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CANCER

Improved antitumor activity of immunotherapy withBRAF and MEK inhibitors in BRAFV600E melanomaSiwen Hu-Lieskovan,1 Stephen Mok,1 Blanca Homet Moreno,1,2 Jennifer Tsoi,3,4 Lidia Robert,1

Lucas Goedert,1 Elaine M. Pinheiro,5 Richard C. Koya,1* Thomas G. Graeber,3,4,6

Begoña Comin-Anduix,6,7 Antoni Ribas1,4,6,7†

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Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the highresponse rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinicaltrial testing the combination of the BRAF inhibitor vemurafenib and the CTLA4 antibody ipilimumab was termi-nated early because of substantial liver toxicities. MEK [MAPK (mitogen-activated protein kinase) kinase] inhibitorscan potentiate the MAPK inhibition in BRAF mutant cells while potentially alleviating the unwanted paradoxicalMAPK activation in BRAF wild-type cells that lead to side effects when using BRAF inhibitors alone. However, thereis the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneicBRAFV600E-driven melanoma, SM1, we tested whether addition of the MEK inhibitor trametinib would enhance theantitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib andtrametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltrationinto tumors, and improved in vivo cytotoxicity. Single-agent dabrafenib increased tumor-associated macrophages andT regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapyresulted in increased melanosomal antigen and major histocompatibility complex (MHC) expression and globalimmune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combinedwith antigen-specific ACT, we tested the combination of dabrafenib, trametinib, and anti-PD1 therapy in SM1 tumors,and observed superior antitumor effect. Our findings support the testing of triple combination therapy of BRAF andMEKinhibitors with immunotherapy in patients with BRAFV600E mutant metastatic melanoma.

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INTRODUCTION

The recent breakthroughs brought by the clinical use of immunecheckpoint inhibition in cancer provide an exciting promise of long-term responses in clinically significant numbers of patients (1–5). Strat-egies to extend this low-frequency event to most patients have becomethe focus of cancer immunotherapy research. In BRAFmutantmelano-ma, the combination of BRAF inhibitors and immunotherapy has beentested in both preclinical models and clinical trials (6–9). This is basedon the targeting of theBRAFV600Edrivermutation, present in about 50%ofmetastaticmelanomas, and the immunosensitization effects of BRAFinhibitors through increased antigen presentation (10–12), antigen-specificT cell recognition (10, 13), homing of immune effector cell to the tumors(12, 14, 15), and improved T cell effector functions (6, 16). However, thebenefit of this combination in preclinical models has been modest (6–9),whereas substantial liver toxicity was observed in the first clinical trialcombining the BRAF inhibitor vemurafenib and the CTLA4 blocking an-tibody ipilimumab (17). Both the improved effector function and the toxi-cities were attributed to the paradoxical activation of the MAPK pathwayby vemurafenib in BRAF wild-type cells (18).

MEK [MAPK (mitogen-activated protein kinase) kinase] inhibitors,on the other hand, can potentiate the antitumor effects in themelanoma

1Division of Hematology-Oncology, Department of Medicine, University of California, LosAngeles (UCLA), Los Angeles, CA 90095, USA. 2Division of Translational Oncology, Carlos IIIHealth Institute, Madrid 28029, Spain. 3Crump Institute for Molecular Imaging, UCLA, LosAngeles, CA 90095, USA. 4Department of Molecular and Medical Pharmacology, UCLA, LosAngeles, CA 90095, USA. 5Merck Research Laboratories, Boston, MA 02115, USA. 6JonssonComprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA. 7Division of SurgicalOncology, Department of Surgery, UCLA, Los Angeles, CA 90095, USA.*Present address: Department of Immunology, Roswell Park Cancer Institute, Buffalo,NY 14263, USA.†Corresponding author. E-mail: aribas@mednet.ucla.edu

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cells (19) and reduce toxicity associated with BRAF inhibitors (18),given their ability to inhibitMAPK signaling in cells with and without aBRAF mutation (20). In addition, MEK inhibitors have demonstratedpotential of immunosensitization by up-regulation of tumor antigen ex-pression and presentation (10, 21), serving as a rational addition to theBRAF inhibitor and immunotherapy combination. However, there istheoretical concern that a MEK inhibitor could dampen immune effec-tor functions, given that in vitro studies have shown impaired T cellproliferation and functions withMEK inhibition (10, 22). Alternatively,when combining with BRAF inhibitors, MEK inhibitors might balancethe potential overreacting effector cells to avoid exhaustion and improvethe tumor microenvironment by influencing the cytokine produc-tion and immunosuppressive cell populations in the tumor micro-environment (20). Using a syngeneic BRAFV600E mutant melanomamouse model (6), we tested the hypothesis that the addition of a MEKinhibitor would enhance the immunosensitization effects of BRAF inhi-bition, with increased antitumor activity and decreased toxicity.

RESULTS

Enhanced in vivo antitumor activity with pmel-1 adoptivecell transfer (ACT), dabrafenib, and/or trametinibWe derived a BRAFV600Emutant murine melanoma SM1, syngeneic tofully immunocompetent C57BL/6 mice, from a spontaneously arisingmelanoma in BRAFV600E transgenic mice (6). Besides the presence ofthe BRAFV600E transversion, SM1 also has CDKN2A gene deletionand BRAF andMITF gene amplification and is onlymoderately sensitiveto vemurafenib (6). Here, we first confirmed the downstream MAPKpathway inhibition of SM1 after treatment with dabrafenib, trametinib,

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or the combination in vitro by down-regulated phosphorylated extra-cellular signal–regulated kinase (pERK) (Fig. 1A). To further explorethe drug effects on effector T cells, we treated gp10025-33–activatedpmel-1mouse splenocytes with serial dilutions of dabrafenib, trametinib,or dabrafenib plus trametinib. Western blot analysis at 24 hours of treat-ment showed paradoxical activation of the MAPK pathway with dabra-fenib alone at medium and high concentrations, evidenced by increasedpERK (fig. S1A). Trametinib alone orwith dabrafenib blocked theMAPKpathway even at low doses. However, in vitro cell viability (MTS) assaywith concentration up to 40 mMdabrafenib and 2 mMtrametinib did notshow any decreased cell viability at 72 hours (fig. S1B).

We then tested the antitumor effects of dabrafenib, trametinib, andthe combination in vivo against established SM1 tumors in syngeneicmice. SM1 tumors established subcutaneously in C57BL/6 mice re-

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sponded to combination therapy of dabrafenib and trametinib, with astatistically significant difference in growth inhibition when comparedwith tumors treated with dabrafenib or trametinib alone or vehicle con-trol (Fig. 1B, P = 0.002, unpaired t test, n = 4, mean ± SD). We thentested the combinatorial effect of dabrafenib, trametinib, and immuno-therapy using the pmel-1 ACT model, which is based on T cells trans-genic for a T cell receptor (TCR) recognizing the murine melanosomalantigen gp100 (23), endogenously expressed by SM1 (Fig. 1C).Myeloid-depleted C57BL/6 mice with bone marrow transplant and establishedsubcutaneous SM1 tumors receivedACT of gp10025-33 peptide–activatedsplenocytes obtained frompmel-1mice.Wild-typeC57BL/6mouse sple-nocytes activatednonspecifically byCD3andCD28were administered asmock ACT controls. Both ACTs were followed by 3 days of high-doseinterleukin-2 (IL-2) injections. In three replicate experiments, the triple

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combination therapy of dabrafenib and tra-metinibwithpmel-1ACTprovided superiorantitumor activity against established SM1tumors with complete tumor regression,which was not observed with pmel-1 ACTplus either BRAF or MEK inhibitor therapyalone or with mock ACTwith both dabrafe-nib and trametinib (Fig. 1D).

Increased effector T cell homing tothe tumors associated with bothdabrafenib and trametinibTo analyze themechanismof improved an-titumor activity with the triple combinationtherapy, we first evaluated the expansionand change in distribution of adoptivelytransferred cells in vivo.Tumors and spleenswere harvested on day 5 after ACT andstained for CD3, CD8, andThy1.1 (expressedby the pmel-1 mice but not the wild-typeC57BL/6 mice). There was a statistically sig-nificant increase of CD3+Thy1.1+ (adoptivelytransferred pmel-1 effector) and CD3+CD8+

(both endogenous andadoptively transferredeffector) cells in the tumors treated withdabrafenib, trametinib, or the combinationof dabrafenib plus trametinib when com-pared to vehicle-treatedmice (unpaired t test,n = 3, mean ± SD, Fig. 2, A and B). On theother hand, effector cells harvested fromthe spleen did not show a statistically sig-nificant difference in distribution betweenthe treatment groups (Fig. 2A). To analyzethe effects on the whole animal, we genet-ically labeled the adoptively transferredcells with the firefly luciferase transgene totrack these cells in vivo using biolumines-cence imaging (Fig. 2C, imaged 5 days afterACT). The quantitative analysis of lucifer-ase activityover time in the livingmice showedpeaked tumor-infiltratingeffectorT cell 5 daysafter ACT. SM1 tumors treated with triplecombination therapy, pmel-1 ACT plusdabrafenib, or pmel-1 plus trametinib showed

Fig. 1. Enhanced in vivo antitumor activity with pmel-1 ACT plus dabrafenib (D) and/ortrametinib (T). (A) Western blot analysis of MAPK pathway. SM1 cells were treated with serial dilu-

tions of D, T, or D + T for 1 and 24 hours. L: low dose [D (0.1 mM)/T (0.005 mM)]. M, medium dose [D(5 mM)/T (0.25 mM)]; H, high dose [D (20 mM)/T (1 mM)]. (B) In vivo tumor growth curves with fourmice in each group (mean ± SD). SM1-bearing C57BL/6 mice were treated with D (30 mg/kg), T(0.15 mg/kg), or the combination via daily oral gavage, starting when tumors were 3 to 5 mm. (C)Schema of pmel-1 ACT model. C57BL/6 mice had myeloid-depleting total body irradiation (TBI)followed by bone marrow transplant (BMT) and SM1 tumor injections. When tumors reached3 mm, 3 million gp10025-33 peptide–activated pmel-1 splenocytes (pmel-1 transgenic mice carryingTCR specific for melanoma antigen gp100) were injected. Wild-type C57BL/6 mouse splenocytes ac-tivated by CD3 and CD28 were mock ACT controls. Both ACTs were followed by high-dose IL-2 for3 days. Daily oral gavage of vehicle control (V), D (30 mg/kg), T (0.6 mg/kg), or the combinationwas started on the day of ACT. i.v., intravenously; s.c., subcutaneously; i.p., intraperitoneally. (D) In vivoSM1 tumor growth curves with three to four mice in each group (mean ± SD), after D, T, and ACT treat-ments. P < 0.0001 by unpaired t test on day 30, pmel-1 ACT + D + T versus pmel-1 ACT + T, or versusmock ACT + D + T.

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a significantly higher accumulation of adoptively transferred effector cellsthan those tumors treated by pmel-1 ACT alone (Fig. 2D, unpaired t test,n = 4, mean ± SD).

No impaired effector function in vivo associatedwith trametinibGiven the concern that MEK inhibitors might impair effector T cellfunction (10), we evaluated the effect of dabrafenib and trametinib onT cell effector function both in vitro and in vivo. When gp10025-33–

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activated pmel-1 mouse splenocytes were exposed in vitro to increasingconcentrations of dabrafenib and trametinib for 72 hours, there was asignificant decrease of interferon-g (IFN-g)–producing effector cells as-sociated with medium and high concentrations of both dabrafenib andtrametinib (Fig. 3A, unpaired t test,n=3,mean± SD). To test the effectsof the combination therapy in vivo, we harvested tumors and spleens5 days afterACTand analyzed the activation state ofT cells. Adoptivelytransferred effectors T cells (CD3+Thy1.1+) collected from mice treatedwith pmel-1 ACT plus trametinib or triple combination therapy did not

Fig. 2. Increased tumor-infiltrating lymphocytes (TILs)withpmel-1ACTplus dabrafenib and/or trametinib in SM1 tumors. (A) Quantification of

versus pmel T). (B) Representative flow data of percentage of CD3+Thy1.1+

TILs are shown. (C) In vivo bioluminescence imaging (BLI) of adoptively

TILs. Splenocytes and TILs harvested on day 5 after ACT were countedand analyzed by flow cytometry for Thy1.1/CD3/CD8 staining (threemice ineachgroup,mean±SD). Percentageof effectors (CD3+CD8+or CD3+Thy1.1+)was shown to be statistically significantly changed by unpaired t test in sev-eral subgroups (CD3+CD8+ TILs: P = 0.049, pmel D versus pmel V; P = 0.02,pmel T versus pmel V; P=0.004, pmelD+T versus pmel V; P=0.035, pmel D+T versus pmel T; CD3+Thy1.1+: P = 0.03, pmel D versus pmel V; P = 0.02, pmelT versus pmel V; P = 0.006, pmel D + T versus pmel V; P = 0.047, pmel D + T

transferred lymphocytes. Pmel-1 transgenic T cells were transduced with aretrovirus–firefly luciferase and used for ACT. Representative figure on day 5depicts four replicate mice per group. (D) Quantification of BLI of serialimages with region of interest (ROI) analysis at the site of tumors (countsper pixel) obtained through day 18 after ACT of luciferase-expressing pmel-1T cells (four mice per group, mean ± SD). On day 5, P = 0.0009, pmel D versuspmel V;P<0.0001, pmel T versuspmel V;P<0.0001, pmelD+T versuspmel V;P = 0.01, pmel T or pmel D + T versus pmel D (unpaired t test, n = 4).

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show statistically significant differences compared to pmel-1 plus vehi-cle control in the ability to respond to short-term ex vivo restimulationwith the gp10025-33 antigen, assessed by IFN-g secretion (Fig. 3, B andC,unpaired t test, n = 3, mean ± SD). We then tested the direct effect ofdabrafenib and trametinib on lymphocyte cytotoxicity in vivo in-dependent of their effects on SM1 tumor cells using an in vivo cyto-toxicity assay, where the targets are syngeneic splenocytes devoid ofthe BRAFV600Emutation and pulsed with gp10025-33 or control peptides(Fig. 3D). Pmel-1 ACT induced potent cytotoxic effects against spleno-cytes pulsed with the gp10025-33 peptide, but not against the controlOVA (ovalbumin) peptide (34% lower gp10025-33 target intensity, P =0.01 pmel-1 V versus mock V, unpaired t test, n = 3, mean ± SD). Thecytotoxicity increasedwith systemic treatment of pmel-1ACTplus dab-

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rafenib, but this was not statistically significant (Fig. 3, E and F, and fig.S1D), and there was no difference in cytotoxic activity with pmel-1 plustrametinib and triple combination therapywhencompared topmel-1ACTplus vehicle control. Therefore, the addition of trametinib to dabrafenib didnot change the functionality of adoptively transferred pmel-1 cells in termsof their ability to release immune-stimulating cytokines and intrinsicantigen-specific lytic activity in vivo.

Improved tumor microenvironment when trametinib wascombined with dabrafenib and ACTTo evaluate the effect of dabrafenib, trametinib, and the combination onother cellular components of the tumormicroenvironment, we harvestedspleens and tumors 5 days after ACT and studied the cell populations by

Fig. 3. Dabrafenib, trametinib, or combination treatment impairs effectorT cell function in vitro but not in vivo. (A) In vitro study of cytokine-

by FACS after 5-hour ex vivo exposure to the gp10025-33 peptide. Percent-age of IFN-g expressing CD3+Thy1.1 cells in the spleen and tumor was nor-

producing function of effector cells. Gp10025-33–activated pmel-1 mouse sple-nocytes were treated at serial dilutions of D, T, or D + T for 72 hours. L, lowdose [D (0.1 mM)/T (0.005 mM)]; M, medium dose [D (5 mM)/T (0.25 mM)]; H,high dose [D (20 mM)/T (1 mM)]. Cells were analyzed by fluorescence-activatedcell sorting (FACS) for CD3/CD8/IFN-g staining. Bar graphs of percentage ofIFN-g expressing CD3+CD8+ cells are shown (mean ± SD). P = 0.002, D M or DH versus D L; P = 0.045, D H versus D M; P = 0.003, T M or T H versus T L; P =0.0002, D + T M or D + T H versus D + T L (unpaired t test, n = 3). (B) In vivoeffect on cytokine production upon antigen restimulation. SM1 tumor–bearingC57BL/6 mice received pmel-1 ACT with or without D and T. On day 5 afterACT, spleens and TILs were isolated for intracellular IFN-g staining analyzed

malized to Pmel + V (mean ± SD). (C) Gating strategy and representative flowdata are shown. (D) Schema of the in vivo cytotoxic T cell assay. C57BL/6 micereceived ACT of 5 × 104 pmel-1 splenocytes and daily D, T, D + T, or vehiclevia oral gavage. On day 5, mice received an intravenous challenge with CFSE(carboxyfluorescein diacetate succinimidyl ester)–labeled target cells (spleno-cytes pulsed with gp10025-33 peptide or control OVA peptide). Gp10025-33–pulsed targets were pulsed with 10 times more concentration of CFSE- thanOVA-pulsed cells. Ten hours later, splenocytes were harvested and analyzedby FACS. (E) Bar graph representation of the in vivo cytotoxicity study result(mean ± SD). P = 0.01, pmel V versus mock V (34% down, unpaired t test,n = 3). (F). Representative flow data are shown.

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multiplex FACS. We first analyzed the myeloid-derived suppressor cells(MDSCs), a heterogeneous group of cells that induce tumor-associatedimmune suppression. MDSCs (Gr+CD11b+) were present at a low levelin SM1 tumors (<5%), and their level increased with antigen-specificACT (fig. S1C). Pmel-1 ACT plus dabrafenib significantly increasedMDSCs in the tumorswhencompared to thepmel-1ACTplus vehicle con-trol group(P=0.02,unpaired t test,n=3,mean±SD),withanonsignificanttrend of decreased MDSCs in the spleen. Trametinib alone or dabrafenibplus trametinib with pmel-1 ACT did not change MDSCs in the tumorsor spleens when compared to pmel-1 ACT plus vehicle.MDSCs consist oftwo major subsets: cells with granulocytic phenotype that express Ly6Gmarker (PMN-MDSC, Ly6CLoLy6GHiCD11b+), and cells with monocyticphenotype expressing Ly6Cmarker (MO-MDSC, Ly6CHiLy6GLoCD11b+)(fig. S1E). These two subsets ofMDSCs have distinct functions in infec-tion, autoimmune diseases, and cancer (24).We examined these two sub-

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sets ofMDSCs, presentedaspercentageofCD11b+cells. InFig. 4 (AandB),there was a significant shift of MDSC subsets in the tumors towardincreasedMO-MDSCs and decreased PMN-MDSCs, associated with dab-rafenib, trametinib, and combination treatments. Whereas in the spleen,other than a decreased percentage of MO-MDSC with pmel-1 ACT plusdabrafenib–treatedmice versus pmel-1ACT plus vehicle control (P= 0.06,unpaired t test, n = 3), there was no significant change among the differenttreatment groups. We then analyzed mature tumor-associated macro-phages (TAMs; F4/80+CD11b+). Both pmel-1 ACT plus dabrafenib andtriple combination therapy significantly increased macrophages in the tu-mors (Fig. 4, C and E), but to a lesser extent with triple combination,whereas no change was seen with pmel-1 ACT plus trametinib. Therewas no significant change in macrophages in the spleen among the dif-ferent treatment groups. Analysis of another immunosuppressive cellpopulation, the T regulatory cells (Tregs; CD4

+CD25+FOXp3+), showed

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Fig. 4. Dabrafenib and trametinib changed the cellular compo-nents of the tumor microenvironment. On day 5 after ACT, spleens

presented as percentage of CD11b+ cells. *P = 0.002, mock D + T versusmock V in tumor (unpaired t test, n = 3). (C) Analysis of macrophages

and tumors were isolated and stained with fluorescent-labeled antibodiesand then analyzed by FACS, with three mice in each group (mean ± SD). (A)MO-MDSC (CD11b+Ly6CHiLy6GLo) presented as percentage of CD11b+ cells.*P = 0.06, pmel D versus pmel V in spleen; P = 0.009, pmel V versus mockV in tumor (unpaired t test, n = 3). (B) PMN-MDSC (CD11b+Ly6CLowLy6GHi)

(F4/80+CD11b+). *P = 0.04, pmel D versus pmel V; P = 0.002, pmel D + Tversus pmel V, both in tumors (unpaired t test, n = 3). (D) Analysis ofTregs (CD4

+CD25+FOXp3+). *P = 0.002, pmel D versus pmel V in tumors(unpaired t test, n = 3). (E) Gating strategy and representative FACS plotsin tumors.

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a significantly increased percentage in tumors with pmel-1 ACT plusdabrafenib treatment (fig. S1F, P = 0.002, unpaired t test, n = 3) butno other significant changewith the other combination therapies in bothtumor and spleen (Fig. 4, D and E). These results indicate that dabrafe-nib, when combined with pmel-1 ACT and IL-2, increases macrophageand Tregs infiltration in the tumor microenvironment, thus providing apotential mechanism for the suboptimal antitumor effect with this com-bination. This effect on macrophage and Tregs can be overcome by theaddition of trametinib. Moreover, both dabrafenib and trametinib canshift the ratio of MDSC subsets from PMN-MDSC to predominantlyMO-MDSC.

Increased immune-related gene expression by bothdabrafenib and trametinib treatmentsTo better understand the impact of dabrafenib and trametinib on thetumor microenvironment, we compared the gene expression profiling

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of SM1 tumors bymicroarray analysis after a 5-day treatment with dab-rafenib, trametinib, or the combinationwith pmel-1 ormockACT.Twoto three replicates were prepared per treatment group after the sampleshad passed quality control by gel electrophoresis (fig. S2A). The expres-sion level of each genewas averaged across samples and used for furtheranalysis. As shown in Fig. 5A by principal components analysis (PCA)and in fig. S2B by hierarchical clustering of global gene expression, thebiological replicates cluster together closely and separate from each otheraccording to the different treatments. Clustering of immune-relatedgenes obtained from the gene ontology (GO) consortium under the GOterm of immune system process (www.geneontology.org; GO:0002376)showed three different patterns of gene expression changes (Fig. 5B).Cluster A genes are up-regulated after treatment with dabrafenib, tra-metinib, or the combination in mice receiving either mock ACT orpmel-1ACT, and includedmelanomaantigensandmanyMAPKpathwaygenes. Also among them are CD274 (PD-L1) and CSF-1R. Cluster B

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Fig. 5. Microarray analysis of tumors treated by dabrafenib, trame-tinib, or the combination of dabrafenib and trametinib with pmel-1

file of the tested samples. (B) Clustering of immune-related genes withanalysis of variance (ANOVA) filter, P < 0.05. Gene names in individ-

ACT or mock ACT. On day 5 after ACT, tumors were isolated and snap-frozen immediately (two to three mice in each group). RNA isolation wasdone after all samples were collected. (A) PCA of gene expression pro-

ual clusters are listed in tables S1 to S3. (C) Clustering of chemokinesand their receptors. (D) Clustering of MDAs and MHC class I and IImolecules.

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genes areup-regulatedbydabrafenib, trametinib, or the combinationwhencombined with antigen-specific pmel-1 ACT only, but not with the mockACT. Many of these genes are major histocompatibility complex (MHC)molecules, cytokines, and chemokines or their receptors (table S2). CD8,granzyme B, and IFN-g are within this group. CD83, a dendritic cell mat-urationmarker (25), CD86, a ligand expressed on antigen-presenting cellsthat binds to CD28 and CTLA4 (26), and CSF-1, are all in this genecluster. Finally, cluster C genes are down-regulated by dabrafenib, tra-metinib, or the combination in mice that received either mock ACT orpmel-1 ACT. Again, this cluster included many MAPK pathway genes,as well as VEGF (vascular endothelial growth factor), a previously reportedtarget of BRAF and MEK inhibition (27, 28). Another interesting gene inthis cluster isCD276 (B7-H3),whichwas reported recently to be associatedwith advanced stage of melanoma progression (29). Further clustering ofsamples according to chemokines and their receptors showed a generaltrend of increased expression of these genes in the dabrafenib-, trameti-nib-, or combination-treated tumors, especially with triple combinationtherapy (Fig. 5C). Expressionof somegenes previously reported to be regu-lated by BRAF inhibitors or mentioned above is shown in fig. S2C.

Increased tumor antigen and MHC expression in tumorstreated by triple combinationOne mechanism wherein tumors can evade the immune attack isthrough decreased tumor antigen or MHC expression. Analysis of

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themicroarray data showed that bothmelanoma antigen andMHC ex-pression are low in mock ACT plus vehicle and pmel-1 ACT plus ve-hicle (Fig. 5D). Dabrafenib and trametinib significantly increasedmelanoma antigen expression in both pmel-1 ACT– and mock ACT–treated tumors, with the highest expression seen after triple combinationtreatment. However, the up-regulation of MHCmolecules was restrictedto the antigen-specific pmel-1 ACT–treated tumors with dabrafenibor/and trametinib, butnotwithmockACTplusdabrafenib and trametinib,indicating that tumor-specific effector cells are important in the mech-anism of MHC up-regulation.

Up-regulation of PD-L1 and enhanced in vivo antitumoractivity with dabrafenib and/or trametinib withPD1 blockadeActivated T cells express PD1, which can bind to PD-L1 ligand up-regulated on tumor cells, and inhibit T cell effector functions. Recently,PD1 and PD-L1 were found to be increased in melanoma tumorsamples from patients treated with BRAF inhibitors (12), and additionof aMEK inhibitor suppressed the production of PD-L1 by in vitro studyof melanoma cell lines (30). The up-regulation of IFN-g, granzyme Bexpression, and PD1 in cluster B indicated increased effector T cell acti-vation and function with dabrafenib and trametinib (Fig. 6A). However,the up-regulation of PD-L1 suggested an adaptive immune resistancemechanism induced by the presence of effector T cells (Fig. 6A). Flow

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Fig. 6. Up-regulation of PD-L1 and triple combination of dabrafenib,trametinib, and PD1 blockade is superior in antitumor effect against

V; P = 0.007, pmel T versus pmel V; P = 0.001, pmel D + T versus pmel V.(C) Expression of PD-L1 on SM1 after 18 hours of stimulation with IFN-g

SM1. (A) Heat map representation of CD8, granzyme B, IFN-g, PD1, and PD-L1 gene expression from microarray data (PD-L1: P = 0.01, mock D + Tversus mock V; P = 0.004, pmel T versus pmel V; P = 0.004, pmel D + Tversus pmel V; P = 0.03, pmel T versus pmel D + T; unpaired t test, n = 3).(B) Percentage of PD-L1–expressing cells in the spleen and tumors 5 daysafter ACT and drug treatments started; three mice in each group (mean ±SD). P = 0.006, mock D + T versus mock V; P = 0.04, pmel D versus pmel

at different concentrations. B16 cells served as a positive control. (D) Invivo SM1 tumor growth curves after D, T, and anti-PD1 treatments; fourmice in each group (mean ± SD). SM1 tumor–bearing C57BL/6 mice re-ceived anti-PD1 (Merck DX400; 200 mg) via intraperitoneal injection every4 days, starting when tumors reached 3 to 5 mm. Daily oral gavage ofvehicle control (V), D (30 mg/kg), T (0.6 mg/kg), or the combination wasstarted on the same day as anti-PD1.

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cytometry analysis of PD-L1 expression of SM1 tumors after 5 days oftreatment was consistent with themicroarray gene expression data (Fig.6B), whereas no significant change was observed in the spleen samples.To test whether the increased IFN-g in the tumormilieu is sufficient forthe up-regulation of PD-L1, we treated SM1 cells with increasing con-centrationsof IFN-g andharvested cells for flowcytometry after 18hours.The result showed that IFN-g (1 mg/ml) up-regulated PD-L1 on the sur-face of SM1 cells bymore than 10-fold, similar to the B16-positive controlcells (Fig. 6C). This up-regulation of PD-L1 provided a rationale for thecombination of PD1 blockade therapy with dabrafenib and trametinib.Immunocompetent C57BL/6 mice with established subcutaneous SM1tumors received PD1 antibody or isotope control (200 mg) via intra-peritoneal injection every 5 days, starting when the tumors reached 4to 6mm2. Consistent with a previous report (8), SM1 is innately resistantto PD1 antibody therapy alone. In three replicate experiments, the com-bined therapy of dabrafenib, trametinib, and anti-PD1 provided superiorantitumor activity against established SM1 tumors compared with anti-PD1 plus either therapy alone or isotope control with both dabrafeniband trametinib (Fig. 6D).

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DISCUSSION

It has been previously reported thatMEK inhibitorsmight be detrimen-tal to T cell responses to cancer mainly on the basis of in vitro studies(10, 22). However, by using an immunocompetent mouse model ofBRAFV600E mutant melanoma, we demonstrate that the addition ofthe MEK inhibitor trametinib significantly improves the antitumor ef-fect of the BRAF inhibitor dabrafenib and two modes of immuno-therapy, ACT and PD1 blockade, via improved effector T cellhoming to the tumors, preserved effector function, increased tumor an-tigen and MHC expression, cytokine release, and attenuated immuno-suppressive cells up-regulated by the BRAF inhibitor in the tumormicroenvironment.

Increased numbers of TILs have been reported in biopsies of patientstreated with BRAF inhibitors (12, 14, 15), with an increase in clonalityafter BRAF inhibition and a better response in those patients who had ahigh proportion of preexisting dominant TCR clones (31), suggestingthat the T cell infiltration may be an antigen-driven recruitment intoregressing tumors. In a xenograft model where a BRAFmutant humanmelanoma cell line was transduced with gp100 and H-2D to allow rec-ognition by gp100-specific pmel-1 mouse T cells, treatment with theBRAF inhibitor vemurafenib significantly increased the tumor infiltra-tion and enhanced the antitumor activity of adoptively transferred Tcells in vivo (27). This increased TIL infiltration was thought to be pri-marily mediated by decreased VEGF production by tumors via C-myc.Analysis of human melanoma biopsies before and during BRAF inhib-itor treatment confirmed the down-regulation of VEGF. Another syn-geneic BRAF mutant melanoma model with PTEN−/− backgroundtested the combination of vemurafenib and PD1 and/or CTLA4 block-ade (9), and showed significantly increased T cell infiltration with ve-murafenib alone. On the contrary, in an inducible BRAF mutantmelanoma model, there was decreased TIL infiltration after BRAF in-hibition (7). In our SM1model, a previous study combining vemurafe-nib and pmel-1 ACT did not show increased TIL infiltration but didshow improved effector function, likely through paradoxical activationof the MAPK pathway in T cells by vemurafenib (6, 16). With dabra-fenib, titrated to higher concentrations to optimize antitumor activity,

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we observed both increased tumor T cell infiltration and improvedfunctions. This is possibly due the different potency and concentrationof the two BRAF inhibitors (we had used a lower dose with vemurafe-nib) (6).We also observed decreasedVEGF expression bymicroarray inthe tumors treatedwith dabrafenib. Trametinib increasedT cell homingto tumors, evidenced by in vivo imaging, ex vivo single tumor cell phe-notyping, and microarray analysis. Evaluation of in vivo effector cellfunction by both cytokine-releasing capacity and cytotoxic activity invivo showed preserved effector functions after treatment with single-agent trametinib or dabrafenib plus trametinib.

One of the suggested mechanisms by which BRAF inhibitors maysensitize tumor to the immune system is through up-regulation ofmelanocyte differentiation antigens (MDAs) and MHC expression inBRAF mutant melanoma (10-12). MEK inhibitors, on the other hand,can increaseMDAexpression in both BRAFmutant andwild-typemel-anoma cells (10, 21). This should result in improved antigen-specific Tcell recognition (10, 13). It has been shown that MDA expression wassignificantly decreased in patients at the time of progression on BRAFinhibitors and restored when subsequent combined BRAF and MEKinhibitors were given (12). Consistent with the previous reports, ourdata suggest increased MDA and MHC expression in tumors treatedby the combination therapy. The up-regulation of MDAs is a drug ef-fect, also seen in dabrafenib and trametinib combination with mockACT. On the other hand, up-regulation of MHC is specific to the com-bination of dabrafenib, trametinib, or dabrafenib plus trametinib withpmel-1, but not withmock ACT, indicating that antigen-specific effectoractivation is crucial for this regulation. Analysis of immune-related genesin themicroarray data indicated other genes that are regulated in similarfashion: up- or down-regulated by drugs in both pmel-1 andmockACT,or up-regulated by drugs in antigen-specific ACT only. Clustering of che-mokines and their receptors showedoverall increased expression ofmanyof these genes in tumors treated with the triple combination.

Despite the theoretical promise of increased tumor-infiltrating Tcells with improved function and increased tumorMDA andMHC ex-pression, the combination of dabrafenib and pmel-1 ACT had onlymodest antitumor effects, with a small but statistically significant dif-ference from mock ACT and pmel-1 ACT with vehicle control. Thisraised the possibility that the immunosuppressive cells (MDSCs, TAMs,and Tregs) in the tumor microenvironment might inhibit effector T cellfunction. One of the main factors that negatively regulate the immunesystem is MDSCs, a heterogeneous population of immature myeloidcells (CD11b+Gr1+ in mice) that are significantly expanded in patientswith cancer and have been shown to correlate negatively with prognosisand overall survival (32).MDSCs have been shown to not only suppressimmune responses but also promote tumor growth and expansion indifferent tumor types (33–37). We observed an interesting shift ofMDSC subtypes in the SM1 tumors, from the PMN-MDSC subset tothe MO-MDSC subset, associated with dabrafenib, trametinib, or com-bination treatment. This was correlated with significantly up-regulatedTAMs (F4/80+CD11b+) andTregs (CD4

+CD25+FoxP3+) after treatmentwith dabrafenib combined with pmel-1 ACT, suggesting a potentialimmune evasion pathway. Microarray data showed concurrent up-regulation of CSF-1, CSF-1R, and the dendritic cell maturation markerCD83. Trametinib, however, did not increase TAMs or Tregs when com-bined with pmel-1 ACT, and further attenuated the effect by dabrafenibwhen combinedwith both dabrafenib and pmel-1 ACT. Thismight haveaccounted for the significantly different antitumor effects of triple com-bination therapy compared to dabrafenib plus pmel-1 ACT.

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Activated T cells express PD1, which in turn binds to its ligand PD-L1 and negatively regulates TCR signaling (38–40). Increased T cell ex-haustionmarkers, includingTIM3, PD1, andPD-L1,were noted in tumorsamples frompatients treatedwithBRAF inhibitors, suggesting apotentialresistance mechanism (12). One in vitro study of melanoma cell linesshowed increasedPD-L1 expressionbyBRAF inhibitor–resistantmelano-ma cells,mediatedby c-JUNandSTAT3 signaling, and additionof aMEKinhibitor suppressed the expression of PD-L1 (30). Our study showed im-proved effector activity manifested by increased IFN-g and granzyme Bexpression, which coincides with the up-regulation of PD1 and PD-L1,unique to the tumor milieu. We also showed that IFN-g is sufficient forthe up-regulation of PD-L1 on SM1, consistent with published reports(39), which also explained why MEK inhibitor–treated tumors also hadelevated PD-L1 expression, and again stressed the importance of the invivo environment to study immune responses. Given that CD8 T cellsare the effectorsofPD1blockade, and theup-regulationofPD-L1 seenwithboth dabrafenib and trametinib treatments when combined with antigen-specific ACT, we tested the triple combination of dabrafenib, trametinib,and anti-PD1 therapy, and observed significant synergy of this combina-tion to inhibit SM1 tumor growth. This antitumor activity was mostsignificant with the triple therapy, but was also notable for the combi-nation of either dabrafenib or trametinib plus anti-PD1 therapies.

Our results are limited to preclinicalmodels and need to be validatedin clinical trials. Several phase 1 clinical trials are ongoing, combiningBRAF andMEK inhibitors with immunotherapies such as anti-CTLA4,anti-PD1, anti–PD-L1, and ACT (20), and our data suggest that thepresence of a MEK inhibitor will improve the effects of the combinedtherapy as opposed to the concern of limiting T cell responses to cancer.

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MATERIALS AND METHODS

Study designThe primary research objective was to evaluate combinatorial strategiesof BRAF inhibitor, MEK inhibitor, and ACT or PD1 blockade. Theoverall study design was a series of controlled laboratory experimentsin mice, as described in the sections below. In all experiments, animalswere assigned to various experimental groups in random. The ex-periments were replicated two to three times as noted. For the ex-periments reporting isolation of TILs, three mice per group were usedfor each experiment, with two to three replicates. All outliers were in-cluded in the data analysis.

Mice, cell lines, and reagentsC57BL/6 mice (Thy1.2, The Jackson Laboratory) and pmel-1 (Thy1.1)transgenic mice were bred and kept under defined-flora, pathogen-freeconditions at the Association for the Assessment and Accreditation ofLaboratoryAnimalCare–approved animal facility of theDivision of Ex-perimental Radiation Oncology, University of California, Los Angeles(UCLA), and used under the UCLA Animal Research Committeeprotocol #2004-159. The SM1 murine melanoma was generated froma spontaneously arising tumor in BRAFV600Emutant transgenicmice aspreviously described (6). The tumor was minced and implanted intoC57BL/6 mice for in vivo experiments. Part of the minced tumor wasplated under tissue culture conditions as SM1 cell line, maintained inRPMI (Mediatech) with 10% fetal calf serum (Omega Scientific), 2 mML-glutamine (Invitrogen), and 1% (v/v) penicillin, streptomycin, andamphotericin (Omega Scientific). Dabrafenib and trametinib were ob-

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tained under amaterial transfer agreement (MTA)withGlaxoSmithKline(GSK).Dabrafenib and trametinibwere dissolved in dimethyl sulfoxide(DMSO; Fisher Scientific) and used for in vitro studies. For in vivostudies, dabrafenib and trametinib were suspended in an aqueous mix-ture of 0.5% hydroxypropyl methyl cellulose (HPMC) and 0.2% Tween80 (Sigma-Aldrich). One hundred microliters of the suspended drugwas administered by daily oral gavage into mice at 30 mg/kg of dabra-fenib and/or 0.6 mg/kg of trametinib when tumors reached 5 mm indiameter. Mouse PD1 antibody (DX400) was obtained under anMTA with Merck.

Cell viability assaysSM1 cells, naïve C57BL/6 splenocytes, or activated pmel-1 splenocyteswere seeded in 96-well flat-bottomplates (5000 cells perwell)with 100mlof 10% fetal calf serummedium and incubated for 24 hours. Serial dilu-tions of dabrafenib, trametinib, orDMSOvehicle control, in cultureme-dium, were added to each well in triplicate and analyzed following theMTS assay (Promega).

Western blottingCells were washed with ice-cold phosphate-buffered saline and thenlysed using a lysis buffer containing 10mMtris (pH7.4), 100mMNaCl,1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mMNa3VO4, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100,10% glycerol, leupeptin (10 mg/ml), aprotinin (60 mg/ml), and 1 mMphenylmethanesulfonyl fluoride. Equal amounts of protein extractswere separated by using 8 or 10% SDS–polyacrylamide gel electropho-resis and then transferred to a polyvinylidene difluoride membrane(Bio-Rad Laboratories Inc.). After blocking for 1 hour in a tris-bufferedsaline containing 0.1% Tween 20 and 5% bovine serum albumin, themembrane was probed with various primary antibodies, followed bysecondary antibodies conjugated to horseradish peroxidase. The immu-noreactivity was revealed by use of a Pierce ECL kit (Thermo Scientific),and the densities of the protein bandswere quantified by ImageJ software.Primary antibodies included pERKThr204/205, ERK, pAKT Ser473, AKT,and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (CellSignaling Technology).

Pmel-1 ACT in vivo modelC57BL/6 mice were treated with lymphoid-depleting (500 cGy) ormyeloid-depleting (900 cGy) total body irradiation followed by bonemarrow transplant and subcutaneous SM1 tumor injection, and thenreceived 5 × 106 gp10025-33 peptide–activated pmel-1 splenocytes intra-venously when tumors reached 3 to 5 mm in diameter as previouslydescribed (29, 30) and daily intraperitoneal administration of 50,000IU of IL-2 for 3 days. Activated splenocytes from wild-type C57BL/6mice were controls. BRAF inhibitor dabrafenib, MEK inhibitor trame-tinib, vehicle, or the combination of dabrafenib and trametinib was giv-en daily by oral gavage from the day of ACT. Tumors were followed bycaliper measurements three times per week.

Flow cytometry analysisSM1 tumors harvested from mice were digested with collagenase(Sigma-Aldrich). Splenocytes and cells obtained from digested SM1 tu-mors were stained with antibodies to CD3 BV605 (clone 17A2), Ly6CFITC (fluorescein isothiocyanate) (clone AL-21), PD-L1/CD274 PE(phycoerythrin) (clone MIH5) (Becton Dickinson Biosciences), CD8aBV421 (clone 53-6.7) (BioLegend), Ly6G (Gr1) PerCP 5.5 (clone

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RB6-8C5), CD11b APC (allophycocyanin) (cloneM1/70), F4/80 Pa-cific Blue/eFluor 450 (clone BM8), CD25 APC (PC61.5), and CD4FITC (RM4-5) (eBioscience), and analyzed with LSR II or FACSCa-libur flow cytometers (Becton Dickinson Biosciences), followed byanalysis using FlowJo software (FlowJo LLC) as previously described(30). Intracellular staining of IFN-gwas done as previously described(30). Intracellular staining of Foxp3 PE (FJK-16s) (eBioscience) wasdone according to the manufacturer’s recommendations.

In vivo cytotoxicity assayThe assay was conducted as previously described (30). In brief, spleno-cytes from naïve wild-type C57BL/6 mice were pulsed with gp10025-33peptide (50mg/ml) or the same amount of control OVA257-264 peptide.After 1 hour of incubation, gp10025-33-pulsed wild-type splenocyteswere labeled with 6 nM CFSE for 10 min at 37°C, whereas controlOVA257-264–pulsed splenocytes were differentially labeled with a 10-fold dilution of CFSE (0.6 nM). Cells were injected intravenously intoexperimental mice at 5 days after pmel-1 ACT. After 10 hours, fivemice per group were sacrificed, and their spleens were examined forthe presence of CFSE-labeled cells. Percent cytotoxic activity wascalculated as number of live gp10025-33–pulsed splenocytes dividedby the number of live OVA257-264–pulsed splenocytes, which were dis-tinguished on the basis of the 10-fold difference in CFSE fluorescenceby flow cytometry.

Bioluminescence imagingPmel-1 splenocytes were retrovirally transduced to express firefly lucif-erase as previously described (29) and used for ACT. Bioluminescenceimaging was carried out with a Xenogen IVIS 200 Imaging System(Xenogen/Caliper Life Sciences) as previously described (22, 23).

Microarray data generation and analysisTotal RNAs were extracted using the RNeasy Micro Kit (Qiagen) fromSM1 tumors. Complementary DNAs were generated, fragmented, bio-tinylated, and hybridized to the GeneChip Mouse 430 V2 Arrays (Af-fymetrix). The arrays were washed and stained on a GeneChip FluidicsStation 450 (Affymetrix); scanning was carried out with the GeneChipScanner 3000 7G (Affymetrix); and image analysis was done with theGeneChip Command Console Scan Control (Affymetrix). Microarrayanalyses were performed in the R statistical programming environment,and Bioconductor suite of packages was used (41). Expression datawerenormalized, background-corrected, and summarized using the robustmulti-array average (RMA) algorithm implemented in the R “affy”package (42). Hierarchical clustering was performed using the Euclide-an distance as the similarity metric with average linkage clustering.Clustering results were visualized by heat maps generated using the R“NMF” package (43).

Statistical analysisDescriptive statistics such as number of observations, mean values, andSD were reported and presented graphically for quantitative measure-ments. Normality assumption was checked for outcomes before statis-tical testing. For measurements such as tumor volume, percentage ofTILs, quantified imaging data, and cytokine expression levels, pairwisecomparisons between treatment groups were performed by unpairedt tests. All hypothesis testingwas two-sided, and a significance thresholdof 0.05 for P value was used. Analyses were carried out using GraphPadPrism (version 6) software (GraphPad Software).

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/279/279ra41/DC1Fig. S1. In vitro study of effects of dabrafenib and trametinib on effector T cell and gatingstrategies.Fig. S2. Microarray analysis quality control, all gene clustering, and expression of interested genes.Fig. S3. Source data of Western blots in Fig. 1A.Table S1. Immune A genes.Table S2. Immune B genes.Table S3. Immune C genes.Table S4. Source data of tumor growth curves in Figs. 1B, 1D, and 6D.

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Acknowledgments:We are grateful to X. Wang from UCLA Statistics Core for her assistance instatistical analysis. We want to thank L. Liu, T. Gilmer, L. Nakamura, and J. Offord from GSK fortheir assistance regarding dabrafenib and trametinib. We also want to thank N. Restifo, Sur-gery Branch, National Cancer Institute, Bethesda, MD, for providing pmel-1 (Thy1.1) transgenicmice, and R. Prins from UCLA for maintaining the pmel-1 mice. Funding: Funded by NIHgrants P01 CA168585 and P50 CA086306, the Dr. Robert Vigen Memorial Fund, the ResslerFamily Foundation, the Wesley Coyle Memorial Fund, and the Garcia-Corsini Family Fund(to A.R.). S.H.-L. was supported by NIH grant T32 CA09297, ASCO (American Society of ClinicalOncology) Young Investigator Award, and Tower Foundation Research Grant. S.M. wassupported by UCLA Final Year Dissertation Fellowship (2014–2015). B.H.M. was supportedby the Rio Ortega Scholarship from the Hospital 12 de Octubre, Madrid, Spain. L.R. wassupported by the V Foundation-Gil Nickel Family Endowed Fellowship in Melanoma Researchand a grant from the Spanish Society of Medical Oncology (SEOM) for Translational Researchin Reference Centers. T.G.G. is supported by the National Cancer Institute/NIH (P01 CA168585and R21 CA169993), an American Cancer Society Research Scholar Award (RSG-12-257-01-TBE), a Melanoma Research Alliance Established Investigator Award (20120279), the NationalCenter for Advancing Translational Sciences UCLA CTSI (Clinical and Translational Science In-stitute) Grant UL1TR000124, and a CONCERN Foundation CONquer CanCER Now Award. J.T. issupported by the NIH Ruth L. Kirschstein Institutional National Research Service Award #T32-CA009120. Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center(JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by NIH awardsCA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School ofMedicine at UCLA. Author contributions: S.H.-L., S.M., B.H.M., L.R., L.G., E.M.P., R.C.K., and B.C.-A.performed the experiments. J.T. and T.G.G. analyzed the gene expression profiling. S.H.-L. and A.R.designed the experiments and wrote the manuscript. Competing interests: E.M.P. is an employeeof Merck. The other authors declare that they have no competing interests. Data and materialsavailability: Dabrafenib and trametinib were obtained through an MTA with GSK. DX400 murinePD1 antibody was obtained through an MTA with Merck. The gene expression profiling is availableat www.ncbi.nlm.nih.gov/geo/ (accession number: GSE64102).

Submitted 24 October 2014Accepted 27 January 2015Published 18 March 201510.1126/scitranslmed.aaa4691

Citation: S. Hu-Lieskovan, S. Mok, B. Homet Moreno, J. Tsoi, L. Robert, L. Goedert, E. M. Pinheiro,R. C. Koya, T. G. Graeber, B. Comin-Anduix, A. Ribas, Improved antitumor activity ofimmunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma. Sci. Transl. Med. 7,279ra41 (2015).

eTranslationalMedicine.org 18 March 2015 Vol 7 Issue 279 279ra41 11

melanomaV600EBRAFImproved antitumor activity of immunotherapy with BRAF and MEK inhibitors in

Pinheiro, Richard C. Koya, Thomas G. Graeber, Begoña Comin-Anduix and Antoni RibasSiwen Hu-Lieskovan, Stephen Mok, Blanca Homet Moreno, Jennifer Tsoi, Lidia Robert, Lucas Goedert, Elaine M.

DOI: 10.1126/scitranslmed.aaa4691, 279ra41279ra41.7Sci Transl Med

-mutated melanoma.BRAFBRAF inhibitions with various immunotherapies in patients with addition, replacing ACT with anti-PD1 in the triple therapy had similar results, supporting the testing of MEK and(ACT) immunotherapy induced complete tumor regression in a manner consistent with immune activation. In

driven melanoma that triple therapy with BRAF and MEK inhibitors together with adoptive cell transfer-V600EBRAFinhibition of BRAF inhibitors while concurrently decreasing the toxicity. They show in a mouse model of

MAPK(mitogen-activated protein kinase) kinase] inhibitors to this combination therapy in an effort to potentiate the test the addition of MEK [MAPKet al.early clinical trial was stopped because of liver toxicity. Hu-Lieskovan

combining a BRAF inhibitor and checkpoint inhibitors was hoped to improve the antitumor response; however, an -mutated melanoma,BRAFCombination therapy is the favored approach to fight drug-resistant cancer. For

Melanoma's triple threat

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MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2015/03/16/7.279.279ra41.DC1

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