BRAF Inhibitors Amplify the Proapoptotic Activity of MEK ...clincancerres.aacrjournals.org/content/clincanres/23/20/6203.full.pdf · the frozen stock.Mycoplasma infection in the cells

Cancer Therapy: Preclinical

BRAF Inhibitors Amplify the ProapoptoticActivity of MEK Inhibitors by Inducing ER Stressin NRAS-Mutant MelanomaHeike Niessner1,Tobias Sinnberg1, Corinna Kosnopfel1, Keiran S.M. Smalley2, Daniela Beck1,Christian Praetorius3,4, Marion Mai3, Stefan Beissert3, Dagmar Kulms3,4, Martin Schaller1,Claus Garbe1, Keith T. Flaherty5, DanaWestphal3,4, InesWanke1, and Friedegund Meier1,3,6

Abstract

Purpose: NRAS mutations in malignant melanoma are asso-ciated with aggressive disease requiring rapid antitumor interven-tion, but there is no approved targeted therapy for this subset ofpatients. In clinical trials, the MEK inhibitor (MEKi) binimetinibdisplayed modest antitumor activity, making combinations arequisite. In a previous study, the BRAF inhibitor (BRAFi) vemur-afenib was shown to induce endoplasmic reticulum (ER) stressthat together with inhibition of the RAF–MEK–ERK (MAPK)pathway amplified its proapoptotic activity in BRAF-mutant mel-anoma. The present study investigated whether this effect mightextent to NRAS-mutant melanoma, in which MAPK activationwould be expected.

Experimental Design andResults: BRAFi increased pERK, butalso significantly increased growth inhibition and apoptosisinduced by the MEKi in monolayer, spheroids, organotypic,and patient-derived tissue slice cultures of NRAS-mutant mel-

anoma. BRAFi such as encorafenib induced an ER stressresponse via the PERK pathway, as detected by phosphorylationof eIF2a and upregulation of the ER stress–related factors ATF4,CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKisuch as binimetinib induced the expression of the proapoptoticprotein BIM and activation of the mitochondrial pathway ofapoptosis, the latter of which was enhanced by combinationwith encorafenib. The increased apoptotic rates caused by thecombination treatment were significantly reduced throughsiRNA knockdown of ATF4 and BIM, confirming its criticalroles in this process.

Conclusions: The data presented herein encourage furtheradvanced in vivo and clinical studies to evaluate MEKi incombination with ER stress inducing BRAFi as a strategy totreat rapidly progressing NRAS-mutant melanoma. Clin CancerRes; 23(20); 6203–14. �2017 AACR.

IntroductionFifteen percent to 25% of all melanoma harbor activating

NRAS mutations. Although not all studies support this notion(1–4), NRAS mutations generally appear to be an independentpredictor of poor survival and are associated with rapid tumorprogression and an increased incidence of brain metastases.Treatment for patients with NRAS mutations is currently limitedto immune checkpoint inhibitors or chemotherapy. Unfortunate-ly, chemotherapy produces only low response rates and no

durable responses, whereas immunotherapy achieves durableresponses, but tend to be slow acting. Thus, there is a high unmetmedical need for fast-acting, first-line therapy options, and alsoeffective second-line therapies after failure of immune checkpointinhibitors in these patients.

In vitro, someNRAS-mutantmelanoma cell lines are sensitive toMEK inhibition (5). In a phase II trial, the MEK inhibitor bini-metinib showed activity in patients with NRAS-mutant melano-ma with an overall response rate of 21% and a median progres-sion-free survival of 3.65 months (6). In a recent phase III study,patients with NRAS-mutant metastatic melanoma without pre-vious therapy or after prior immunotherapy were treated with theMEKi binimetinib or chemotherapy (DTIC; ref. 7). Overallresponse rates were 15% for binimetinib versus 7% for DTIC.Even though an overall survival benefit was not observed forbinimetinib versus DTIC, binimetinib displayed a clear benefitover DTIC in patients with prior immunotherapy (median pro-gression-free survival 5.5 vs. 1.6months).Moreover, in a phase Ib/II studyof theMEKi binimetinib in combinationwith theCDK4/6inhibitor LEE011 partial responses were achieved in 33% ofpatients with NRAS-mutant metastatic melanoma. However, thiscombination was associated with frequent adverse events (8).Altogether, these clinical data suggest that the MEKi binimetinibprovides a backbone for combination treatments with othertargeted therapies or immunotherapies.

Several groups observed that the BRAFi vemurafenib inducesboth, inhibition of theMAPK signaling pathway and induction of

1Department of Dermatology, Oncology, University Medical Center, T€ubingen,Germany. 2Department of Tumor Biology, Moffitt Cancer Center and ResearchInstitute, Tampa, Florida. 3Department of Dermatology, Carl Gustav CarusMedical Center, TU Dresden, Dresden, Germany. 4Center for RegenerativeTherapies Dresden, TU Dresden, Germany. 5Massachusetts General HospitalCancer Center, Harvard Medical School, Boston, Massachusetts. 6NationalCenter for Tumor Diseases (NCT), Partner Site Dresden, Germany.

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

D.Westphal, I. Wanke, and F. Meier contributed equally as senior authors for thearticle.

Corresponding Author: Heike Niessner, Section of Dermatooncology, Lieber-meisterstr. 20, 72076 T€ubingen, Germany. Phone: 49-707-1298-0803; Fax: 49-70-7129-5187; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0098

�2017 American Association for Cancer Research.

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ER stress in the setting of activating BRAF mutations (9, 10). ERstress is caused by multiple factors such as Ca2þ imbalance,hypoxia, nutrient deprivation or perturbation of protein glyco-sylation, leading to the accumulation of unfolded proteins in theER that can then trigger the unfolded protein response (UPR;refs. 11–13). The UPR pathway is initiated by three sensors, thePKR-like ER kinase (PERK), activating transcription factor 6(ATF6), and inositol-requiring enzyme 1 (IRE1) that are normallykept in check by binding to the chaperone protein glucose-regulated protein 78 (GRP78), but are released upon ER stress.The UPR reduces the accumulation of unfolded proteins byinducing UPR gene transcription, reducing global protein syn-thesis, and stimulating ER-associated protein degradation torestore normal ER function. If normal ER function cannot berestored, the UPR switches modes from pro-survival to proapop-tosis (13–15).

In our previous study, we demonstrated that the BRAFi vemur-afenib raised cytosolic Ca2þ levels, suppressed the ER chaperoneprotein GRP78 and induced phosphorylation of the a-subunit ofthe eukaryotic initiation factor 2 (eIF2a) downstream of PERK.These effects led to an increased expression of ER stress–relatedgenes, including nuclear protein 1 (NUPR1), activating transcrip-tion factor 4 (ATF4), and growth arrest and DNA-damage–induc-ible transcript 3 (DDIT3/CHOP; ref. 9). In cooperation withincreased expression of proapoptotic BIM, these activities inducedintrinsic apoptosis.

Next to inhibition of the MAPK pathway, ER stress inductionthus appeared to be a secondary event of vemurafenib thatremarkably enhances its proapoptotic activity in BRAFV600-mutant melanoma.

In this study, we explored whether BRAFi-induced ER stresscould be used to amplify the proapoptotic activity of MEKi inNRAS-mutant melanoma, despite the paradoxical activation ofthe MAPK pathway in this setting.

Materials and MethodsIsolation and culture of human cells

The use of human skin tissues was approved by the MedicalEthics committee of the University of T€ubingen. Experiments

were performed in accordance with the Declaration ofHelsinki.

Melanoma cell lines were kindly provided by M. Herlyn(451Lu, Mel1617, WM1346, and WM1366), A. Bosserhoff(MelJuso) or purchased from the ATCC (SKMel19, SKMel147).Melanoma cell lines, cells isolated from excised melanomametastases, melanocytes, keratinocytes, and fibroblasts wereisolated and cultured as described previously (16–18). Allmelanoma cell lines were used within 3 months of thawingthe frozen stock. Mycoplasma infection in the cells was regularlychecked using a Venor GeM Classic Mycoplasma Detection Kit(Minerva Biolabs).

Melanoma spheroids were generated and prepared via the"hanging drop" method as described previously (19).

Establishment of resistant cell linesThe resistant cell lines were generated by continuous treat-

ment with increasing concentrations of encorafenib or/andbinimetinib for 5 months, starting at 0.1 mmol/L and increasingthe concentration with every passage up to 10 or 5 mmol/L,respectively.

Signaling pathway inhibitors and treatmentsThapsigargin and QVD-OPH were purchased from Sigma.

Dabrafenib, encorafenib, binimetinib, trametinib, and Raf265were purchased from Selleck Chemicals, while vemurafenib wasfrom Roche. The combination indices (CIs) showing the syner-gism between various BRAF and MEK inhibitor combinationswere calculated using COMPUSYN based on the methodologyand stratification of Chou (20) and displayed in SupplementaryTable S1. All chemicals were dissolved in dimethylsulfoxide(DMSO) and added directly to the cell culture medium.

Anchorage-independent (long-term) growth assayAnchorage-independent growth was assessed as described ear-

lier (21).

Growth inhibition assayMelanoma cell lines and cells from melanoma biopsies were

tested for growth inhibition using the 4-methylumbelliferylheptanoate (MUH) assay as described previously (21). Briefly,cells were treated with inhibitors or with DMSO (at the highestcombined DMSO concentration of the inhibitors) for 72 hoursand were then incubated with 100 mg/mL MUH in PBS for 1hour at 37�C. The absolute fluorescence intensity at lex of 355nm and lem of 460 nm was measured using a Fluoroskan II(Labsystems).

Cell-cycle analysis to measure apoptotic cellsCell-cycle analyses were described previously (21).

Western blottingTotal protein was extracted from cell pellets, separated by

SDS-PAGE (15–60 mg/lane), blotted onto PVDF membranes(Roche) and probed with antibodies listed in SupplementaryTable S2. Proteins were visualized with secondary peroxidase–conjugated antibodies (Cell Signaling Technology) and theECL detection system (Thermo). Band intensities for peIF2ain comparison with eIF2 were quantified using the ImageJsoftware.

Translational Relevance

Patients with NRAS-mutant metastatic melanoma oftenhave aggressive disease requiring a fast-acting, effective ther-apy. TheMEK inhibitor binimetinib shows an overall responserate of 15% in patients with NRAS-mutant melanoma, pro-viding a backbone for combination strategies. Our studiesdemonstrate that in NRAS-mutant melanoma, the antitumoractivity of the MEK inhibitor binimetinib is significantlypotentiated by the BRAF inhibitor encorafenib through induc-tion of ER stress, leading to melanoma cell death by apoptoticmechanisms. Encorafenib combined with binimetinib waswell tolerated in a phase III trial showing potent antitumoractivity in BRAF-mutant melanoma, making rapid evaluationin NRAS-mutant melanoma imminently feasible. These dataprovide a mechanistic rationale for evaluation of binimetinibcombinedwith encorafenib in clinical studies on patients withNRAS-mutant metastatic melanoma.

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Clin Cancer Res; 23(20) October 15, 2017 Clinical Cancer Research6204




Cell death ELISACell lysates were prepared from monolayer and spheroid

cultures. Nucleosomal enrichment in the cytosolic fractionduring apoptotic cell death was determined with the CellDeath Detection ELISA Kit (Roche) according to the manufac-turer's instructions and was calculated as: absorbance of samplecells/absorbance of control cells. The enrichment factor of 2corresponds to 10% apoptotic cells as determined by FACSanalysis.

Gene expressionQuantitative real-time PCR (RT-PCR) was performed with the

LightCycler 480 (Roche) using the Kapa SYBR Fast System (VWR).RNA extraction (MACHERY-NAGEL) and reverse transcription(Invitrogen) were performed according to the manufacturer'sinstructions. Twenty-five ng cDNA was used for PCR analyses.Primer sequences are listed in Supplementary Table S3. PCRproduct specificity was verified by melting curve analysis.

Organotypic skin cultureMelanoma cells were seeded onto human dermal recon-

structs as described previously (18). Reconstructs were culturedfor 4 days, treated with the inhibitors for another 10 daysbefore fixation with 4% formalin and embedment in paraffin.Sections were stained with either hematoxylin and eosin (H&E)or a a-Ki67 antibody (DAKO #M7240) and the UltraViewUniversal Alkaline Phosphatase Red Detection kit from Ven-tana (Tucson).

Tumor slice culturesPatient tissue samples were expanded in a patient-derived

xenograft (PDX) mouse model by implanting the digestedtumor tissue subcutaneously, as described previously (22).After expansion, tumors were excised and cut with the LeicaMicrotome VT1200S into 400-mm slices. The slices were treatedfor 4 days with BRAF and MEK inhibitors in quadruplicatesand then analyzed by confocal microscopy. H&E stainings ofthe two NRAS-mutant human and corresponding mousetumors were performed (Supplementary Fig. S6A), revealinga similar appearance and structure of the matching tumortissues.

Immunofluorescence staining and confocal microscopyImmunofluorescence staining was performed as described pre-

viously (23) using a confocal laser scanning microscope (Leica)and the antibodies listed in Supplementary Table S2. Cell nucleiwere stained with YO-PRO-1 (Thermo).

Transmission electron microscopyElectron microscopy was performed as described previously

(9).

Transfection experimentsGenes were knocked down with pre-validated, siRNAs

against ATF4 (Qiagen, SI03019345) and BIM (Cell SignalingTechnology, 6518). As control siRNA a non-silencing, lucifer-ase-specific siRNA (Biomers) was used. Cells were transfectedwith 25 nmol/L siRNA using RiboxxFect (Riboxx) according tothe manufacturer's instructions and were cultured for 6 hoursbefore drug treatment.

Statistical analysisGraphPad Prism version 7.0 (GraphPad Software) was used

for statistical analysis. P value calculation and significancedetermination were performed using one-way ANOVA fol-lowed by the Tukey's multiple comparisons test. P values<0.05 were considered statistically significant, with �, P <0.05; ��, P < 0.01; ��, P < 0.001; and ��, P < 0.0001.

ResultsMEKi combined with BRAFi inhibit ERK phosphorylation aswell as cell growth and induce apoptosis in NRAS-mutantmelanoma cells

To investigate whether BRAFi increased the antitumor activityof MEKi in NRAS-mutant melanoma, we tested a range of mel-anoma cell lines for ERK phosphorylation, growth inhibition andapoptosis induction following treatment with MEKi or/andBRAFi.

As described previously (24), BRAFi (encorafenib, dabrafenib,and vemurafenib) and the panRAFi (Raf265) increased ERKphosphorylation in NRAS-mutant melanoma cells (Supplemen-tary Figs. S1A and S2A). However, theMEKi binimetinib was ableto counteract this paradoxical activation of the MAPK pathway,resulting in complete inhibition of ERK phosphorylation (Fig. 1Aand Supplementary Fig. S2A).

In short-term proliferation assays, encorafenib treatment had aminimal effect on the growth of NRAS-mutant melanoma celllines (SKMel147, WM1366, MelJuso, WM1346) or patient-derived NRAS-mutant melanoma cells, while binimetinib causedmoderate growth inhibition (Fig. 1B and Supplementary Fig.S1B). The combination of encorafenib plus binimetinib achievedsynergistic growth inhibition (Supplementary Table S1) withgrowth-inhibitory rates of >80% (Fig. 1B and Supplementary Fig.S1B). In BRAF-mutant melanoma cells (SKMel19, Mel1617,451Lu), encorafenib or binimetinib alone led to amarked growthinhibition of 75% that was slightly enhanced by combination ofboth inhibitors (Fig. 1B and Supplementary Fig. S1C). Of note,consistent with the paradoxical hyperactivation of the MAPKkinase pathway, BRAFi alone caused acceleration of growth inNRAS-mutant melanoma cell lines in a long-term growth assay,which could be inhibited by addition of a MEK inhibitor (Sup-plementary Fig. S1D).

Cell-cycle analysis of NRAS-mutant melanoma cells revealedthat binimetinib but not encorafenib increased the sub-G1

fraction (apoptotic fraction; Fig. 1C and Supplementary Fig.S1E). The combination of binimetinib plus encorafenib furtherenhanced the number of apoptotic cells up to 40%. A nucle-osomal enrichment assay of cytosolic fractions during apopto-sis showed similar results (Fig. 1D): moderate apoptosis afterbinimetinib treatment alone (fourfold enrichment) that wasfurther enhanced (nearly 10-fold enrichment) with the com-bination of binimetinib plus encorafenib. Both apoptosisassays revealed high apoptosis rates in BRAF-mutant melanomacells with encorafenib or binimetinib alone that were furtherincreased with combination treatment (Fig. 1C and D andSupplementary Fig. S1F).

Of note, combination of binimetinib with Raf265, also led toincreased growth inhibition and apoptosis induction in NRAS-and BRAF-mutant melanoma cells compared with the MEKiand panRAFi single treatments (Supplementary Fig. S2B andS2C).

ER-Stress Induction Sensitizes NRAS-Mutant Melanoma to MEKi

www.aacrjournals.org Clin Cancer Res; 23(20) October 15, 2017 6205




Of relevance for clinical translation of these findings, otherBRAF and MEK inhibitors such as dabrafenib and trametinibcaused similar growth inhibition and apoptosis in NRAS-mutantand BRAF-mutant melanoma cells, respectively (SupplementaryFig. S3A–S3C and Supplementary Table S1).

Together, these data illustrate that BRAFi augment the growthinhibiting and apoptosis-inducing effects of MEKi in NRAS-mutant melanoma cells.

Binimetinibplus encorafenibdoesnot induce apoptosis in cellsfrom normal human skin

In fibroblasts, melanocytes, and keratinocytes, encorafenibtreatment increased ERK phosphorylation, which was partial-ly, and in keratinocytes completely, inhibited with the com-bination treatment (Supplementary Fig. S4A). In contrast withmelanoma cells (Fig. 1B and Supplementary Fig. S1B), growthinhibition rates of normal skin cells after binimetinib or/and

encorafenib treatment remained below 40% (SupplementaryFig. S4B). Furthermore, cell-cycle analyses indicated that apo-ptosis was not induced in these cells with neither treatment(Supplementary Fig. S4C). Our data together with the corre-sponding clinical data (25) show that the effects of BRAFi andMEKi treatment on normal skin cells are tolerable.

MEKi combined with BRAFi display antitumor activities inspheroids, organotypic and tissue slice cultures

To test whether binimetinib plus encorafenib also affectssurvival of NRAS-mutant melanoma cells in a more physio-logical context, we first assessed apoptotic cell death of mela-noma cells in a 3D melanoma spheroid model. As observedin the NRAS-mutant monolayer culture (Fig. 1D), in the spher-oid culture, binimetinib but not encorafenib induced apoptosisthat was further enhanced by the combination treatment(Fig. 1E). In BRAF-mutant cells, binimetinib and encorafenib

Figure 1.

The MEKi binimetinib combined withthe BRAFi encorafenib leads to stronggrowth inhibition and apoptosisinduction in NRAS-mutant melanomacells. A, Whole-cell lysates fromNRAS- or BRAF-mutant melanomacells treated with encorafenib or/andbinimetinib or DMSO as a control for24 hours were subjected to Westernblot analysis to detect pERK, ERK, andb-actin. The experiment shown is arepresentative of three independentexperiments. B, Melanoma cells weretreated with encorafenib or/andbinimetinib for 72 hours. Thepercentage of growth inhibition wascalculated, normalized to the DMSO-treated control. One representativeexperiment of two is shown (mean �SD of quadruplicates). C, Melanomacells were treated with the inhibitorsor DMSOas a control for 72 hours. Cell-cycle analysis was performed anddisplayed in a bar graph withapoptotic cells (sub-G1 fraction) inblack (mean � SD of duplicates fromthree independent experiments).D and E, Melanoma cells grown inmonolayer (D) or spheroids (E) weretreatedwith the inhibitors or DMSO for48 hours. Nucleosomal enrichment incytosolic fractions (apoptotic celldeath)was calculated anddisplayed ina bar graph (mean � SD of triplicatesfrom three independent experiments).P values <0.05 were consideredstatistically significant. � , P < 0.05;��, P < 0.01; ��, P < 0.001; and �� ,P < 0.0001.

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alone induced apoptosis that was further increased by thecombination treatment.

Melanoma cells were seeded onto human dermal reconstructsand treated with encorafenib or/and binimetinib. Histologicalsections of these reconstructs were stained with H&E to identifymelanoma cells (cells with dense, blue nucleus and small cyto-plasm) invading the dermis. In addition, sections were stained forKi67 to visualize proliferating melanoma cells. In NRAS-mutantmelanoma cells, encorafenib did not inhibit proliferation orinvasive tumor growth. Binimetinib inhibited proliferation butnot invasion of the tumor cells into the skin reconstructs, whilebinimetinib plus encorafenib completely abolished proliferationand invasive tumor growth (Fig. 2A). In BRAF-mutant melanomacells, encorafenib and binimetinib were effective as mono- orcombination treatment.

To demonstrate the efficacy of this combination treatment onNRAS-mutant melanoma metastases in tissue context (Supple-mentary Fig. S6A), tumor tissue excised from two patients withNRAS-mutant melanoma was expanded in a mouse PDX modeland then cut into slices. After treatment of the sliceswithMEKi andBRAFi, the tumor cells in these tissue slice cultures displayedproliferation inhibition and apoptosis induction compared withthe untreated controls (Fig. 2B). Furthermore, in monolayer cellcultures derived from the same tumor tissue, MEKi combinedwith BRAFi significantly enhanced growth inhibition (>80%)compared with MEKi alone (Fig. 2C). These results underscorethe therapeutic potential of this combination treatment forpatients with NRAS-mutant melanoma.

BRAFi induces ER swelling and upregulation of ER stress-related transcription factors in NRAS-mutant melanoma cells

We next determined whether BRAFi caused ER stress in NRAS-mutant melanoma, as we previously described for BRAF-mutantmelanoma (9).

Using electron microscopy, we found that dabrafenib,vemurafenib, and in particular, encorafenib induced morpho-logical features of ER stress with significant dilation of the ER inboth NRAS-mutant and BRAF-mutant cells (Fig. 3A). Similareffects on the ER were observed with the combination ofencorafenib plus binimetinib, while binimetinib alone did notappear to affect the ER.

Upon ER stress, the sensor PERK is activated, leadingto phosphorylation of eIF2a. Encorafenib alone or in combi-nation with binimetinib caused a 1.5- to twofold increase inpeIF2a in both NRAS-mutant melanoma cell lines, whilepeIF2a was increased seven- to 12-fold in the BRAF-mutantmelanoma cell line (Fig. 3B).

Encorafenib and more pronounced encorafenib plus binime-tinib also induced expression of the ER stress–related transcrip-tion factor genes ATF4, CHOP, and NUPR1 downstream of eIF2aphosphorylation in the NRAS-mutant melanoma cells (Fig. 3C).In BRAF-mutant cells, treatment with encorafenib alone or incombination increased expression of these genes. Similar resultswere obtained with dabrafenib and trametinib (SupplementaryFig. S3D). In addition, combination of Raf265 and binimetinibalso led to increased expression of the ER stress–related transcrip-tion factors (Supplementary Fig. S2D). Expression of NUPR1,ATF4, and CHOP proteins was elevated with encorafenib andencorafenib plus binimetinib in NRAS- and BRAF-mutant mela-noma cells, whereas binimetinib alone only minimally affectedlevels of these ER stress factors (Fig. 3D).

In addition to activation of the PERK pathway, ER stress alsoinduces activation of the ATF6 and IRE1 pathway, which can bemonitored by cleavage of ATF6 and by phosphorylation of IRE1a.Interestingly, in contrast with the classical ER stress inducerthapsigargin, encorafenib, and/or binimetinib treatment did notcause phosphorylation of IRE1a or cleavage of ATF6 (Supple-mentary Fig. S5).

Altogether, these data indicate that BRAFi trigger an ER stressresponse via the PERK pathway in melanoma cells.

Apoptosis induction by binimetinib combined withencorafenib ismediatedby caspase 9 and caspase 3activation inNRAS-mutant melanoma cells

So far, we have determined that BRAFi cause ER stress and boostapoptosis induced byMEKi in NRAS-mutant melanoma cells. Wethus wanted to further investigate the underlying mechanismsinvolved in apoptosis induction.

Inhibition of caspase activity with the pan-caspase inhibitorQVDalmost completely abrogated apoptosis induction caused bytreatment with binimetinib alone or in combination with encor-afenib in the NRAS-mutant cells, and also inhibited apoptosisinduced by any treatment in the BRAF-mutant cells (Fig. 4A).

Performing Western blot analysis, we next characterized theactivation of members of the intrinsic caspase cascade. In NRAS-mutantmelanoma cells, processing of caspase 9, caspase 3 and thecaspase product PARP was observed in response to binimetinibtreatment alone, but was much more distinct when combinedwith encorafenib (Fig. 4B). In contrast, encorafenib treatmentalone did not show processing of these proteins. In BRAF-mutantmelanoma cells, all 3 treatment strategies caused cleavage ofcaspase 9, caspase 3, and PARP. These results indicate that thecombination of binimetinib plus encorafenib induces apoptosisvia the intrinsic mitochondrial pathway in NRAS-mutant mela-noma cells.

Binimetinib combined with encorafenib upregulate theproapoptotic proteins BIM and PUMA in NRAS-mutantmelanoma cells

Proapoptotic Bcl-2 proteins like BIM, PUMA, and NOXAmediate the activation of the intrinsic mitochondrial apoptosispathway. Induction of BIM by MEKi has been reported (26),prompting us to investigate whether BIM is induced in NRAS-mutant melanoma cells following MEKi or/and BRAFi. InNRAS-mutant melanoma cells, binimetinib alone and in com-bination with encorafenib increased the expression of the threeBIM isoforms, whereas encorafenib alone showed no effect onany BIM isoform (Fig. 4C). In BRAF-mutant melanoma cells,binimetinib, or/and encorafenib resulted in an increase of thethree BIM isoforms. Analogous with BIM, binimetinib aloneand in combination with encorafenib caused downregulationof the antiapoptotic proteins MCL-1 and BCL-XL and theproapoptotic protein NOXA in NRAS-mutant cells, whereasencorafenib had a minimal effect on these Bcl-2 proteins(Supplementary Fig. S7).

PUMA has been reported to mediate ER stress-induced apo-ptosis (27, 28). In both NRAS- and BRAF-mutant melanomacells, elevated levels of PUMA were detected following treat-ment with encorafenib or encorafenib plus binimetinib but notwith binimetinib alone, suggesting that PUMA is upregulatedby encorafenib through ER stress induction (Fig. 4C). In accor-dance with this, PUMA expression was also increased in NRAS-






mutant tissue slice cultures after treatment with BRAFi andMEKi compared with the untreated controls (SupplementaryFig. S6B).

Together these data indicate that in NRAS-mutant melanomacells the MEKi binimetinib induces the proapoptotic protein BIMand the BRAFi encorafenib induces the proapoptotic protein

Figure 2.

MEKi combinedwithBRAFi inhibit invasive tumor growth in organotypic skin cultures and lead to apoptosis induction and reduction in proliferation inpatient-derivedtumor slice cultures.A,Melanoma cells were seeded onto organotypic skin reconstructs, incubated for 4 days and then treated for 10 dayswith DMSO (control) or 0.1(BRAF-mutant cells) and 1mmol/L (NRAS-mutant cells) encorafenib or/and binimetinib. Fixed tissue sectionswere stainedwithH&Eor Ki67. Anorange linemarks thetumor front. T, tumor cells; D, dermis; Scale bar, 10 mm. B, Tumor tissue from patients with NRAS-mutant melanoma was expanded in a PDX model, sliced, and thentreated with the indicated inhibitor combinations. After 4 days, the slices were stained with YO-PRO-1 (nuclei marker), Ki67 (proliferation marker), or cleaved PARP(apoptosis marker) and analyzed by confocal microscopy. Images of one representative per treatment group are shown. The percentage of apoptotic andproliferating cells was quantified and displayed in a bar graph (error bars represent SD of four random sites in each tumor per treatment group). C, NRAS-mutantpatient-derivedmelanoma cellswere treatedwith the indicated inhibitors for 72 hours. The percentage of growth inhibitionwas calculated, normalized to the DMSO-treated control (mean � SD of quadruplicates).

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Figure 3.

BRAF inhibitors alone and in combination with MEK inhibitors induce ER swelling, eIF2a phosphorylation, and expression of NUPR1, ATF4, and CHOP.A, Electron microscopy of melanoma cells treated with dabrafenib (1 mmol/L), vemurafenib (6 mmol/L), encorafenib (1 mmol/L), or/and binimetinib (1 mmol/L)or DMSO for 6 hours. The cell nuclei are shown in blue. The endoplasmic reticulae are shown in green and additionally marked by red arrows; scalebar, 0.5 mm; n ¼ 2. B, Whole-cell lysates from melanoma cells treated with the inhibitors or DMSO as a control for the indicated times were subjected toWestern blot analysis to detect peIF2a, eIF2a, and b-Actin. Experiment shown is a representative of two independent experiments. Quantificationof the peIF2a band intensities in comparison with eIF2a is displayed in the bottom row. C, Melanoma cells were treated with the inhibitors for 18 hours.Real-time PCR results show levels of NUPR1, ATF4, and CHOP mRNA expression in comparison with DMSO-treated cells. All samples were normalized to TBPmRNA (mean � SD of triplicates from three independent experiments). D, Melanoma cells were treated with the inhibitors or DMSO for 24 hours. Whole-celllysates were subjected to Western blot analysis of NUPR1, ATF4, and CHOP in comparison with b-actin as loading control. Experiment shown is arepresentative of three independent experiments.






Figure 4.

Binimetinib- and encorafenib-induced apoptosis is mediated by the proapoptotic BH3-only protein BIM and ATF4. A, Melanoma cells were pretreated for 1 hourwithout or with 1 mmol/L pan-caspase inhibitor QVD-OPH and then treated with encorafenib or/and binimetinib or DMSO as control for 72 hours. Cell-cycle analysiswas performed and displayed in a bar graph with apoptotic cells (sub-G1 fraction) in black (mean � SD of duplicates from three independent experiments). B,Melanoma cells were treatedwith the inhibitors or DMSO for the indicated time points.Western Blot analysis of whole-cell lysateswas performed to detect cleavageof caspase 3 and 9 aswell as PARP compared with b-actin as a loading control. Experiment shown is a representative of two independent experiments. C,Melanomacellswere treatedwith the inhibitors or DMSO for 24 hours.Western Blot analysis ofwhole-cell lysateswas performed to detect BIM isoforms (EL, L, and S) andPUMAcompared with b-actin as a loading control. Experiment shown is a representative of two independent experiments. (Continued on the following page.)

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PUMA, possibly causing the marked apoptosis seen in NRAS-mutant cells after combination treatment.

ATF4 and BIM play important roles in apoptosis induction bybinimetinib and encorafenib

Wepreviously showed that both, the proapoptotic protein BIMand the ER stress–related factor ATF4 play relevant roles inapoptosis induction by the vemurafenib in BRAF-mutant mela-noma cells (9).

In NRAS-mutantmelanoma cells treated with binimetinib plusencorafenib, knockdown of BIM by siRNA inhibited upregulationof all three BIM isoforms (Fig. 4D) and significantly suppressedapoptosis (Fig. 4G).

In NRAS-mutantmelanoma cells treated with binimetinib plusencorafenib, siRNA-mediated knockdown of ATF4 reduced notonly ATF4 itself (Fig. 4E), but also other ER-stress related factorssuch as CHOP and NUPR1 as well as the proapoptotic proteinPUMA (Fig. 4F). Furthermore, ATF4 knockdown significantlyinhibited apoptosis in these cells (Fig. 4G).

These findings indicate that the proapoptotic protein BIM andthe ER stress-related factor ATF4 are important factors in NRAS-mutant melanoma cells during induction of apoptosis by bini-metinib plus encorafenib.

NRAS-mutant cells resistant to encorafenib plus binimetinibshow hyperactivation of the MAP kinase pathway andadaptation to ER stress

We established NRAS-mutant WM1366 melanoma cell linesresistant to encorafenib, binimetinib or the combination of bothdrugs.

Double-drug–resistant cells showing neither growth inhibitionnor apoptosis following treatment with encorafenib or/and bini-metinib were obtained after 5 to 6 months of treatment (Fig. 5Aand B). Interestingly, single-drug–resistant cells were partiallysensitive to the combination treatment in terms of growth inhi-bition and apoptosis induction (Supplementary Fig. S8A andS8B), proposing the use of upfront combination therapy inNRAS-mutant melanoma.

Double-drug–resistant cells displayed a strong increase inphosphorylated ERK, demonstrating hyperactivation of theMAPK pathway that could not be counteracted by treatment withbinimetinb plus encorafenib (Fig. 5C). In double-drug resistantcells treated with binimetinib plus encorafenib, expression ofATF4, CHOP, andNUPRwas only slightly increased and BIM andPUMA were only marginally altered (Fig. 5C and D and Supple-mentary Fig. S8C). Furthermore, combination treatment hardlyled to PARP cleavage in these cells (Fig. 5C), consistent with theobserved lack of apoptotic cell death.

These data illustrate the relevance and interplay of significantER stress induction by BRAFi and robust MAP kinase pathwayinhibition byMEKi to induce efficient apoptosis in NRAS-mutantmelanoma cells.

DiscussionFinding effective therapies for patients with aggressive NRAS-

mutant melanoma is one of the current major aims in the field ofmelanoma research. In a recent phase III study (7), the MEKibinimetinib was superior to chemotherapy with dacarbazine butfailed to establish an overall survival benefit. However, the dem-onstrated antitumor activity of binimetinib provides a solid basisfor combination treatment strategies in patients with NRAS-mutant metastatic melanoma.

The present study shows for the first time that BRAF inhibitorscan augment the antitumor effects of MEK inhibitors in NRAS-mutant melanoma. Although BRAF inhibitors like encorafenibproduce MAPK activation, co-administration of a MEK inhibitorcan not only overcome this effect, but also lead to greater cellkilling than observed with MEK inhibition alone. The potentgrowth-inhibiting and proapoptotic effects of BRAFi combinedwith MEKi on tissue slice cultures of metastases derived fromNRAS-mutant melanoma patients further supports the potentialclinical relevance of the combination strategy in this patient groupgiven the inclusion of additional components of the tumormicroenvironment.

This study also provides thefirst insight into themechanisms ofBRAF and MEK inhibitor activity in NRAS-mutant melanomacells. ER stress induction by BRAFi together with MAPK pathwayinhibition by MEKi culminate in the activation of the mitochon-drial apoptosis–inducing pathway (Fig. 6). The decisive role of theER stress and MAPK pathways during BRAFi and MEKi treatmentis further strengthened by the observed hyperactivation of theMAPK pathway, abrogation of ER stress induction and lack ofapoptosis in treated double-drug–resistant NRAS-mutant mela-noma cells.

First data on the mechanisms that underlie ER stress inductionby BRAFi were provided byMa and colleagues (10). They reportedthat BRAFi enhance binding of themutant BRAFV600E protein tothe chaperone protein GRP78, thereby reducing GRP78 bindingto PERK and promoting activation of PERK. The data from ourstudy demonstrate that BRAFi also cause activation of the PERKsub-pathway in NRAS-mutant melanoma, indicating that ERstress may be initiated in a similar manner in a BRAFwt setting.The fact that BRAFi such as vemurafenib can also bind to BRAFwt(29) would support this hypothesis.

Cerezo and colleagues (30) reported on the development of thesmall-molecule HA15 that displayed antitumor activity in bothBRAF-mutant and NRAS-mutant melanoma cell lines and inmelanoma xenograft mouse models. HA15 appears to interactdirectly with the chaperone protein GRP78, leading tomelanomacell death by significantly increasing ER stress and activating theUPR. Analogous to our findings with BRAFi in BRAF-mutant (9)and NRAS-mutant melanoma (this study), HA15 caused rapidphosphorylation of the downstream target of PERK, eIF2a, andexpression of the downstream transcription factors ATF4 and

(Continued.)D,Melanoma cellswere transfectedwith siCtrl or siRNAdirected against BIMbefore treatmentwith encorafenib plus binimetinib or DMSO.Western blotanalysis of whole-cell lysates was performed after 24 hours to detect BIM isoforms compared with b-actin as a loading control. The experiment shown is arepresentative of three independent experiments. E and F, Melanoma cells were transfected with siCtrl or siRNA directed against ATF4 before treatment with orwithout (DMSO) encorafenib plus binimetinib. E,Western blot analysis ofwhole-cell lysateswas performed after 24 hours to detect ATF4 comparedwith b-actin as aloading control. Experiment shown is a representative of three independent experiments. F, After 18 hours, real-time PCR was performed to measure ATF4, NUPR1,CHOP, and PUMA mRNA expression in comparison with DMSO-treated cells. All samples were normalized to TBP mRNA (one representative experiment ofthree is shown, mean � SD of triplicates). G, Melanoma cells were transfected with control, siRNA directed against BIM or siRNA directed against ATF4 beforetreatment with encorafenib plus binimetinib or DMSO. Cell-cycle analysis was performed, and the sub-G1 fraction was displayed in a bar graph (mean � SD ofduplicates from three independent experiments). P values <0.05 were considered statistically significant. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; and �� , P < 0.0001.






CHOP. In addition to activating the PERK pathway, HA15also caused activation of the ATF6 and IRE1 branches of the ERstress pathway, whereas the action of the BRAFi encorafenibwas confined to the PERK branch. Activation of individual armsof the UPR has also been described for other cellular stresses,indicating that different factors can trigger unique ER stresspathway profiles (31).

Altogether, these findings highlight the key role of the ER stressaxis/UPR pathway in melanoma and strengthen the idea that ERstress inducers could be useful in melanoma treatment.

Interestingly, HA15 induced both apoptosis and autophagy,culminating in cell death that could be blocked by QVD andsiRNA against the autophagy protein LC3 (30). In our system,BRAFi alone was not capable of inducing apoptosis in NRAS-mutant melanoma. However, combinations of BRAFi and MEKidisplayed apoptosis rates higher than those achieved by theMEKialone, suggesting that ER stress induced by BRAFi leads to apo-ptosis when combined with additional cell death triggers. Long-term ER stress has been shown to cause apoptotic cell deaththrough ATF4-CHOP–mediated induction of proapoptotic orsuppression of antiapoptotic members of the Bcl-2 family (28,32), the guardians of the mitochrondrial pathway of apoptosis.The proapoptotic Bcl-2 proteins BIM, PUMA, and NOXA areknown to play a role in ER stress-mediated apoptosis (27). In

our study, encorafenib treatment increased the expression ofATF4, CHOP, and NUPR1 and induced the expression of PUMA.Furthermore, silencing ATF4 using siRNA reduced transcription ofCHOP, NUPR1, and PUMA, resulting in diminished apoptosisfollowing treatment with encorafenib plus binimetinib. This isconsistent with two reports showing that siRNA against ATF4reduces NUPR1 expression and that ATF4-CHOP–mediated ER-stress causes neuronal apoptosis via PUMA induction (28, 33).

Our data demonstrate that binimetinib induces cell deaththrough the mitochondrial pathway of apoptosis in NRAS-mutant melanoma. Binimetinib caused activation of the caspase9 and caspase 3 and cleavage of PARP. The apoptotic activity ofbinimetinib was enhanced by combination treatment with encor-afenib, possibly via increased expression of PUMA, as discussedabove. Binimetinib did not induce PUMA, but increased expres-sion of BIM (all three isoforms). Importantly, knockdown of BIMusing siRNA diminished apoptosis induction by the combinationtreatment.

In agreement with our data, inhibition of the MAPK pathwaywas found to increase the expression of BIM and to induceapoptosis in melanoma cells (26). In accordance, Jiang andcolleagues (34) showed that increased expression of the shortBIM isoform, BIM S, is a key mechanism underlying apoptosisinduction by the BRAFi PLX4720.

Figure 5.

NRAS-mutant cells resistant to thecombination of encorafenib andbinimetinib show hyperactivation ofthe MAP kinase pathway andadaptation to ER stress. Melanomacells resistant or sensitive toencorafenib plus binimetinib weretreated with or without (DMSO) thiscombination. A, After 72 hours, thepercentage of growth inhibition wascalculated, normalized to the DMSO-treated control. One representativeexperiment of two is shown (mean �SD of quadruplicates). B, After72 hours, cell-cycle analysis wasperformed and displayed in a bargraph with apoptotic cells (sub-G1

fraction) in black (mean � SD ofduplicates from three independentexperiments). C, After 24 hours,Western blot analysis of whole-celllysates was performed to detectpERK, ERK, ATF4, BIM EL, L, S, PUMA,cleaved PARP and PARP comparedwith b-actin as a loading control.Experiment shown is a representativeof two independent experiments.D, After 18 hours, real-time PCR wasperformed to measure ATF4, NUPR1,CHOP mRNA expression incomparison with DMSO-treated cells.All samples were normalized toTBP mRNA (one representativeexperiment of two is shown, mean �SD of triplicates).

Niessner et al.





Altogether, we have shown that MEKi induces proapoptoticBIM and that BRAFi induces proapoptotic PUMA, which togetherpromote apoptosis in NRAS-mutant melanoma.

In summary, we demonstrate that BRAFi cause ER stress inNRAS-mutant melanoma cells, thereby sensitizing these cells toMEKi-induced apoptosis. This study thus provides a rationale forinvestigating MEKi and ER stress inducing BRAFi in furtheradvanced in vivo studies followed by clinical studies on patientswith NRAS-mutant melanoma. ER stress inducers such as thapsi-gargin have been shown to induce severe skin irritation, salivaryhypersecretion, gastroenteritis, vomiting, and in severe cases leadto death (35). Instead, we observed no detrimental effects on theviability of normal human fibroblasts or melanocytes with BRAFiand MEKi combinations. In addition, hyperactivation of theMAPK pathway in keratinocytes caused by encorafenib was abol-ished in combination with the binimetinib. These findings areconsistent with data from a phase III trial showing that in BRAF-mutant melanoma patients the frequency of hyperproliferativeskin lesions including keratoacanthomas or squamous cell carci-nomas is lower under combined encorafenib and binimetinibtherapy compared with encorafenib monotherapy (36). In addi-tion, grade 3/4 events and the discontinuation rate due to adverseevents are also lower in the combination compared with themonotherapy. Numerous clinical trials and real-world experienceconfirm that combinations of BRAFi (dabrafenib, vemurafenib,encorafenib) with MEKi (trametinib, cobimetinib, binimetinib)have a favorable safety profile and are generally well tolerated inthe long run (37–39).

Disclosure of Potential Conflicts of InterestC. Garbe reports receiving speakers bureau honoraria from and is a consul-

tant/advisory board member for Amgen, Bristol-Myers Squibb, MSD, Novartis,Philogen and Roche, and reports receiving commercial research grants fromBristol-Myers Squibb, Novartis and Roche. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: H. Niessner, K.S.M. Smalley, D. Beck, C. Garbe,I. Wanke, F. MeierDevelopment of methodology: H. Niessner, T. Sinnberg, D. Beck, D. Kulms,I. Wanke, F. MeierAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Niessner, T. Sinnberg, C. Kosnopfel, M. Mai,D. Kulms, M. Schaller, D. Westphal, I. Wanke, F. MeierAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Niessner, C. Praetorius, S. Beissert, M. Schaller,C. Garbe, K.T. Flaherty, D. Westphal, I. Wanke, F. MeierWriting, review, and/or revision of the manuscript: H. Niessner, T. Sinnberg,K.S.M. Smalley, C. Praetorius, S. Beissert, D. Kulms, M. Schaller, C. Garbe,K.T. Flaherty, D. Westphal, I. Wanke, F. MeierAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Niessner, D. Kulms, C. Garbe, D. Westphal,F. MeierStudy supervision: H. Niessner, F. Meier

AcknowledgmentsWewould like to thank Theresia Schneider, Renate Nordin, Astrid Odon and

Birgit Fehrenbacher for outstanding assistance in electron microscopy.

Grant SupportThe authors of this publicationwere supported by theUniversity of T€ubingen

(fort€une 2224-0-0; to H. Niessner), the Federal Ministry of Education andResearch (FKZ 031A423B; to C. Praetorius, D. Kulms, and F. Meier) and theNational Center for Cancer/NCTProgramand InfrastructureGrant (to F.Meier).K.S.M. Smalley is supported by NIH grants R01 CA161107-01 and P50CA168536.

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.

Received January 11, 2017; revised May 25, 2017; accepted July 11, 2017;published OnlineFirst July 19, 2017.

Figure 6.

Proposed mechanism of MEK andBRAF inhibitor–dependent inductionof apoptosis in NRAS-mutantmelanoma cells. BRAF inhibitors suchas encorafenib activate the MAPKpathway (increase of pERK) inNRAS-mutant melanoma cells. Inaddition, they induce ER stress,thereby activating the downstreamERstress pathway, includingphosphorylation of eIF2a andexpression of ER stress-related genesand proteins such as ATF4, NUPR, andCHOP as well as the proapoptotic Bcl-2 family member PUMA. Inhibitors oftheMAPKpathway such asbinimetinibare able to counteract the increasedERK phosphorylation by BRAFi andalso cause upregulation of theproapoptotic Bcl-2 family memberBIM. Both Bcl-2 proteins affect themitochondrial pathway of apoptosis,causing an activation of caspasesto induce PARP cleavage andultimately apoptosis in NRAS-mutantmelanoma cells.






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2017;23:6203-6214. Published OnlineFirst July 19, 2017.Clin Cancer Res Heike Niessner, Tobias Sinnberg, Corinna Kosnopfel, et al. by Inducing ER Stress in NRAS-Mutant MelanomaBRAF Inhibitors Amplify the Proapoptotic Activity of MEK Inhibitors

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