in this class such as dabrafenib andencorafenib, whereas other activating BRAF mutants function as

Kinase activity inhibition IC50 [µmol/L]

% of Target binding at 1 µmol/L

BRAF 0.0015 99.7

BRAFV600E 0.001 99.9

CRAF 0.0004 99.8

RAF709

Figure 1.

RAF709 exhibits highly selectiveactivity targeting both BRAF andCRAF kinases. Chemical structure ofRAF709. Activity of RAF709 againstBRAF, CRAF, and BRAFV600E

presented as IC50 values measured inin vitro biochemical assays andpercentage of target binding at1 mmol/L of RAF709 in a binding assayas part of the KINOMEscan humankinase panel profiling. RAF709 kinaseselectivity profile is representedon thehuman kinase phylogenetic tree;targets bound are marked with redcircles, with the size of the circlesproportional to percentage of bindingto each kinase.

Antitumor Properties of Novel RAF Inhibitor RAF709

www.aacrjournals.org Cancer Res; 78(6) March 15, 2018 1539




RAS dependent heterodimers with CRAF (8). These inhibitors areinactive against RAF dimers because they have much reducedaffinity for the second protomer when the first protomer isdrug-bound. We used the cellular system developed by Yao andcolleagues to examine the activity of RAF709 in inhibiting RAFmonomers anddimers in tumor cells harboring BRAFV600E (A375)or KRASG13D (HCT116; Fig. 2A; Supplementary Fig. S1). At a

concentration of 1 mmol/L, encorafenib induces maximum acti-vation of pMEK/pERK in HCT116 cells and at this concentrationwe presume that one protomer from each active RAF dimer isoccupied,while the secondprotomer is free tophosphorylateMEK(8). Encorafenib has a very slow-off rate (25) and therefore excessdrug can be washed out while maintaining maximal activationthrough occupancy on the first protomer. Following wash-out,

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Figure 2.

RAF709 is an effective inhibitor of BRAFmonomers and RAF dimers.A,HCT116 (KRASG13D) cells were treatedwith DMSOor 1 mmol/L encorafenib for 1 hour. After thefirst RAF site was occupied by encorafenib, encorafenib was then washed off from the cells and replaced with dabrafenib or RAF709 across a dose rangefor 1 hour. A375 (BRAFV600E) cells were treated with dabrafenib or RAF709 across a dose range for 1 hour. pERK levels were assessed by Western blot analysis(Supplementary Fig. S1) and quantified to generate the dose inhibition curves and IC50 values in A375 and SK-MEL-30 cells, representing the compound inhibition ofBRAFV600E monomer second site and wild-type RAF dimer second site, respectively. Potency of the inhibitor in inhibiting RAF dimer relative to monomer isrepresented as the ratio of dimer second-site pERK IC50 versus monomer second-site pERK IC50. B, A375 cells expressing the doxycycline (Dox)-induciblep61BRAFV600E were maintained in culture media without or with doxycycline for 2 days, then treated with DMSO, dabrafenib, or RAF709 at the concentrationsindicated for 2 hours. Cell lysateswere prepared forWestern blot analysis of protein levels of BRAFV600E, pMEK, and pERK. GAPDHwas included as a loading control.For cell proliferation assays, cells were maintained in culture media without or with doxycycline for 2 days, followed by treatment with dabrafenib or RAF709 for5 additional days across a dose range to determine IC50. C, HCT116 cells were treated with DMSO or RAF709 at indicated concentrations for 1 hour; 1 mmol/Ldabrafenib treatment was included for comparison. BRAF/CRAF dimerization was assessed by immunoprecipitating BRAF or CRAF, followed by Western blotanalysis of BRAF and CRAF. Levels of pMEK and pERK inwhole-cell lysates (WCL)were determined byWestern blot analysis. GAPDH level was included as a loadingcontrol.

Shao et al.





cells were treatedwith a dose range of either dabrafenib or RAF709for an additional hour. Phosphorylation of MEK and ERK weredetermined byWestern blot analysis (Supplementary Fig. S1) andquantified to determine the dose response (Fig. 2A). In A375(BRAFV600E) cells, both dabrafenib and RAF709 showed robustactivity inhibiting mutant BRAF monomer–driven ERK activationwith IC50s of 5 nmol/L and 44 nmol/L, respectively. In compar-ison, in HCT116 cells (KRASG13D) pretreated with encorafenib,RAF709 exhibited equipotent activity inhibiting RAF dimer–driv-en signaling with a pERK IC50 of 79 nmol/L, whereas dabrafenibshowed approximately 100-fold higher IC50 of 3 mmol/L. Thesame experiment was carried out in SK-MEL-30 (NRASQ61K) cellswith similar results: RAF709hadan IC50 of 0.139mmol/L (3.2-foldshift) and dabrafenib had an IC50 of 3.7 mmol/L (>700-fold shift;Supplementary Fig. S2). These data suggest that RAF709 inhibitsboth RAF monomers and dimers with similar potency.

To further test the activity of RAF709 in inhibiting dimerizedRAF, we took advantage of the p61BRAFV600E mutant thatsignals as a constitutive dimer (26) and asked whether RAF709would remain active in cells expressing the p61BRAFV600Emutant.We generated A375 cells expressing p61BRAFV600E in a doxycy-cline-inducible system, and measured the activity of RAF709 ininhibiting pathway signaling and proliferation in the absenceor presence of doxycycline induction (Fig. 2B). The activity ofdabrafenib was significantly reduced by the expression of the p61V600E constitutive dimer, leading to more than 1,000-fold doseresponse shift in both signaling and growth inhibition. In con-trast, RAF709 activity was minimally affected by p61 V600E withonly approximately 5-fold IC50 shift in both pMEK/pERK inhi-bition and inhibition of proliferation. These results furtherstrengthen the hypothesis that RAF709 is a potent inhibitor ofboth RAF monomers and dimers.

We then investigated how RAF709 would affect B/CRAF het-erodimerization and downstream signaling in the KRASG13D

HCT116. B/CRAF dimerization in the cells was assessed byimmunoprecipitating one RAF isoform followed by Westernblotting with the second isoform. Both co-IP studies showed thatRAF709 treatment led to a dose-dependent induction of B/CRAFheterodimerization in HCT116, but inhibited MEK and ERKphosphorylation, in line with the ability of RAF709 to effectivelyinhibit the RAF dimers (Fig. 2C). In contrast, dabrafenib inducedB/CRAF dimerization and increased both MEK and ERK phos-phorylation. These data demonstrate that RAF709 exhibits amode of inhibition distinct from the class of RAF monomerinhibitors. Its activity inhibiting both RAFmonomers and dimerssuggests it should be effective in treating tumors harboring BRAFor RAS mutations.

RAF709 selectively inhibits oncogenic signaling andproliferation in tumor cells with BRAF, NRAS, or KRASmutations with minimal paradoxical activation

Wenext examined the activity of RAF709 and dabrafenib in celllines harboring BRAFV600, NRAS, or KRASmutations (Fig. 3A). InA375 cells, both dabrafenib and RAF709 inhibited MEK and ERKphosphorylation to near completion at 0.05 and 0.5 mmol/L,respectively. In contrast, in the three cell lines harboring eitherNRAS or KRAS mutation, dabrafenib treatment at 0.05 and0.5 mmol/L led to an increase in MEK and ERK phosphorylation,and only at 5 mmol/L showed modest inhibition. In comparison,RAF709 showed dose-dependent inhibition of MEK and ERKphosphorylationwithout apparent pathway activation in all three

RAS-mutantmodels (minimal activationof pMEK in IPC-298 andHCT116). The ability of RAF709 to inhibit pathway signaling wascomparable in cells harboring different RAS mutations and thosewith the BRAFV600mutation, reaching near complete inhibition ofpMEK and pERK at 0.5 mmol/L.

We next determined the antiproliferation activity of RAF709and dabrafenib in these cell lines. In line with the signaling data,dabrafenib showed the most potent antiproliferative activity inBRAFV600E A375 cells (IC50 ¼ 0.003 mmol/L), approximately100-fold less potent in NRASmut IPC-298 cells (IC50 ¼0.27 mmol/L), and very little activity in the two KRASmut cell lines(Calu-6 IC50 ¼ 23 mmol/L, HCT116 IC50 ¼ 17 mmol/L; Fig. 3B).In contrast, RAF709 showed less IC50 dose shift in RASmut modelscompared with the BRAFmut model and exhibited dose-dependent growth inhibition in all cell lines examined, with IC50

values of 0.13 mmol/L, 0.07 mmol/L, 1.4 mmol/L, and 0.98mmol/Lin A375, IPC-298, Calu-6, and HCT116, respectively. We alsoexamined the mechanism by which RAF709 inhibited tumor cellproliferation. Cell-cycle analysis by FACS in both Calu-6 andHCT116 after 48 hours of RAF709 treatment indicated thatRAF709 led to cell-cycle arrest in the G1 phase with a concomitantincrease of cells in sub-G1, indicating cell death (SupplementaryFig. S3A). In both Calu-6 and HCT116 cells, the maximum G1

induction was observed at 1.1 mmol/L with increasing percent ofcell death at�3.3 mmol/L. In agreement with the cell-cycle results,Western blot analysis of Calu-6 and HCT116 cells following 48hours of inhibitor treatment showed inhibition of MEK and ERKphosphorylation, and induction of p27 and cleaved-PARP byRAF709 in a dose-dependent manner (Supplementary Fig. S3B).

To confirm the anticancer activity of RAF709 observed inRASmut cells is mediated by on-target inhibition of the RAF kinasefunction, we expressed a constitutively active variant of MEK1,MEK DD (MEK1 S217D/S221D) under doxycycline regulation inCalu-6 cells, and examined whether MEK DD expression couldrescue RAF709 activity in Calu-6 cells (Fig. 4A and B). WithoutMEK DD expression (�Dox), RAF709 showed dose-dependentinhibition of MEK and ERK phosphorylation and concomitantincrease in cPARP (Fig. 4A). In comparison, RAF709was unable tosuppress constitutively activatedMEK signaling (þDox) up to thehighest concentration tested at 10 mmol/L. Corresponding to thesignaling data, cell growth in the absence of MEK DD inductionwas sensitive to RAF709 inhibition but insensitive in the presenceof MEK DD expression (Fig. 4B). Overexpression of MEK DDalone had no impact on the growth of Calu-6 cells and these datasupport that the activity RAF709 exhibited in RASmut cells ismediated by on-target inhibition of the oncogenic RAF/MEK/ERKsignaling.

RAF709 exhibits increased potency in cancer cell linesharboring BRAF or RAS mutations

We next profiled the antiproliferative activity of RAF709 in abroad panel of genetically characterized human cancer cell linemodels (18). Using an IC50 value of 5 mmol/L based on in vivoexposure (Cav) at well-tolerated doses, we determined thenumber of sensitive (IC50 < 5 mmol/L) and insensitive (IC50 >5 mmol/L) cell lines (Fig. 5A). Cells harboring BRAF, KRAS, orNRASmutations exhibited significantly increased sensitivity com-paredwith those that are wild-type, with P values of 2.41� 10�26,4.08 � 10�4, and 1.73 � 10�7, respectively (Fisher exact test).

Selective sensitivity of RAS- and BRAF-mutant tumor cells wasthe desired pharmacologic profile for RAF709 and we were






encouraged to observe a relative lack of sensitivity of cells withwild-type genotypes. To further evaluate the pharmacologic pro-file, we compared the RAF709 sensitivity profile to RAF and MEKinhibitors that have been evaluated clinically (Fig. 5B; Supple-mentary Table S5). In this analysis, RAF709 profile appeared tomatch best with the highly selective MEK inhibitor trametinib,which showed a similar activity toward BRAF-, KRAS-, and NRAS-mutant cells and with dabrafenib, RAF709 shared sensitivity ofBRAF-mutant cell lines. While the difference in potency betweenRAF709 and dabrafenib is striking, dabrafenib also appears to bemore potent than RAF709 in a biochemical assay using purifiedrecombinant BRAFV600E (IC50 ¼ 1 nmol/L for RAF709 and0.07 nmol/L for dabrafenib; ref. 25) and the relationship betweenbiochemical and cellular potency can be impacted by multiplefactors. Comparing RAF709 to sorafenib and RAF265, both rel-atively nonselective Type II RAF inhibitors is quite revealing.Sorafenib has no activity against RAS- or BRAF-mutant cells andRAF265has amixed profile, with several wild-type cells showing asimilar degree of sensitivity as RAS and BRAFmutants. These dataare consistent with the clinical activity observed for each com-pound: sorafenib's pharmacology is driven mainly by activityagainst angiogenic receptor tyrosine kinases (e.g., VEGFR), andwhile RAF265 demonstrated some activity in patients with

mutant tumors, its activity is limited by what are likely off-targettoxicities (15, 27).

RAF709 demonstrates antitumor activity in tumors harboringRAS mutations in vivo

Following the evaluation of RAF709 activity in human cancercell lines,we investigatedboth signaling inhibition andantitumorefficacy of RAF709 in the KRASmut Calu-6 model in vivo (Fig. 6).Nude mice bearing Calu-6 xenograft tumors were treated with asingle dose of RAF709 across a wide dose range (from 10 to200 mg/kg). Tumor tissues were then collected at multiple timepoints postdose to determine pMEK levels. As shown in Fig. 6A,RAF709 treatment led to inhibition of MEK phosphorylation in adose-dependent manner both in degree and in duration. RAF709at 100mg/kg and 200mg/kgwas able to suppress pMEK to greaterthan 50% formore than 16 hours.We subsequently evaluated theantitumor efficacy of RAF709 in the same tumor xenograftmodel.Tumor-bearing animals were dosed with vehicle, RAF709 at 10,30, 100, or 200 mg/kg, administered orally every day (qd) for 19days. Antitumor activity was determined by assessing percentageof tumor volume in the treatment groups versus that in vehicle-treated (% T/C) or percentage of tumor regression compared withthe starting volume (% regression). In line with pMEK inhibition,

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50

0-3 -2 -1 -0 -1 -3 -2 -1 -0 -1

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Conc. Log [mmol/L] Conc. Log [mmol/L]

Figure 3.

RAF709 inhibits oncogenic signalingand proliferation in tumor cells withBRAF, NRAS, and KRAS mutations,with minimal paradoxical activation.A, Cell lines harboring different BRAFor RAS mutations were treated withDMSO, dabrafenib, or RAF709 at theindicated concentrations for 2 hours.Inhibition of MEK or ERKphosphorylation was measured byWestern blot analysis. B, Growthinhibition of cell lines after 5 days oftreatment by RAF709 (green curve)or dabrafenib (blue curve) wasdetermined and IC50 values arepresented in the table.

Shao et al.





RAF709 treatment resulted in dose-dependent antitumor activitystarting from 30 mg/kg (Fig. 6B). Treatment with 30 mg/kg ofRAF709 led to a 52% T/C, while treatment at 100 and 200 mg/kgresulted in tumor regressions of 47% and 88%, respectively, inline with the more durable pathway inhibition at the two higherdose levels. All treatment groups were well tolerated with nobody weight loss at the end of the efficacy study, no signs oftoxicity ormortality were observed (data not shown). Doses up to300 mg/kg daily were also tolerated in additional studies (datanot shown); however, higher doses resulted in significant bodyweight loss.

To further assess the antitumor activity of RAF709, we per-formed a large-scale in vivo screen of RAF709 efficacy in 23 PDXmodels derived from patient non–small cell lung cancer(NSCLC) tumors. Comprehensive molecular profiling of NSCLCshowed that the RAS/RAF/MEK pathway is frequently altered inthis tumor type, including mutations in KRAS, NRAS, and BRAF(The Cancer Genome Atlas, 2014). We have previously reportedthat the NSCLC PDX models we established showed goodconcordance with clinical samples with respect to the genomiclandscape (20). We therefore used these models to assess clinicalpotential of RAF709 in the treatment of NSCLC. Our screenadopted a "one animal per model per treatment" approach asdescribed by Gao and colleagues to determine RAF709 responseat the population level. Tumor response is presented as awaterfall plot of best average percentage change in tumor volumewith RAF709 treatment, and tumors were annotated for theirmutation status of RAS or BRAF (Fig. 6C). RAF709 dosed at60 mg/kg or 200 mg/kg (!) daily led to tumor growth inhibi-tion in a subset of NSCLC PDX tumors, in which tumors thatharbor mutation of BRAF, NRAS, or KRAS were enriched amongthe better responders. One of the BRAFmut tumors, HLUX1323,harbors a D595N mutation that has been shown to activatesignaling mediated through RAF dimerization (8). For compar-ison, the chemotherapeutic agent paclitaxel dosed at a clinicallyrelevant dose (1.5 mg/kg weekly, i.v.) showed less antitumoractivity across the PDX models, and its activity was not associ-ated with BRAF/RAS mutation. These data further support thatRAF709 demonstrates significant antitumor activity in tumorsdriven by either oncogenic BRAF or RAS.

RAF709 in combination with MEK inhibitor led to enhancedantitumor activity

RAF709 demonstrated significant activity in a subset of RAS-mutant tumors both in vitro and in vivo. To better understand theunderlying biology of response, we examined the activity ofRAF709 in inhibiting the pathway signaling in Calu-6 versus aninsensitive model HPAF-II (IC50 ¼ 14 mmol/L, SupplementaryFig. S4), a pancreatic adenocarcinoma cell line harboring a G12DKRAS mutation. We compared pMEK and pERK levels followingtreatment with RAF709 in both cell lines at 2 and 24 hoursposttreatment. In Calu-6, RAF709 showed strong inhibition ofMEK/ERK phosphorylation at 0.5 mmol/L at both 2 and 24 hours(Fig. 7A). In HPAF-II, RAF709 also showed inhibition of ERKphosphorylation at 0.5 mmol/L at 2 hours; however, it was unableto sustain the level of inhibition up to 24 hours at 0.5 and5 mmol/L. Similar pathway rebound in these cells was observedwith the MEK inhibitor, trametinib, where its ability to inhibitpERK was reduced at 24 hours compared with 2 hours. Pathwayrebound observed with MEK inhibitors in KRAS-mutant cells hasbeen attributed to reactivation of CRAF (10, 13). We thereforehypothesized that combining RAF709with trametinib could leadtomore sustained pathway inhibition inHPAF-II. Supporting thishypothesis, the combination of 0.5 mmol/L RAF709 and1 nmol/Ltrametinib led to a stronger andmore durable inhibition of pERKcompared with either agent alone at the same concentration(Fig. 7A). This combination also led to induction of apoptosisas judgedby the appearance of cleaved PARP (cPARP) at one tenthof the concentration required for either single agent. Followingthis observation, we next determined whether combination treat-ment also led to enhanced antiproliferative activity. Growthinhibition of HPAF-II cells was measured following treatmentwith RAF709 or trametinib as single agent, or with the twoinhibitors in combination, across a wide dose range. Isobolo-grams and synergy scores were generated to assess the combina-tion activity as described previously (19). As shown in Fig. 7B,RAF709 in combination with trametinib had a synergistic effecton inhibiting HPAF-II cell growth, with a synergy score of 11.1.These data suggest that RAF709 antitumor activity in KRAS-mutant tumors could be further enhanced by combining witha MEK inhibitor.

pERK

ERK

Cleaved PARP

GAPDH

MEK

RAF709 [μmol/L]-Dox +Dox

0 103.3

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th

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-dox+dox

Figure 4.

RAF709 demonstrates selectiveanticancer activity in KRASmut NSCLCcells. A, Calu-6 cells expressingdoxycycline-inducible constitutivelyactive MEK (MEK DD) were treatedwith or without doxycycline for 2 days,followed by DMSO or RAF709treatment at indicated concentrationsfor 24 hours. MEK/ERKphosphorylation and PARP cleavagewere determined by Western blotanalysis.B,Growth inhibition of Calu-6cells by RAF709 without (blue curve)or with (red curve) doxycycline (Dox)-inducible expression of MEK DD wasmeasured after 3 days of inhibitortreatment across a dose range. IC50

values are indicated in the table.






Sensitive (#) 41 23 17 20

Resistant (#) 15 77 27 264

2.41E-25 4.08E-04 1.73E-07 1

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WT NRASmut KRASmut BRAFmut

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Figure 5.

RAF709 exhibits greater antiproliferative activity in cancer cell lines harboring BRAF or RAS mutations. A, Dot plots of IC50 values for growth inhibition in352 human cancer cell lines by RAF709 following 3 days of inhibitor treatment. The dotted line represents an IC50 of 5 mmol/L, which was used as acutoff for cell line sensitivity based on in vivo drug exposure from pharmacology studies (Cav � 5 mmol/L at 100 mg/kg). The number of sensitive and resistantcell lines to each inhibitor among BRAFmut, KRASmut, NRASmut, or wild-type (WT) cells is indicated below the graph. A Fisher exact test was performedto determine the statistical significance of inhibitor activity in BRAF- or RAS-mutant cell lines versus WT cell lines. B, The antiproliferative activity ofRAF709 was compared with the selective MEK inhibitor trametinib, selective RAF inhibitor dabrafenib, and the relatively nonselective RAF inhibitorssorafenib and RAF265. As in A, each dot represents a different tumor cell line. Purple dots, BRAF-mutant cells; pink dots, KRAS-mutant cells; green dots,NRAS-mutant cells; and black dots, wild-type RAS and BRAF cells.

Shao et al.





We next assessed whether the combination activity of RAF709and trametinib could be extended in vivo. To evaluate the effect ofthe combination treatment on signaling inhibition versus eitheragent alone, nude mice bearing HPAF-II xenograft tumors weretreated with a single dose of RAF709 at 100 mg/kg, trametinib at0.3 mg/kg or two inhibitors combined. DUSP6mRNA levels, as ameasurement of pathway activity, was determined in tumorsamples collected at multiple time points postdose. As shownin Fig. 7C (left), RAF709 treatment led to 83% inhibition ofDUSP6 at 4 hours postdose compared with vehicle control;however, this inhibition was not durable as demonstrated by theincreased levels of DUSP6 at 16 and 24 hours postdose. Similarly,trametinib treatment led to a partial and transient inhibition of

DUSP6. In contrast, the combination of RAF709 and trametinibled to a more sustained DUSP6 inhibition, showing greater than80% of inhibition even at 16 hours postdose. We next evaluatedantitumor efficacy of the different treatments in the same tumorxenograft model. Tumor-bearing animals were dosed with vehi-cle, RAF709 at 100mg/kg once daily, trametinib at 0.3mg/kg oncedaily, or a combination of both for 10 days. In line with DUSP6inhibition, the combination of RAF709 and trametinib treatmentresulted in greater antitumor activity than either of the singleagents alone, resulting in 33% regression as compared with 40%T/C or 54% T/C by RAF709 or trametinib, respectively (Fig. 7C,center). It should be noted, however, that this combination doseand regimenmay not be tolerated long-term since onemouse had

NSCLC Patient-derived tumor xenograft (PDX) models BRAF GOF Mut KRAS GOF Mut NRAS GOF Mut Wild type

RAF709 Paclitaxel

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Figure 6.

RAF709 demonstrates antitumor activity in tumors harboring RASmutations in vivo.A, Tumor samples were collected at the indicated time points following a singledose of vehicle or increasing doses of RAF709 in the Calu-6 tumor-bearing animals to determine pMEK levels. pMEK levels are represented as the ratio ofpMEK/total MEK in the treatment group compared with vehicle control at each time point. B, Calu-6 tumor xenograft growth inhibition was measured followingtreatment with vehicle or RAF709 across four dose levels. Tumor volume is represented as the mean tumor volume from 6 animals per treatment group� SEM. C, In vivo antitumor activity of RAF709 was assessed in a panel of 23 NSCLC PDX models, presented as the percentage of change in tumor volume at thetime of measurement compared with initial tumor volume. Positive values indicate tumor growth and negative values indicate tumor regression. RAF709 wasdosed orally once daily at 60 mg/kg or 200 mg/kg (!). Each tumor is annotated for BRAF or RAS mutation status. Tumor response to paclitaxel in the samepanel of PDX models was included for comparison.






to be euthanized due to dose-limiting body weight loss and theaverage bodyweight loss in the groupwas 10% (right). These datasupport that combination of RAF709 with MEK inhibitor trame-tinib could lead to more durable pathway inhibition and higherantitumor activity; however, alternative dose schedules will needto be adopted for longer term treatment.

DiscussionDespite the clinical activity demonstrated by RAF and MEK in-

hibitors in BRAFV600-mutant melanoma, these inhibitors are rela-tively ineffective in mutant RAS-driven cancers, tumors in whichthere remains a high unmet clinical need. Recent advances havebeen made in developing drugs against the KRAS G12C–mutantprotein directly through covalent inhibition (3, 4). However, devel-opment of this class of molecules into potential therapeutics would

require further improvement in cellular potency and exploration ofcombinations that could alleviate receptor tyrosine kinase (RTK)activation and/or downstream pathway reactivation. Inhibition ofRAF downstream of mutant RAS has been a long-standing goal;however, RAF inhibitors such as dabrafenib and vemurafenib,induce paradoxical activation in RASmut tumors and this has beenattributed to their reduced affinity for the second site of BRAF-CRAFdimer after occupancy of the first protomer site, leading to increasedRAF dimerization and transactivation (8). The structural basis forthe differential effects of RAF inhibitors has recently been describedby Karoulia and colleagues (28). The model they developed basedpartially on X-ray cocrystal structures with BRAF and a set of ATP-competitive RAF inhibitors, indicates that the pharmacology isdriven by a combination of drug-induced dimerization, which isdependent on the position of the alpha-C helix, and the potenti-ation of RAF/RAS-GTP and RAF/MEK interactions by the inhibitor.

pERK

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]

Growth inhibition (% of untreated)


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709

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]

Isobologram(50% of inhibition)

Loewe synergy score = 11.1


RAF

709

[μm

ol/L

]

Excess inhibition (% of untreated)

Loewe CI at 50% inh = 0.032

0.05

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)

Figure 7.

RAF709 in combination with a MEK inhibitor provides enhanced antitumor activity in KRASmut tumors. A, The ability of RAF709 single agent to inhibit MEK/ERKphosphorylation in Calu-6 and HPAF-II, and in combination with trametinib in HPAF-II, was assessed by Western blot analysis following 2- and 24-hourtreatment. Cleaved PARPwasmeasured as amarker for cell death. B,Antiproliferative combination activity of RAF709 and trametinib in HPAF-II cells was assessed.Left, dose matrix representing percentages of growth inhibition relative to DMSO by RAF709, trametinib, and the combination following 5 days of treatment.Middle, excess inhibition values representing the deviation between the combination effect and the calculated additivity effect of the two single agents usingthe Loewe model. The calculated Loewe synergy score is indicated. Right, isobologram analysis of the dose matrix data. Blue line, data points; red line, additivity.The calculated Loewe combination index (CI) at 50% growth inhibition is indicated. C, In vivo activity of RAF709 and trametinib as single agents or incombination in the HPAF-II xenograft model. Left, signaling inhibition following a single dose of the treatment as measured by the DUSP6 mRNA levels.DUSP6 levels are represented as the percentage change in comparison with the vehicle group after normalization to a control gene RPLPO. In vivo tumorgrowth (center) and tolerability (right) following 10 days of treatment (n ¼ 6 mice/group); treatment groups and activity are indicated.

Shao et al.





The development of so-called pan-RAF inhibitors with the goalof treating RAS-mutant tumors has been described in two recentreports; however, in addition to inhibiting RAF isoforms, thesecompounds also exhibit activity against many other kinases. Forexample, the ability to target SRC signaling was attributed to theantitumor efficacy of CCT196969 and CCT241161 in drug-resis-tant BRAFmut melanomas with upregulated RTK signaling (29).However, as with LY3009120, RAF265, BGB-283, and sorafenib,the spectrumof off-target activities of these inhibitorsmay impacttheir ability to potently inhibit RAF at tolerated doses. To addressthe need for a highly selective RAF inhibitor with the ability toinhibit pathway signaling in RASmut tumors, we have developedRAF709, an inhibitor with low to subnanomolar potency target-ing RAF isoforms and a high level of selectivity against otherkinases. Similar to the pan-RAF inhibitor LY300912, RAF709exhibits a type II binding mode in the RAF ATP pocket with theDFG loop out and aC helix-in conformation. Consistent withother ATP-competitive inhibitors, RAF709 stabilizes B/CRAFdimerization; however, unlike other RAF inhibitors, it demon-strates equipotency at inhibiting both RAFmonomer- and dimer-driven signaling in cellular assays. The ability of RAF709 toeffectively inhibit RAF dimer activity leads to its minimal para-doxical activation and antitumor activity in not only BRAFV600 butalso RASmut tumors. RAF709 demonstrates enriched activity inBRAF- and RAS-mutant tumors in a large in vitro cancer cell linepanel and in in vivo PDX screens.

In addition to the most prevalent BRAF V600 mutations, otherBRAFmutations havebeen identified across different tumor types.Studies of NSCLC clinical samples revealed that non-V600 BRAFmutations represented 50%–75%of all BRAFmutations (30, 31).These non-V600 mutations have been experimentally character-ized as activating or kinase-impaired, and all could activatepathway signaling in a CRAF dimerization–dependent manner(8, 32, 33). The ability of RAF709 to inhibit RAF dimers suggeststhat it would be active in tumors that harbor these BRAF muta-tions. Indeed RAF709 has shown antiproliferative activity incancer cell lines that harbor non-V600 mutation with or withouta cooccurring KRAS mutation (Supplementary Table S5), includ-ing Hey-A8 (KRASG12D/BRAFG464E, IC50 ¼ 0.63 mmol/L),MDA-MB-231 (KRASG13D/BRAFG464V, IC50 ¼ 3.4 mmol/L), andNCI-H1666 (BRAFG466V, IC50 ¼ 3.97 mmol/L). In comparison,the BRAF monomer inhibitor dabrafenib had IC50>30 mmol/L inall three cell line models. In addition, RAF709 showed in vivoantitumor activity in HLUX1323, a NSCLC PDX model thatharbors BRAFD594Nmutation, leading to 26% of tumor shrinkage(Fig. 6). Our data suggest that RAF709 could be effective for thetreatment of tumors driven by atypical BRAF mutations that donot respond to the current BRAFV600 inhibitors.

Data fromour cell line and PDXpanels suggest that tumors withBRAF or RAS mutations are more sensitive to RAF709 treatment.Within these molecular subtypes, however, sustained pathwayinhibition is required for optimal antitumor activity. This hypoth-esis is supported by our findings that combination of RAF709 andtrametinib led tomore durable pathway inhibition and anticanceractivity comparedwith either agent alone. Thesedata are consistentwith reports from the functional genomic screens that demonstrat-ed that targeting CRAF could sensitize response to MEK inhibitorsin KRAS-mutant tumors (10, 13). In addition to targetingmultiplesignaling nodes within the MAPK pathway tomaximize both leveland duration of signaling inhibition, combining RAF709 withinhibitors targeting the upstream RTK activation such as EGFR

(34, 35), bypass mechanisms such as YAP1 (36) or targetingprosurvival BCL2 family members (37), may further enhanceefficacy and reduce the development of drug resistance.

Thus, we have developed a highly selective pan-RAF inhibitorthat is equipotent at inhibiting RAF monomers and dimers. As aresult, RAF709 demonstrates antitumor activity in both BRAF-and RAS-mutant tumors. RAF709 is orally bioavailable andexhibits a pharmacodynamic/efficacy relationship, which indi-cates that sustained target inhibition leads to tumor regressionin vivo. The activity of RAF709 in KRASmut tumors could be furtherenhanced by combination with a MEK inhibitor. The mousexenograft studies described here provide only a rough estimateof the therapeutic index of RAF709 and detailed histopathologicstudies have not been conducted. However, highly selective RAFdimer inhibitors like RAF709 provide the opportunity to effec-tively test the therapeutic potential of RAF inhibition in atypicalBRAF-mutant and RAS-mutant tumors, without the risk of inhi-biting off-target kinases as observed with previous inhibitors inthis class. Ongoing clinical trials with LXH254, which is structur-ally related to RAF709, will answer this critical question.

Disclosure of Potential Conflicts of InterestN. Keen is a chief scientific officer and has a commercial research grant from

Bicycle Therapeutics Ltd. M.P. Dillon has ownership interest (including patents)in Novartis. W.R. Sellers has ownership interest (including patents) in NovartisPharmaceuticals and Peloton Pharmaceuticals, is a consultant/advisory boardmember for Sanofi Pharmaceuticals, Servier Pharmaceuticals, Astex Pharmaceu-ticals,Merck-SeronoPharmaceuticals, and Ideya Pharmaceuticals. D.D. Stuart is adirector at Novartis and has ownership interest (including patents) in Novartis.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: W. Shao, Y. Feng, G. Nishiguchi, J. Tellew, J. Haling,V.R. Polyakov, R. Zang, P. Amiri, M. Singh, N. Keen, M.P. Dillon, E. Lees,S. Ramurthy, D.D. StuartDevelopment of methodology: Y.M. Mishina, Y. Feng, J. Haling, R. Aversa,R. Zang, P. Amiri, D.D. StuartAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Feng, V.G. Cooke, S. Rivera, Y. Wang, F. Shen,L.A. Mathews Griner, R. Zang, M. Singh, S. Ramurthy, D.D. StuartAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Shao, Y.M. Mishina, Y. Feng, G. Caponigro,Y. Wang, F. Shen, J.M. Korn, L.A. Mathews Griner, J. Haling, V.R. Polyakov,R. Zang, M. Hekmat-Nejad, P. Amiri, M. Singh, N. Keen, M.P. Dillon, E. Lees,S. Ramurthy, D.D. StuartWriting, review, and/or revision of the manuscript: W. Shao, Y.M. Mishina,Y. Feng, G. Caponigro, V.G. Cooke, A. Rico, J. Haling, V.R. Polyakov, M. Singh,E. Lees, W.R. Sellers, D.D. StuartAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.M. Mishina, Y. Feng, G. Caponigro, S. Rivera,J. Tellew, R. Zang, M. Singh, S. Ramurthy, D.D. StuartStudy supervision:W.Shao, V.G.Cooke, R. Zang,M. Singh, E. Lees, S. Ramurthy,W.R. Sellers, D.D. Stuart

AcknowledgmentsThe authors thank Brent Appleton, Ann Van Abbema, Laura Tandeske,

Hanne Merritt, Sylvia Ma, and Hanneke Jansen for scientific and technicalsupport during the early discovery phase of this project. We would also like toacknowledge Novartis Institutes for Biomedical Research for funding.

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 July 7, 2017; revisedNovember 22, 2017; accepted January 11, 2018;published OnlineFirst January 17, 2018.






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Shao et al.




2018;78:1537-1548. Published OnlineFirst January 17, 2018.Cancer Res Wenlin Shao, Yuji M. Mishina, Yun Feng, et al. BRAF

orInhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS Antitumor Properties of RAF709, a Highly Selective and Potent

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