Dual MAPK Inhibition Is an Effective Therapeutic Strategy ... · each mouse prior to imaging, 50 mL of luciferin was injected intraperitoneally. Quantiﬁcation of signal intensity

Translational Cancer Mechanisms and Therapy

Dual MAPK Inhibition Is an Effective TherapeuticStrategy for a Subset of Class II BRAF MutantMelanomasMatthew Dankner1,2, Mathieu Lajoie1,3, Dan Moldoveanu1,4, Tan-Trieu Nguyen1,3,Paul Savage1,2, Shivshankari Rajkumar1,3, Xiu Huang5, Maria Lvova5, Alexei Protopopov5,DanaVuzman5,6,7, DavidHogg8,MoragPark1,2,3,Marie-ChristineGuiot9,10, KevinPetrecca9,Catalin Mihalcioiu11, Ian R.Watson1,3, Peter M. Siegel1,2,3, and April A.N. Rose12

Abstract

Purpose: Dual MAPK pathway inhibition (dMAPKi) withBRAF andMEK inhibitors improves survival in BRAF V600E/Kmutant melanoma, but the efficacy of dMAPKi in non-V600BRAF mutant tumors is poorly understood. We sought tocharacterize the responsiveness of class II (enhanced kinaseactivity, dimerization dependent) BRAF mutant melanoma todMAPKi.

Experimental Design: Tumors from patients with BRAFwild-type (WT), V600E (class I), and L597S (class II)metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of mel-anoma cell lines with class IIa (activation segment) or IIb(p-loop) mutations and compared these with WT orV600E/K BRAF mutant cells. Cell lines and PDXs weretreated with BRAFi (vemurafenib, dabrafenib, encorafenib,and LY3009120), MEKi (cobimetinib, trametinib, andbinimetinib), or the combination. We identified 2 patients

with BRAF L597S metastatic melanoma who were treatedwith dMAPKi.

Results: BRAFi impairedMAPK signaling and cell growth inclass I and II BRAF mutant cells. dMAPKi was more effectivethan either singleMAPKi at inhibiting cell growth in all class IIBRAF mutant cells tested. dMAPKi caused tumor regression intwo melanoma PDXs with class II BRAF mutations and pro-longed survival of mice with class II BRAF mutant melanomabrain metastases. Two patients with BRAF L597S mutantmelanoma clinically responded to dMAPKi.

Conclusions: Class II BRAF mutant melanoma is growthinhibited by dMAPKi. Responses to dMAPKi have beenobserved in 2 patients with class II BRAF mutant melanoma.These data provide rationale for clinical investigation ofdMAPKi in patients with class II BRAF mutant metastaticmelanoma. Clin Cancer Res; 24(24); 6483–94. �2018 AACR.

See related commentary by Johnson and Dahlman, p. 6107

IntroductionBRAF is a constituent of theMAPK signaling pathway and is one

of themost commonlymutated oncogenes in human tumors (1).

The most prevalent BRAF mutations occur at codon V600, con-stitutively activating BRAF's kinase domain and enhancingMAPKsignaling (2). Given the importance of this hyperactivated path-way in cancer, several MAPK inhibitors have been developed fortargeted treatment of V600 BRAFmutant tumors, including BRAFinhibitors (BRAFi; vemurafenib, dabrafenib, and encorafenib)and MEK inhibitors (MEKi; cobimetinib, trametinib, and bini-metinib; refs. 3, 4). BRAFi and MEKi used as single agents, or incombination, have been shown to improve survival in BRAFV600mutant melanoma and non–small cell lung cancer (NSCLC;refs. 4–6).

Data from large-scale sequencing efforts have identifiedmany additional hotspot BRAF mutations existing outside ofthe V600 codon (1, 7). Recently, a new classification system ofBRAF mutations has been proposed (8, 9). V600 mutations arereferred to as class I BRAF mutations and signal constitutively asRAS-independent monomers. Class II mutations are also BRAF-activating, but signal as RAS-independent dimers (9–11). Here-in, we draw a distinction between class II BRAF mutationsbased on their location; class IIa mutations occur within theactivation segment (i.e., L597, K601), and class IIb mutationsoccur within the glycine-rich p-loop (i.e., G464, G469; Fig. 1A).Class III is comprised of "low activity" or kinase-dead BRAFmutations (9, 12).

It has been previously reported that only tumors with class IBRAF mutations are sensitive to approved BRAFi (10). However,

1Goodman Cancer Research Centre, McGill University, Montr�eal, Qu�ebec,Canada. 2Department of Medicine, McGill University, Montr�eal, Qu�ebec, Canada.3Department of Biochemistry, McGill University, Montr�eal, Qu�ebec, Canada.4Department of General Surgery, McGill University, Montr�eal, Qu�ebec, Canada.5KEW Inc., Cambridge, Massachusetts. 6Division of Genetics, Department ofMedicine, Brigham and Women's Hospital and Harvard Medical School, Boston,Massachusetts. 7Broad Institute of Harvard and MIT, Cambridge, Massachusetts.8Princess Margaret Cancer Centre, Toronto, Ontario, Canada. 9Department ofNeurology and Neurosurgery, McGill University, Montr�eal, Qu�ebec, Canada.10Department of Pathology, McGill University, Montr�eal, Qu�ebec, Canada.11McGill University Health Centre, McGill University, Montr�eal, Qu�ebec, Canada.12Division of Medical Oncology, Department of Medicine, University of Toronto,Toronto, Ontario, Canada.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: April A.N. Rose, University of Toronto, Princess Mar-garet Cancer Center, OPG Building, 700 University Avenue, Work Station7W460, Toronto, Ontario M5G 1Z5, Canada. Phone: 514-862-6423; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-3384

�2018 American Association for Cancer Research.

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several other studies report that cell lines endogenously expres-sing non-V600 BRAFmutants are sensitive to BRAFi (13–15). Thisevidence, combined with case reports of patients with BRAF non–V600-expressing tumors responding to BRAFi suggests that theestablished paradigm for non-V600BRAFmutantsmay be incom-plete (8, 13, 16, 17).

In this study, we use cell lines and patient-derived xenograft(PDX) models and report on clinical responses in 2 patientsto demonstrate that dual MAPK pathway inhibition (dMAPKi)with approved BRAFi þ MEKi is an effective therapeutic strat-egy for some patients with class II BRAF mutant melanoma.These results provide the rationale for clinical trials to assessthe efficacy of dMAPKi in these patients.

Materials and MethodsSequencing of patient samples and PDX models

A next-generation sequencing–based test was performed bythe CANCERPLEX assay (18). The CANCERPLEX data analysispipeline was applied to report single-nucleotide variants, inser-tions, deletions, structural variants, and copy-number varia-tions. For each patient tumor, the reported mutations in theprimary metastatic tumor sample and the PDX sample wereintersected to identify common variants. Variant allele frequen-cies (VAF) were compared and plotted using R (www.R-project.org). Variants of interest were manually reviewed in BAM filesusing IGV (19).

Cell growth assaysFor long-term growth assays, cells were seeded into 12-well

plates and treated with inhibitors at the following concentrationsfor 10- and 15-day assays, respectively: vemurafenib (1,500 and2,000 nmol/L), dabrafenib (150 and 300 nmol/L), encorafenib(150 and 300 nmol/L), LY3009120 (100 nmol/L), cobimetinib(25 nmol/L), trametinib (5 nmol/L), and binimetinib (50 and100 nmol/L). Media with drug were replaced every 4 to 5 days. Atexperimental endpoint (10 days for Fig. 2A; Supplementary Fig.S1C, 15 days for Fig. 2B and C), cells were fixed in 10% formalin,incubated in crystal violet (Sigma-Aldrich; Cat # HT90132-1L),and washed in water. Five representative images were taken ofeach well and quantified using Scion Image Software. Positivepixel count was acquired from these images, representing the areacovered by tumor cells. Experiments were repeated in 3 wells perexperiment and performed in triplicate for a total of 9 wells.

In vivo experimentsFor subcutaneous tumor growth experiments, 5 � 105 tumor

cells were injected bilaterally. For cranial tumor growth experi-ments, 1 � 105 tumor cells were injected into the right frontallobe using a guide screw technique (20). All in vivo subcuta-neous and cranial PDX experiments were performed withpassage 2 or earlier, or passage 5 or earlier, respectively. Forsubcutaneous xenografts, tumors were measured with calipers(ASICSA; cat # 19600). For brain metastasis measurements,lesions were measured with IVIS Spectrum (Perkin Elmer). Foreach mouse prior to imaging, 50 mL of luciferin was injectedintraperitoneally. Quantification of signal intensity was per-formed with Living Image software. For cranial injection experi-ments, treatment was initiated when all mice exhibited cleardetectable lesions by IVIS Spectrum imaging. Mice were treatedby daily oral gavage with vehicle of hydroxypropyl methylcel-lulose, trametinib (LC Laboratories T-8123) at 0.5 mg/kgmouse body weight, dabrafenib at 25 or 50 mg/kg, as indicatedin the figure legends (LC Laboratories D-5699), encorafenib(Array Biopharma) at 75 mg/kg, and binimetinib (Array Bio-pharma) at 15 mg/kg. All animal studies and protocols werepreapproved by the McGill Comparative Medicine and AnimalResources Centre.

Patient informationPatient clinical information and tissue were received after

obtaining written-informed consent from patients in accordancewith the Declaration of Helsinki and after studies were approvedby an Institutional Review Board.

ResultsClassifying BRAF mutations in melanoma

To assess the prevalence of class II mutations across tumortypes, we accessed the AACR GENIE project (21). Within thisdataset, among tumor types with at least 5 BRAF mutant tumorspresent, the prevalence of BRAF mutations varied substantiallyfrom 0.4% in breast cancer to 40.4% melanoma (Fig. 1B).Among melanoma samples, class I mutations comprised65.9% of all BRAF mutations, whereas class II and III comprised11.4% and 9.5%, respectively (Fig. 1C). A further 13.2% weremutants of unknown function that did not belong to any of thethree classes. A similar distribution of class II and III BRAFmutations were observed in The Cancer Genome Atlas (TCGA)melanoma dataset (22). Class II mutations occurred within theactivation segment (i.e., L597, K601; class IIa) and in theglycine-rich P-loop (i.e., G464, G469; class IIb; Fig. 1A). Anadditional subset of class II mutations is comprised of BRAFfusions (class IIc) that have also been reported to signal asRAS-independent BRAF dimers (10, 23, 24). All non-V600mutations identified in the AACR GENIE dataset with knownfunction are indicated in Supplementary Table S1.

It has been reported that class III BRAF mutations are com-monly associated with RAS mutations in melanoma (9). Indeed,we found that 47% of class III mutant melanoma within theGENIE dataset coexpressed activating RAS mutations (Supple-mentary Table S1; Fig. 1D). In contrast, we found that class IImutant tumors were similar to class I mutant tumors, in that theyrarely coexpressed activating RAS mutations (2.8% and 1.4%,respectively). These data support the notion that BRAF class IImutations, like class I mutations, are kinase activating in a RAS-independent manner.

Translational Relevance

Class II BRAFmutations are commonly recurringmutationsthat confer enhanced BRAF activity andMAPK pathway hyper-activation akin to class I (V600E/K) mutations. In this study,we use various melanoma cell lines and patient-derived xeno-graft models that endogenously express class II BRAF muta-tions to demonstrate that these tumors are indeed sensitive totargeted therapy with dual BRAF þ MEK inhibition. Further-more, we present data on 2 melanoma patients with class IIBRAF mutations that achieved objective clinical responses toBRAF þ MEK inhibition. This represents a viable therapeuticstrategy for this emerging subgroup of patients and warrantsfurther investigation in clinical trials.

Dankner et al.

Clin Cancer Res; 24(24) December 15, 2018 Clinical Cancer Research6484



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In datasets published before the widespread approval ofBRAFi and MEKi, melanoma patients with BRAF V600 muta-tions who did not receive MAPKi had worse prognosis thanthose with BRAF wild-type (WT) tumors (25). We asked wheth-er melanoma patients with other, potentially targetable muta-tions also experienced poor prognosis. Indeed, metastatic mel-anoma patients with class II/III and/or NRAS mutations in theTCGA dataset experienced inferior overall survival comparedwith patients with class I mutations (Fig. 1E). The improvedsurvival of melanoma patients with class I BRAF mutations dueto the development of targeted therapies highlights the need for

the identification of similarly effective targeted therapy strate-gies for patients with class II/III BRAF mutant and NRASmutantmelanoma (4).

Development and characterization of WT, class I, and class IIBRAF mutant PDX models

We established PDXs from 4 patients with metastatic melano-ma, including 2 with class II BRAF mutations (both BRAFL597S; Fig. 3A). All PDXs retained similar genomic landscapescompared with the tumor from which they were derived. Geno-mic analyses included copy-number alterations (CNA; Fig. 3B;

0

10

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K601 (n = 13) D594 (n = 11)

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Figure 1.

Classification of BRAF mutations in cancer. A, Lollipop plot from the AACR GENIE tumor sequencing dataset representing the incidence of BRAF mutations foundin melanoma samples (n ¼ 785). Class IIa and IIb mutations are indicated in blue and purple, respectively, and class III mutations are indicated in red. B, Incidenceof BRAF mutations in different tumor types in the AACR GENIE dataset. Only those cancer types with greater than 5 samples harboring BRAF mutations wereincluded in this analysis: melanoma (n¼ 785), thyroid (n¼ 410), histiocytosis (n¼ 24), small bowel (n¼ 69), colorectal (n¼ 2081), gastrointestinal neuroendocrine(n ¼ 92), carcinoma of unknown primary (CUP; n ¼ 367), NSCLC (n ¼ 2985), non-melanoma skin cancer (n ¼ 198), endometrial (n ¼ 552), cervical (n ¼ 148),leukemia (n ¼ 344), bladder (n ¼ 638), non-Hodgkin lymphoma (NHL; n ¼ 189), glioma (n ¼ 977), pancreatic (n ¼ 455), ovarian (n ¼ 934), prostate (n ¼ 752),hepatobiliary (n ¼ 386), esophagogastric (n ¼ 528), soft tissue sarcoma (n ¼ 635), and breast (n ¼ 2193). C, Prevalence of BRAF mutation classes amongBRAF mutant tumors in common cancer types in the AACR GENIE tumor sequencing dataset: melanoma (n ¼ 317), colorectal (n ¼ 230), and NSCLC (n ¼ 162).D, Cooccurrence of RAS-activating mutations with different BRAF mutant classes in the AACR GENIE tumor sequencing dataset melanoma cohort.E, Survival analysis of metastatic melanoma patients whose tumors expressed BRAF wild type (WT)/NRAS WT (n ¼ 88), BRAF class I V600E/K (n ¼ 149),and class II/III and/or NRAS mutant (mt; n ¼ 101). In comparison between BRAF class I V600E/K and BRAF class II/III and/or NRAS mt; P ¼ 0.021, HR: 1.49, 95%confidence interval, 1.06–2.09. Data were obtained from updated survival analysis of the melanoma The Cancer Genome Atlas (TCGA) dataset.

Dual MAPK Inhibition for Class II BRAF Mutations

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Supplementary Fig. S2A), somatic missense variants (Fig. 3Cand D), and VAFs (Supplementary Fig. S2B). An exception tothis trend was the expected discrepancy in the CNA and VAFbetween the clinical specimen and GCRC2073 PDX (Supplemen-tary Fig. S2A and S2B). This was due to a low purity of the patientsample that can be seen in the representative hematoxylin andeosin (H&E) from this specimen (Fig. 3E). Importantly, theknown driver mutations that result in gain (BRAF, NRAS, RET)or loss of function (PTEN, ARID2, and CDKN2A) were conservedin the corresponding PDXmodels (Fig. 3D). Immunohistochem-ical staining of three PDX models and corresponding patienttissues revealed that PDXs maintain similar expression of mela-noma markers (Melan-A, BRAF V600E, HMB-45) compared withtheir tumor of origin (Fig. 3E). Taken together, these profilesdemonstrate the high fidelity of these PDX model systems to themetastatic tumor from which they were derived.

We also obtained a variety of cancer cell lines bearingWT, class Imutant, and class IImutant BRAF. Among these cell lines andPDXmodels, cooccurring RAS mutations were present in 2 of 7melanoma cell lines that expressed class II BRAF mutations(Supplementary Table S2). This is consistent with the notion thatactivating RASmutations are commonly found in class III but lessfrequently in class I or class II BRAF mutant melanomas (Fig. 1C;ref. 9). Both class II BRAF mutant melanoma cell lines thatcoexpressed activating RAS mutations were of the class IIb type.Class IIb–activating mutations have been reported to enhancemutant BRAF:CRAF dimerization (26), and therefore, the pres-ence of an activating RAS mutation may facilitate their signalingcapacity in this manner.

Class II BRAFmutant cancer cells respond to single-agent BRAFior MEKi

Clinically indicated BRAFi, such as vemurafenib and dabrafe-nib, cause paradoxical activation of the MAPK pathway in cellswith WT BRAF (27, 28), but it is unclear whether the same is truefor class II BRAF mutant tumors (7, 10, 11). Therefore, we usedcells derived from the aforementioned PDX and cell line models(Supplementary Table S2) to determine whether cells with class IImutations are responsive to BRAFi or MEKi in vitro.

Short-term treatment with MEKi (cobimetinib and trameti-nib) universally inhibited the MAPK pathway, irrespective ofBRAF class (Fig. 2A; Supplementary Fig. S3A). Short-termtreatment with BRAFi (vemurafenib, dabrafenib) induces par-adoxical activation of the MAPK pathway in BRAF WT cells,whereas class I (BRAF V600) and IIa (K601E and L597S)mutant cells exhibit marked inhibition of the MAPK pathway(Fig. 2B; Supplementary Fig. S3A). In contrast, the class IIbmutant cancer cells tested were neither paradoxically activatednor inhibited by single-agent BRAFi (Fig. 2B). This result high-lights the marked difference between class IIa and class IIb cellswith respect to their biochemical response to BRAFi. Thesedifferences may be based on the location of the mutationwithin the BRAF protein or by the RAS mutation status of thecell lines tested (Supplementary Table S2).

One of the key determinants of BRAFi efficacy is the speed ofpERK recovery following drug treatment (10). We sought tocompare the dynamics of pERK recovery between melanomacells of different BRAF mutant classes treated with physiolog-ically relevant doses of encorafenib. Encorafenib is an emergingBRAFi that is a promising candidate to become a front-linetargeted therapy for class I BRAF mutant melanoma (3). Cells

were treated with encorafenib for 1 hour, washed with drug-freemedia, and then lysed at defined time points after washout. InWM3918 BRAF WT cells, we observe paradoxical activation ofthe MAPK pathway at 1 hour on treatment, which returns tobaseline levels within minutes after treatment (Fig. 2C). InA375 class I BRAF mutant melanoma cells, we observe strongpERK inhibition that recovers to baseline levels by 8 hours afterremoval of drug. In class IIa mutant cells, pERK levels returnedto baseline at earlier time points (1–2 hours) following drugremoval. By contrast, pERK levels in class IIb HMV-II and M619melanoma cells are not significantly decreased by encorafenibafter 1 hour of treatment. These data indicate that class I and, toa lesser extent, class IIa mutant BRAF dimers are effectivelyinhibited by single-agent encorafenib, whereas WT and class IIbBRAF dimers are not.

Another important indicator of MAPKi efficacy is the extentto which the inhibitory signal is propagated to downstreameffector molecules. Such signals include cell-cycle regulatorssuch as Cyclin D1 (CCND1; ref. 29), which in turn phosphor-ylates the retinoblastoma (Rb) tumor-suppressor protein topromote cell survival and proliferation (30). In addition tothese transcriptionally regulated targets of the MAPK pathway,ERK is itself a kinase that phosphorylates and stabilizes anumber of effector proteins with critical functions, includingFRA-1 (31).

We examined the effects of either MEKi (trametinib; Fig. 2D)or BRAFi (encorafenib; Fig. 2E) on these downstream effectorsof the MAPK pathway. Trametinib inhibited phosphorylationof ERK, FRA-1, and Rb. Total levels of CCND1, FRA-1, and Rbwere also diminished in all cell lines treated with trametinib.Encorafenib similarly inhibited these downstream signalingcomponents to a comparable extent in class I and class IIamutant melanoma cells. This demonstrates that cell prolifera-tion and survival pathways are inhibited in class IIa mutantcells treated with either BRAFi or MEKi.

Next, we sought to determine whether class II BRAF mutantcells were growth inhibited by these targeted therapies usingstandard BRAFi or MEKi doses that achieved >50% growthinhibition of BRAF V600 mutant cells in short-term prolifera-tion assays (Supplementary Fig. S3B and S3C). In clonogenicgrowth assays, MEKi effectively inhibited the growth of class Iand IIa mutant cancer cells and, to a lesser extent, BRAF WT andclass IIb cells (Fig. 4A). BRAFi inhibited growth of class I and IIaBRAF mutant melanoma cells but did not significantly impairthe growth of WT and class IIb mutant cancer cells (Fig. 4A).Representative images of clonogenic assays from each class areshown in Supplementary Fig. S4A. Although class I BRAFmutant cells responded similarly to all 3 BRAFi, we observeda marked contrast in class IIa mutant cells between the marginalefficacy seen with vemurafenib and stronger inhibition of cellproliferation in the presence of dabrafenib and encorafenib.Class IIb mutant cells were not significantly growth inhibitedby single-agent BRAFi (Fig. 4A).

LY3009120 (LY), a pan-RAF andBRAFdimer inhibitor that is inearly stage clinical development, was also tested to assess itsefficacy in class II BRAF mutant cells. LY inhibited pERK in classI and II cell lines at low doses, but induced modest paradoxicalactivation in the BRAFWT cell line,WM3918 (Supplementary Fig.S1A).Using short-term cell growth assays,wedetermined thedoseof LY3009120 that achieved >50% growth inhibition of BRAFV600 mutant cells at 2 days (Supplementary Fig. S1B). In

Dankner et al.





clonogenic growth assays, LY3009120 at this dose (100 nmol/L)moderately inhibited growth ofWM3918 but substantially inhib-ited growth of class I and II BRAFmutant cells, including the classIIb cell line, HMV-II (Supplementary Fig. S1C and S1D). Thesedata demonstrate that although LY3009120 is only marginallyeffective in BRAFWT cells, itmay also be effective for patientswithclass II BRAF mutant tumors.

Enhanced efficacy of dMAPKi in class II BRAF mutantcancer cells

To assess efficacy of a combined therapeutic strategy usingBRAFi and MEKi, cells were treated with the standard clinicalBRAFi/MEKi combinations (vemurafenib/cobimetinib, dabrafe-nib/trametinib, encorafenib/binimetinib). We observe augment-

ed growth inhibition when either dabrafenib, encorafenib, orLY3009120 were added to a MEKi in class I or IIa BRAF mutantcells (Fig. 4A; Supplementary Fig. S1C and S1D). AlthoughdMAPKi did significantly inhibit the growth of BRAF WT cellscomparedwithDMSO, combinedBRAFiþMEKiwas less effectivethan single-agent MEKi in most WT cells. In particular, weobserved significantly enhanced growth of BRAF WT cells treatedwith vemurafenib (SkMel2 P¼ 0.009; WM3918 P¼ 0.005; CHL1P¼0.024) or dabrafenib (SkMel2 P¼0.047,WM3918P¼0.001)in addition to a MEKi, compared with MEKi alone (Fig. 4A).Encorafenib did not significantly enhance the growth of any BRAFWT cells treated with a MEKi. Conversely, encorafenib signifi-cantly inhibited the growth of binimetinib-treated triple WTCHL1 cells (P ¼ 0.047). In class IIb mutant cell lines, we

Figure 2.

BRAFi and MEKi effectively inhibit MAPK signaling in class IIa BRAF mutant cells. Immunoblots of BRAF WT (green border), class I BRAF mutant (black),class IIa BRAF mutant (blue), and class IIb BRAF mutant (purple) cells treated with (A) single-agent MEKi cobimetinib (cobi; 5 and 50 nmol/L) andtrametinib (tram; 1 and 10 nmol/L) for 1 hour or (B) single-agent BRAFi vemurafenib (vemu; 100 and 1,000 nmol/L) and dabrafenib (dab; 10 and 100 nmol/L)for 1 hour. C, Immunoblots of BRAF WT (green border), class I BRAF mutant (black), class IIa BRAF mutant (blue), and class IIb BRAF mutant (purple)cells treated for 1 hour with encorafenib (300 nmol/L), which were then washed 3 times in prewarmed media and replaced with drug-free media. Cellswere lysed at the following time points after washout: 0 minute, 5 minutes, 30 minutes, 60 minutes, 120 minutes, 480 minutes, and 1,440 minutes. D and E,Immunoblots against the indicated proteins in BRAF WT (green border), class I BRAF mutant (black), class IIa BRAF mutant (blue), and class IIb BRAFmutant (purple) cells treated for 24 hours (hrs.) with (D) DMSO or trametinib (5 nmol/L), or (E) DMSO or encorafenib (300 nmol/L).






consistently observed further growth inhibition only with encor-afenib, but not with vemurafenib, when added to a MEKi (Fig.4A).

Next, we sought to directly compare the effects of specific BRAFiwhen added to the same MEKi. To do so, we tested each BRAFi incombination with either trametinib or binimetinib. In long-termgrowth assays where all cells were grown in the presence oftrametinib � BRAFi (Fig. 4B; Supplementary Fig. S4B), vemur-afenib potentiated the growth of BRAFWT, NRASmutant SkMel2and GCRC1987 cells, and all class IIb mutant cells tested. Mean-while, vemurafenib modestly augmented growth inhibition ofclass I A375 and class IIamutant cells. Dabrafenib potentiated thegrowth of trametinib-treated SkMel2 and GCRC1987 cells butinhibited the growth of all trametinib-treated class I, IIa, and IIbcells, with the sole exception of class IIb mutant MDA-MB-231breast cancer cells, which were unaffected by the addition ofdabrafenib. When added to trametinib, encorafenib potentlyinhibited the growth of BRAF WT, NRAS mutant SkMel2 cells,

and all class I, IIa, and IIb BRAF mutant cells tested (Fig. 4A).Similar BRAFi effects were observed when binimetinib was usedas the MEKi (Fig. 4C; Supplementary Fig. S4C).

In all classes of cells, 48-hour treatment with trametinib led tosustained inhibition of ERK phosphorylation (Fig. 4D). In class IBRAF mutant melanoma, dMAPKi further impairs the MAPKpathway (32, 33). Therefore, we asked if the addition of BRAFiwill have a similar effect inMEKi-treated class II cells. In class I andclass IIa mutant cells, the addition of any BRAFi further exacer-bated ERK inhibition. Meanwhile, in class IIb mutant cells, onlyencorafenib led tomore profoundERK inhibition than trametinibalone (Fig. 4D).

dMAPKi induces regression of twomelanoma PDXs expressingclass II BRAF mutations

To determine whether BRAFi þ MEKi combinations causetumor regression in vivo, we used our melanoma PDX modelsbearing class IIa, BRAF L597S mutations (GCRC2015 and

PDX ID Primary site Tumor loca�on BRAF Status Treatment history

GCRC1987 Melanoma Brain Wild-Type Untreated

GCRC2015 Melanoma Brain L597S Untreated

GCRC2073 Melanoma Brain V600ELGX818/MEK162- par�al response in all systemic (non-brain) metastases

GCRCMel1 Melanoma Inguinal lymph node L597S Untreated

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MAPK pathway BRAF p.L597SBRAF p.R239QBRAF p.V600ENRAS p.Q61RNF1 p.P678LMLK4 p.D420N

PI3K/AKT/mTOR PIK3R3 p.D11NPIK3CG p.G242SPIK3C2G p.P1234SPREX2 p. D312NAKT1 p.L28FPTEN p.R130fsRPTOR p.A523V

RTK Signaling BTK p.D531EPHB1 p.A872VFLT1 p.L709SKDR p.P1355LPDGFRB p.S1092LRET p.S649LROS1 p.T1147SROS1 p.P1941LIGF1R p.R1353H

DNA Damage BARD1 p.A421SBRCA1 p. R1347GBRCA2 p. R2520QFANCA p.L179FFANCL p.G313EPALB2 p.S133TPOLD1 p.R331QPRKDC p.3398ARAD17 p.R301*DAXX p.E452del

Melanoma drivers ARID1A p.S949PARID1B p.H89delARID1B p.G315delARID1B p.G319delARID1B p.S41delARID1B p.Q121delARID2 p.E74fsARID2 p.N1507DARID5B p.419SCDKN2A p.P81L

GCRC1987 GCRC2073 GCRC2015 GCRCMel1(WT) (Class I) (Class IIa) (Class IIa)

Figure 3.

Characterization of metastatic melanoma PDX models. A, Characteristics of patients whose tumors were used to establish PDXs. B, Copy-number analysis ofthe GCRC2015 (BRAF L597S brain metastasis) and GCRCMel1 (BRAF L597S lymph node metastasis) patient tumor and first-passage mouse xenograft. C, Totalnumber of identified mutations in GCRC 1987, 2073, 2015, and Mel1 patient tumor tissue (blue) and first-passage xenografts (red) in 435-gene panel sequencing.D, Spectrum of mutations in clinically actionable genes in patient and first-passage xenografts of GCRC 1987, 2073, 2015, and Mel1. Green ¼ gain of function,red ¼ loss of function, and blue ¼ mutation of unknown significance. E, Representative images of patient brain metastasis and matching PDX materialembedded into a tissue microarray and stained for H&E, Melan-A, BRAF V600E, and HMB-45.

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GCRCMel1). Tumorswere implanted subcutaneously and treatedwith vehicle or MAPKi.

In the GCRCMel1 model, treatment with either single-agentdabrafenib or single-agent trametinib was insufficient to induceshrinkage in any of the tumors, but 17 of 19 (89%) tumors treatedwith dabrafenib þ trametinib had shrunk by day 4 (Fig. 5A).This result was corroborated with immunoblots that demonstrat-ed decreased pERK in dabrafenib þ trametinib 4-day treatedtumors, compared with vehicle-, dabrafenib-, or trametinib-treated tumors (Fig. 5E). Both dabrafenib and trametinib, whenused as single agents, were capable of delaying the growth ofGCRCMel1 tumors over time. Meanwhile, tumors treated withdMAPKi were significantly more growth inhibited than tumorstreated with either single agent (Fig. 5C). By day 15, all tumors inthe study had begun to progress, implying that they had acquiredresistance to MAPKi (Fig. 5C). Phosphorylated ERK levels wereuniform between all arms at experimental endpoint (Fig. 5E).

Resistant dabrafenib þ trametinib-treated tumors demonstratedincreased expression of the HER3 receptor tyrosine kinase (RTK),aswell as increased pAKT andpCRAF (Fig. 5E), implying potentialmechanisms of resistance to dMAPKi in class II mutant tumors.

In the GCRC2015 model, after 4 days of treatment, 83.3%(10/12) of vehicle-treated tumors were progressively growing.Trametinib monotherapy induced tumor shrinkage in 75%(8/12) of subcutaneous tumors. Meanwhile, dMAPKi with dab-rafenib and trametinib induced tumor shrinkage in 100%(13/13)of tumors (Fig. 5B). Immunoblot analysis revealed that earlyinto treatment, dabrafenib augmented the inhibitory effect oftrametinib on the MAPK pathway (Fig. 5F). All treatment groupseventually began to acquire MAPK inhibitor resistance, as evi-denced by the reactivation of pERK (Fig. 5F) and increasing tumorgrowth (Fig. 5D) at the experimental endpoint of 14 days.However, 88.9% (8/9) of dabrafenib þ trametinib comparedwith 0%(0/8) of trametinib-treated tumorsmaintained anoverall

Figure 4.

Dual MAPKi effectively inhibits the growth of class II BRAF mutant cancer cells in vitro. A, Quantification of 10-day cell growth clonogenic assay using cell linesendogenously expressing class I mutant BRAF (black), WT BRAF (green), class IIa mutant BRAF (blue), and class IIb mutant BRAF (purple) that were treated withBRAFi vemurafenib (vemu; 1,500 nmol/L), dabrafenib (dab; 150 nmol/L), encorafenib (enco; 150 nmol/L), cobimetinib (cobi; 25 nmol/L), trametinib (tram; 5 nmol/L),and binimetinib (bini; 50 nmol/L). In comparisons between DMSO and BRAFi, MEKi, or BRAFi þ MEKi, � represents P < 0.05, �� represents P < 0.0005, and# represents not significant. B and C, Quantification of 15-day cell growth assay, with cell lines expressing class I mutant BRAF(black), WT BRAF (green),class IIamutant BRAF (blue), and class IIbmutant BRAF (purple) treatedwith (B) trametinib (tram; 5 nmol/L), or (C) binimetinib (bini; 100 nmol/L), plus vemurafenib(vem; 2,000 nmol/L), dabrafenib (dab; 300 nmol/L), or encorafenib (enco; 300 nmol/L). In comparisons between MEKi and BRAFi þ MEKi, � represents P < 0.05,�� represents P < 0.0005, and # represents not significant. For A, B, and C, adjacent scale bars represent positive pixel count (i.e., area covered by cancercells) at quantification compared with either (A) DMSO or (B and C) MEKi controls. Representative images from A, B, and C for each condition, taken atexperimental end point, are shown in Supplementary Fig. S4. D, Immunoblots of class I BRAF mutant (black), BRAF WT (green), class IIa BRAF mutant (blue),and class IIb BRAF mutant (purple) cells treated with DMSO, the MEKi trametinib (tram), or tram in combination with BRAFi vemurafenib (vemu), dabrafenib(dab), and encorafenib (enco), at the same doses as in B.






reduction in tumor size at endpoint. Immunoblots fromGCRC2015-resistant tumors demonstrate the same resistancemechanisms as the GCRCMel1 model, in that RTKs (HER2 andHER3) were upregulated, coinciding with increased pAKT in allMAPKi-treated tumors. In both PDX models, we only observedenhanced pCRAF in tumors that had acquired resistance todMAPKi with dabrafenib þ trametinib (Fig. 5E and F). Together,this suggests that activation of CRAF is a mechanism of resistancethat is unique to dMAPKi in class II BRAF mutant melanoma.

Treatment with encorafenib, binimetinib, or encorafenib þbinimetinib produced similar results to dabrafenibþ trametinib,causing shrinkage of 8% (1/12), 25% (3/12), and 67% (8/12) ofGCRC2015 tumors, respectively, whereas all of the vehicle tumors

were progressively growing by day 4 (Supplementary Fig. S5A).Immunoblot analysis of tumors treated for 4 days demonstratedthat both encorafenib and binimetinib robustly inhibit ERKphosphorylation as single agents, whereas the encorafenib þbinimetinib combination further inhibited pERK compared witheither agent alone (Supplementary Fig. S5B). Both encorafeniband binimetinib, when used as single agents, delayed GCRC2015tumor growth. Combined encorafenib þ binimetinib elicitedtumor shrinkage and more significant tumor growth delay com-pared with either single agent (Supplementary Fig. S5C).

Importantly, the patient from whom the GCRCMel1 PDX wasderived presentedwith stage IV (M1a)metastaticmelanoma,withdisease involving the inguinal lymphnodes,muscle, and adjacent

Figure 5.

dMAPKi induces tumor regression in class II BRAF L597S mutant PDX melanoma models. A, Waterfall plot demonstrating responses of individual GCRCMel1tumors grown subcutaneously; treatment was initiated when tumors reached an average volume of 180 mm3. Mice were treated with vehicle (V; n¼ 18), dabrafenib(D; 50 mg/kg, n ¼ 16), trametinib (T; 0.5 mg/kg, n ¼ 16), or dabrafenib (50 mg/kg) þ trametinib (DT; 0.5 mg/kg; n ¼ 19). N ¼ 4 tumors were removedfrom each cohort at day 4. B, Waterfall plot demonstrating responses of individual GCRC2015 tumors grown subcutaneously; treatment was initiated whentumors reached an average volume of 460mm3. Mice were treated with vehicle (V; n¼ 12), trametinib (T; 0.5mg/kg, n¼ 12), or dabrafenib (50mg/kg)þ trametinib(DT; 0.5 mg/kg; n ¼ 13). N ¼ 4 tumors were removed from each cohort at day 4. C, Growth curves plotted from GCRCMel1 subcutaneous tumor measurements.V vs. D: P ¼ 0.2, V vs. T: P ¼ 0.0164, V vs. DT: P < 0.0001, D vs. T: P ¼ 0.4, D vs. DT: P < 0.0001, and T vs. DT: P < 0.0001. D, Growth curves plotted fromGCRC2015 subcutaneous tumor measurements. V vs. T: P ¼ 0.0018, V vs. DT: P ¼ 0.0002, and T vs. DT: P ¼ 0.0001. E, Immunoblots against the indicatedproteins from GCRCMel1 tumor lysates at 4 and 14 days on treatment. Dab, dabrafenib; Tram, trametinib. F, Immunoblots against the indicated proteins fromGCRC2015 tumor lysates at 4 and 14 days on treatment. Dab, dabrafenib; Tram, trametinib. G, Patient GCRCMel1 scan results before and after treatment withdabrafenib (dab)þ trametinib (tram). Prior to treatment, target lesions #1 and #2 measured 1.8� 1.8 cm and 2.4� 1.7 cm, respectively. After 6 weeks of treatment,target lesions #1 and #2 measured 1.0 � 0.8 cm and 1.8 � 1.2 cm, respectively. Target lymph node lesions are delineated by green cross hairs and outlined in red.Together, this represents a 33% reduction in size according to RECIST 1.1 criteria.

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soft tissues. This patient was treated with dabrafenibþ trametiniband achieved an objective radiographic response, with a 34%reduction in tumor size at 2 months on treatment (Fig. 5G). Afterseveral months of treatment, the patient began to experience drugtoxicity (pyrexia, hepatotoxicity) despite dose reductions andwasswitched to immunotherapy. These observations of an objectivepartial response provide proof-of-principle demonstrating thatdabrafenib þ trametinib has clinical activity in class IIa BRAFmutant melanoma.

BRAF þ MEK inhibition is effective in class II BRAF mutantbrain metastases

GCRC2015 was derived from amelanoma brain metastasis. Inlight of recent data indicating that dMAPKi can effectively shrink

brainmetastases in patients with BRAF V600Emutant melanoma(34), we asked whether dabrafenib þ trametinib would be sim-ilarly effective in class II BRAF mutant brain metastases. TheGCRC2015 PDX model was propagated as an intracranial xeno-graft and infected with pHIV-Luc-ZsGreen virus to allow longi-tudinal bioluminescence imaging in vivo (Fig. 6A). We observedthat both trametinib monotherapy and dabrafenib þ trametinibslowed growth of GCRC2015 intracranial tumors. At the exper-imental endpoint, the change in in vivo bioluminescence from thetime treatment was initiated was significantly smaller in thedabrafenib þ trametinib group compared with trametinib orvehicle (Fig. 6B; Supplementary Fig. S6A). Indeed, after 9 daysof treatment, the average size of the intracranial tumors fromdMAPKiwas smaller than tumors treatedwith trametinib alone or

Figure 6.

dMAPKi improves survival in class II BRAF L597S mutant melanoma brain metastases. A, Experimental pipeline outlining development of PDX models ofbrain metastasis. Brain metastatic tissue is retrieved from the operating room and grown in the flank of immunocompromised mice. The resulting tumors areenzymatically dissociated and injected into the brains of new mice and are labeled with pHIV-Luc-ZsGreen for longitudinal imaging in vivo. These tumorcells were then used for in vivo treatment experiments. B, GCRC2015 PDX-expressing pHIV-Luc-ZsGreen were injected intracranially. Tumor growth wasmonitored with in vivo bioluminescent imaging. Representative images are shown from three treatment groups: vehicle (n ¼ 6), trametinib (0.5 mg/kg, n ¼ 7),dabrafenib (dab; 50 mg/kg) þ trametinib (tram; 0.5 mg/kg; n ¼ 6) at day 9. C and D, IHC was performed for pERK on 5 brain metastases per treatment arm.Quantification of staining was performed for nuclear pERK positivity (C), and representative images (D) are shown. � , P < 0.05. E, Mice were injected intracraniallywith GCRC2015 cells and treated with vehicle (V; n ¼ 11), trametinib (T; 0.5 mg/kg; n ¼ 12), or dabrafenib (D; 25 mg/kg) þ T (0.5 mg/kg; n ¼ 11). Mice weremonitored until they showed signs of neurologic decompensation or poor body condition, at which point they were recommended for euthanasia by blindedanimal health technicians. Overall survival for each group is presented in a Kaplan–Meier plot. T vs. V: HR, 0.723, 95% confidence interval (CI), 0.253–2.064; DT vs.V: HR, 0.272, 95% CI, 0.107–0.697; and DT vs. T: HR, 0.376, 95% CI, 0.167–0.848. F, A patient presenting with BRAF L597S metastatic melanoma was treatedwith dabrafenib (dab) þ trametinib (tram). Before- and on-treatment scan results are shown, demonstrating a profound response in the brain, lung, and adrenal.






vehicle (Supplementary Fig. S6B and S6C). Furthermore, immu-nohistochemistry for pERK revealed decreased staining in dabra-fenibþ trametinib, but not in trametinib-treated brainmetastases(Fig. 6C and D). In longer-term survival analyses, trametinibalone did not significantly prolong survival compared with vehi-cle treatment of mice bearing class II BRAF mutant melanomabrain metastases (Fig. 6E). However, dMAPKi treatment didsignificantly improve survival of mice compared with eithertrametinib monotherapy or vehicle. Finally, we retrospectivelyidentified a patient with class II BRAF (L597S) mutant melanomawith brain metastases. This patient received treatment with dab-rafenib þ trametinib and experienced a dramatic response inmetastases in the brain, lung, liver, and adrenal gland (Fig. 6F).After 4 months of treatment, this patient developed progressivebrain metastases. She went on to receive additional brain radio-therapy and immunotherapy but eventually died of her disease.These observations provide further validation that dMAPKi caninduce objective responses in visceral and brain metastases ofpatients with class II BRAF mutant melanoma.

DiscussionWe initiated this study after encountering 2melanoma patients

with BRAF L597S mutations in clinical practice. At the time oftheir presentation, little was known about class II BRAFmutationsand their responsiveness to targeted therapy. We sought to bettercharacterize this emerging class of BRAF mutant tumors and toinquire whether patients with class II BRAFmutantmelanoma areresponsive to therapeutic intervention with approved targetedtherapies.

Data from colorectal cancer (35) or NSCLC (36) indicate thatpatients with non-V600 BRAF mutations tend to experienceimproved overall survival than those with V600 BRAF muta-tions. In contrast, we report here that advanced melanomapatients with potentially targetable NRAS and/or class II/IIIBRAF mutations experience worse survival than those with classI BRAF mutations. This finding highlights the need for improvedtherapeutic strategies for melanoma patients with non-V600BRAF mutations.

We draw the distinction between class IIa mutations within theactivation segment and class IIbmutations within the glycine-richp-loop. Class IIa and IIbmutations have been shown to engenderenhanced kinase activity that is RAS-independent and dimeriza-tion-dependent (10). However, the class IIa and IIb mutant cellstested are unique in terms of their sensitivity to single-agentBRAFi: class IIa BRAF mutant cells were sensitive to single-agentBRAFi, whereas class IIb BRAF mutant cells were not. The differ-ential sensitivities of class IIa and IIb BRAF mutants to approvedBRAFi may be due, in part, to the ability of BRAFi to moreeffectively inhibit the second protomer of a class IIa BRAFmutantdimer than that of IIb BRAF dimers. Alternatively, although it isclear that BRAF L597S and K601E signal predominantly as dimers(10), class IIa mutant BRAF may harbor some degree of mono-meric signaling capacity. It is also possible that class IIa mutantsmore readily form BRAF homodimers, whereas class IIb mutantsform BRAF:CRAF heterodimers, rendering them less sensitive toBRAFi (26). Interestingly, we found that activated CRAF is acommon resistance mechanism to dMAPKi in our two class IIBRAF L597S mutant PDX models; this result suggests thatacquired resistance to dMAPKi with BRAFi and MEKi in class IIBRAFmutant melanoma results from a shift from primarily BRAF

homodimer-driven MAPK signaling toward BRAF:CRAF hetero-dimer or CRAF homodimer-driven MAPK signaling.

Although all three BRAFi augmented MEKi-mediated growthinhibition in class IIa mutant cells, encorafenib was the onlyBRAFi that consistently augmented MEKi-mediated growth inhi-bition in class IIbmutant cells. The contrast between vemurafenib,dabrafenib, and encorafenib in this context may be due todifferences in eliciting paradoxical activation of the MAPK path-way. This results from differential ability of each inhibitor to bindand inhibit the second protomer of a BRAF dimer. It has beenshown that significantly higher concentrations of vemurafenib arerequired to inhibit BRAF dimers, compared with encorafenib anddabrafenib (10, 37). Moreover, recent findings have demonstrat-ed the efficacy of encorafenib when used in combination withMEKi in NRAS mutant melanoma through an ER stress pathway(38). This may explain the sensitivity we observe in NRASmutantSKMel2 cells treated with MEKi þ encorafenib, highlightingunique properties of encorafenib that may support its broaderutility amongmelanomapatients. The encorafenibþbinimetinibcombination has been shown to provide a significant survivaladvantage over vemurafenib in BRAF V600E/K mutant melano-ma, with a favorable safety profile (3). Therefore, this combina-tion is promising for patients with class II BRAF mutations andperhaps even some NRAS mutant melanoma patients.

It has been proposed that non-V600 BRAF mutant melanomaare sensitive to single-agent MEKi, prompting an ongoing trialrecruiting non-V600 mutant melanoma patients for treatmentwith trametinib (39, 40). Since these initial observations, severalclinical trials investigating single-agent MEKi have failed to yieldsustained clinical benefit in a variety of indications (41–43). InBRAF V600 mutant melanoma, trametinib has a much loweroverall response rate (22%) than single-agent BRAFi (48%–51%;ref. 4). These data suggest that the more effective therapeuticapproach of approved agents for class II BRAFmutant melanomawould be combination therapy including a clinically viable BRAFi(i.e., encorafenib) plus a MEKi. Moreover, in addition to thepotential for enhanced efficacy with dMAPKi, these combinationregimens are frequently better tolerated than either BRAFi orMEKialone (3, 44).

In this study, we established two PDX models of BRAF L597Smutant melanoma in order to assess their sensitivity to MAPKi.Importantly, the PDX models established herein adequatelyretain the genetic features of their tumors of origin. We demon-strate in both class II BRAF mutant PDX models that dMAPKiaugments inhibition of the MAPK pathway and impairs tumorgrowth of class II BRAFmutant melanoma compared with single-agent therapy. Single-agent MEKi produced only short-lived sta-ble disease in the GCRC2015 BRAF L597S subcutaneous PDXmodel and only modestly slowed progression of the GCRCMel1model, whereas the addition of BRAFi to MEKi resulted insustained partial responses in the majority of tumors in bothmodels. As an important proof of principle, we show that single-agent encorafenib is able to robustly inhibit the MAPK pathwayand slow tumor growth in the GCRC2015 PDX model. Further-more, we report a partial response to dabrafenib þ trametinib inpatient GCRCMel1, corroborating the results from the aforemen-tioned PDX studies.

Although our results indicate that dMAPKi is superior tosingle-agent MAPKi for class II BRAF L597S mutant melano-mas, the duration of growth inhibition with dMAPKi was lessthan we have observed in class I BRAF mutant melanoma (45).

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This implies that melanoma patients with class II BRAF muta-tions may be less responsive to dMAPKi than those with class IBRAF mutations. Moreover, both of our PDX models bore aBRAF L597S mutation, and as such it is unknown at this pointwhether other common class IIa mutations (i.e., L597Q/R/V,K601N/T) would derive equivalent benefit from dMAPKi invivo. As such, investigation into combinations of dMAPKi withantibody drug conjugates (46), ERK inhibitors (47), and immu-notherapies is also warranted for class II BRAF mutant mela-noma. Further investigation of therapies targeting the resistancepathways we identified in both PDX models, such as RTKs,PI3K/AKT signaling, and CRAF, may also be beneficial inpreventing or delaying resistance to dMAPKi.

The patient from whom the GCRC2015 PDX was derivedpresented with brain metastases. Cytotoxic chemotherapieshave minimal effect in intracranial metastatic disease, in partdue to limitations of the blood–brain barrier (48). However,emerging data espouse the efficacy of systemic immuno-therapies and MAPKi in the management of brain metastaticmelanoma (34, 48). In this study, dMAPKi provided a signif-icant survival advantage to mice with class II BRAF L597Smutant brain metastases, whereas single-agent MEKi did not.Therefore, we speculate that a similar approach can be appliedfor melanoma patients with class II BRAF mutant tumors,including those with brain metastases. This is further sup-ported by a patient with BRAF L597S brain-metastatic mela-noma who experienced a major intracranial response todabrafenib þ trametinib.

In summary, we have provided in vitro, in vivo, and clinicalevidence indicating that dMAPKi effectively impairs the growthof subsets of non-V600, class II BRAF mutant melanoma. Thesedata provide intriguing preclinical rationale to support thedevelopment of clinical trials to investigate BRAFi þ MEKicombinations in patients with class II BRAF mutations.

Disclosure of Potential Conflicts of InterestD. Hogg reports receiving speakers bureau honoraria from EMD Serono,

and is a consultant/advisory board member for Bristol-Myers Squibb, EMDSerono, Merck, Novartis, and Roche. C. Mihalcioiu reports receiving speak-ers bureau honoraria from Novartis, and is a consultant/advisory boardmember for Bristol-Myers Squibb, Merck, Novartis, and Roche. No poten-tial conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Dankner, C. Mihalcioiu, A.A.N. RoseDevelopment of methodology: M. Dankner, D. Vuzman, A.A.N. RoseAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M.Dankner, D. Moldoveanu, T.-T. Nguyen, P. Savage,S. Rajkumar, M. Lvova, A. Protopopov, D. Hogg, M. Park, M.-C. Guiot,K. Petrecca, C. Mihalcioiu, A.A.N. RoseAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Dankner, M. Lajoie, S. Rajkumar, X. Huang,D. Vuzman, I.R. Watson, A.A.N. RoseWriting, review, and/or revision of the manuscript: M. Dankner, M. Lajoie,D. Moldoveanu, S. Rajkumar, X. Huang, D. Vuzman, D. Hogg, M. Park,M.-C. Guiot, K. Petrecca, C. Mihalcioiu, I.R. Watson, P.M. Siegel, A.A.N. RoseAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):M.Dankner,M. Park, C.Mihalcioiu, A.A.N. RoseStudy supervision:M. Park, C.Mihalcioiu, I.R.Watson, P.M. Siegel, A.A.N. Rose

AcknowledgmentsThe authors are grateful to the patients who donated the tissues studied in

this work. The authors acknowledge technical assistance from the McGill/GCRC Histology core facility and the McGill Comparative Medicine andAnimal Resources Centre (CMARC). The authors acknowledge the supportand assistance of Valentina Mu~noz Ramos and Margarita Souleimanova inbiobanking and sample collection. The authors are thankful to Array Bio-pharma for providing encorafenib and binimetinib used in in vivo studies.The authors are thankful to Juan Canale, Karen Stone, Vasilios Papavasiliou,Matthew Annis, and William Muller for animal support. The authors aregrateful to Nicholas Hayward, Antoni Ribas, Wilson Miller, and DavidDankort for providing cell lines used in this study. The authors thankmembers of the Siegel laboratory for thoughtful discussions and criticalreading of the article.

This research has been supported by a grant from the DOD (CA-140389;to P.M. Siegel). M. Dankner acknowledges support from the McGill UniversityMD/PhD program and the Brain Tumour Foundation of Canada. I.R. Watson isfunded by grants from the Melanoma Research Alliance (MRA—Grant#412429), the V Foundation (Grant #2016-023), and the Canadian Instituteof Health Research (CIHR—Grant # PJT-152975). P.M. Siegel is a McGillUniversity William Dawson Scholar. A.A.N. Rose acknowledges a DavidCornfield Melanoma Fund Award.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedNovember 11, 2017; revisedMarch 28, 2018; accepted June 6, 2018;published first June 14, 2018.

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Dankner et al.




2018;24:6483-6494. Published OnlineFirst June 14, 2018.Clin Cancer Res Matthew Dankner, Mathieu Lajoie, Dan Moldoveanu, et al. Subset of Class II BRAF Mutant MelanomasDual MAPK Inhibition Is an Effective Therapeutic Strategy for a

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