BRAF and MEK Inhibitors Increase PD-1-Positive Melanoma Cells … · Figure 1. Melanoma cells express PD-1. A, Box plots of PD-1, PD-L1, and PD-L2 Affymetrix mRNA expression values

Translational Cancer Mechanisms and Therapy

BRAF and MEK Inhibitors Increase PD-1-PositiveMelanoma Cells Leading to a PotentialLymphocyte-Independent Synergism withAnti–PD-1 AntibodyMartina Sanlorenzo1,2,3, Igor Vujic4,5, Arianna Floris1, MauroNovelli2, Loretta Gammaitoni6,Lidia Giraudo6, Marco Macagno1,6, Valeria Leuci1,6, Ramona Rotolo1,6, Chiara Donini1,Marco Basiric�o6, Pietro Quaglino2, Maria Teresa Fierro2, Silvia Giordano1,7, Maria Sibilia3,Fabrizio Carnevale-Schianca6, Massimo Aglietta1,6, and Dario Sangiolo1,6

Abstract

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor mel-anoma-infiltrating lymphocytes, providing the rationale forcurrent combinatorial trials with anti–PD-1 antibody. A por-tion of melanoma cells may express PD-1, and anti–PD-1antibody could have a direct antitumor effect. Here, we explorewhether BRAF/MEKi modulate rates of PD-1þ melanoma cells,supporting an additional—lymphocyte-independent—basisfor their therapeutic combination with anti–PD-1 antibody.

Experimental Design:With data mining and flow cytometry,we assessed PD-1, PD-L1/2 expression on melanoma cell lines(CCLE, N ¼ 61; validation cell lines, N ¼ 7) and melanomatumors (TCGA, N ¼ 214). We explored in vitro how BRAF/MEKi affect rates of PD-1þ, PD-L1/2þ melanoma cells, andcharacterized the proliferative and putative stemness features ofPD-1þ melanoma cells. We tested the functional lymphocyte-independent effect of anti–PD-1 antibody alone and in com-

bination with BRAF/MEKi in vitro and in an in vivo immuno-deficient murine model.

Results: PD-1 is consistently expressed on a small subset ofmelanoma cells, but PD-1þ cells increase to relevant rates duringBRAF/MEKi treatment [7.3% (5.6–14.2) vs. 1.5% (0.7–3.2),P ¼ 0.0156; N ¼ 7], together with PD-L2þ melanoma cells[8.5% (0.0–63.0) vs. 1.5% (0.2–43.3), P ¼ 0.0312; N ¼ 7]. PD-1þ cells proliferate less than PD-1� cells (avg. 65% less; t¼ 7 days)and are preferentially endowed with stemness features. In vivo, thedirect anti-melanoma activity of PD-1 blockage as monotherapywas negligible, but its association with BRAF/MEKi significantlydelayed the development of drug resistance and tumor relapse.

Conclusions: BRAF/MEKi increase the rates of PD-1þ melano-ma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies withanti–PD-1 antibody. Clin Cancer Res; 24(14); 3377–85. �2018 AACR.

IntroductionMetastatic melanoma is still deadly, despite novel immuno-

modulatory and protein kinase inhibitor therapies. In preclinicalstudies, combinations of anti–PD-1 antibody and target therapywith BRAF/MEK inhibitors (BRAF/MEKi) had synergistic effects,explained by an increased number and activity of tumor-infiltrat-

ing lymphocytes (1, 2). This increase of tumor-infiltrating lym-phocytes following BRAF/MEKi treatment is well documented(3, 4), but the tumors may evade the immune system throughexpressionof programmeddeath-receptor-ligand 1 (PD-L1) and2(PD-L2). These ligands bind and activate the programmed death-receptor 1 (PD-1) on T-lymphocytes and suppress the antitumorresponse (5), whereas its blockage—by anti–PD-1 antibody—restores the antitumor effect.

However, it was suggested that PD-1 is "ectopically" expressedalso on melanoma cells, and that its activation could promotetumor growth (6, 7). The biological relevance of these findings isstill not clear, but PD-1þ melanoma cell subsets were found topreferentially express tumor-initiating determinants (6, 7). Suchputative cancer stem cells could contribute to the development ofdrug resistance and tumor relapse (8–10), which is a main issuefor patients treated with BRAF/MEKi (11–13). In fact, after aninitial rapid anti-tumor response, most patients experience dis-ease progression despite ongoing treatment (11–13). Therefore,there is the need to elucidate the relevance of PD-1þ melanomacells during BRAF/MEKi treatment, and to define therapeuticapproaches, which could contrast the development of resistanceto target therapies.

Here, we evaluate the "ectopic" melanoma-intrinsic PD-1expression and show that PD-1þ and PD-L2þ melanoma cells

1Department of Oncology, University of Turin, Turin, Italy. 2Department ofMedical Sciences, Section of Dermatology, University of Turin, Turin, Italy.3Institute of Cancer Research, Department of Medicine I, Comprehensive CancerCenter, Medical University of Vienna, Vienna, Austria. 4The RudolfstiftungHospital, Department of Dermatology, Vienna, Austria. 5Department of Derma-tology, Medical University of Vienna, Vienna, Austria. 6Division of MedicalOncology—Experimental Cell Therapy, Candiolo Cancer Institute, FPO—IRCCS,Candiolo, Turin, Italy. 7CancerMolecular Biology, CandioloCancer Institute, FPO-IRCCS, Candiolo, Turin, Italy.

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

Corresponding Author: Martina Sanlorenzo, University of Turin, Candiolo Can-cer Institute, FPO-IRCCS, Km 3,95, SP142, 10060 Candiolo (Turin), Italy. Phone:39 011 9933521; Fax: 39 011 9933522; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-1914

�2018 American Association for Cancer Research.

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increase during BRAF/MEKi treatment, sensitizing tumor cells todirect anti–PD-1 antibody effects, thus delaying the developmentof resistance to target therapy.

Materials and MethodsCancer cell line encyclopedia

We extracted from the cancer cell line encyclopedia (CCLE) themRNA expression values of the 61 available melanoma cell lines(http://www.broadinstitute.org/ccle, access September 2015).

The Cancer Genome AtlasWedownloaded fromTheCancerGenomeAtlas (TCGA)portal

the clinical data (clinical data, pathology report) and the mRNAsequencing data (gene) of the 470 melanoma samples included(https://portal.gdc.cancer.gov/, access September 2015). ThemRNA sequencing data were available for 469 samples. Geneswith FPKM values >0.1 were considered expressed (14). Wematched each mRNA sequencing file with the correspondingclinical and histologic data to divide the melanomas in primary,regional metastases and distant metastases and to exclude fromfurther analyses all the samples with histologic evidence ofimmune infiltrate.

Cell linesMelanoma cell lines SKMEL2, SKMEL5, SKMEL28 were

obtained from NCI-Frederick Cancer/DCTD Tumor Repositoryin 2011, and A375 (ATCC CRL-1619) from the ATCC in2013. Cell line identity was performed by the bank of originusing morphology, karyotyping, and PCR-based approaches.Mycoplasma detection was performed after cell thawing by theUniversal Mycoplasma Detection Kit ATCC (ATCC-30-1012K)according to the manufacturer's instructions. Cell lines for experi-mentswereobtained from theoriginal cryopreserved golden stockand experiments performed immediately after and for no longerthan 6 months, no further cell identification was performed.SKMEL2, SKMEL5, and SKMEL 28 were maintained in RPMI(Sigma Aldrich) supplemented with 10% FBS (Gibco BRL).Melanoma cell line A375 cell was maintained in DMEM (GibcoBRL) with the addition of 2mmol/L glutamine and 15% FBS(Gibco BRL). All the cells were propagated at 37�Cunder 5%CO2.Cells were passaged and harvested from flasks using Accutasesolution (Gibco BRL).

Patient-derived samplesPatient-derived melanoma cell cultures (mMel2, mMel3,

mMel7, and mMel11) were generated from surgical biopsies ofmetastatic/locally advancedmelanoma, before any systemic treat-ment (December 2010–June 2012). All patients provided consentunder institutional review board approved protocols. Technical

procedures andmelanoma cell cultures were previously described(10, 15). Mycoplasma detection was performed by MycoplasmaPCR Detection Kit (Applied Biological Materials Inc., MICRO-TECH s.r.l.) according to the manufacturer's instructions. Thetest was done after cell thawing/just before the experimentexecution. All the experiments were performed on patient-derived cell culture of not more than 24-week-old.

Generation of hOct4.eGFP transduced cell linesThe previously described lentiviral vector (14) was transduced

in melanoma primary cells resuspended in fresh KODMEM-F12(GibcoBRL)with 10%FBSadding virus-conditionedmediumat adose of 400 ng P24/100,000 cells. The lentiviral vector pRRL.sin.PPT.hOct4.eGFP.Wpre (LV-Oct4.eGFP) was obtained as previ-ously described (15). Briefly, the hOct4-eGFP cassette fromphOct4.eGFP1 vector (ref. 16; kind gift from Dr. Wei Cui, IRDB,Imperial College, London) was cloned into the transfer vectorpRRL.sin.PPT.hPGK.eGFP.Wpre (ref. 17; kindly provided byDr. Elisa Vigna, IRCCS Candiolo/University of Turin, Italy) inplace of the hPGK.eGFP cassette. After 16 hours, cells werewashedtwice and grown for a minimum of 10 days to reach steady-stateeGFP expression and to rule out pseudotransduction before flow-cytometry analysis. Technical procedure including transductioncontrols were previously described (10).

DrugsThe BRAF inhibitor dabrafenib (GSK2118436) and the MEK

inhibitor trametinib (GSK1120212) were purchased from Sell-eckchem. The anti–PD-1 antibody is the inVivoMAb anti-humanPD-1(CD279), Clone: J110 from Bio X Cell. The isotype controlantibody is the inVivoMAb mouse IgG1 isotype control, Clone:MOPC-21 from Bio X Cell. Drugs were used accordingly toprevious reports (7, 18, 19).

Flow cytometryAnalyses of melanoma cells were performed using a CyanADP

cytometer (BeckmanCoulter s.r.l.). Thefluorochrome-conjugatedmonoclonal antibodies included anti-PD-L1 PE (clone MIH1);anti-PD-L2 APC (clone MIH18); anti–PD-1 APC (clone MIH4)from BD Biosciences. The negative staining threshold was estab-lished by the addition of an isotype-matched control tube.

In vitro proliferation assay and CFSE stainingTo evaluate the proliferation rate, cells had been labeled with

5(6)-Carboxyfluorescein diacetateN-succinimidyl ester (CFSE),for which fluorescence intensity decreased by half at each celldivision per kit protocol (Sigma-Aldrich). Briefly, the CFSE dyesolution was prepared accordingly to the number of cells tostain and added to the previously washed cell pellet. After a first15-minutes incubation at 37�C, cells were washed once withculture medium added with 10% heat-inactivated serum andincubated in culture medium added with 10% heat-inactivatedserum for 30 minutes at 37�C. An aliquot of these labeled andcounted cells was read on a Flow Cytometry Cyan (Cyan ADP,Beckman Coulter s.r.l.) and analyzed using Summit Software(Daki Cytomation, Heverlee, Belgium) to set the baselinefluorescence level. The remaining cells were seeded in cultureunder experimental conditions. After 4 and 7 days, the reduc-tion in fluorescence was quantified by flow cytometry. In case ofdrug treatments, treated cells were compared with untreatedcells which were also labeled with the same dye.

Translational Relevance

BRAF and MEK inhibitors lead to increased rates of mela-noma cells "ectopically" expressing PD-1, supporting a lym-phocyte-independent antitumor effect of anti–PD-1 antibody.This provides further rationale for BRAF and MEK inhibitors/anti–PD-1 antibody combination therapies in metastatic mel-anoma patients.

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Clin Cancer Res; 24(14) July 15, 2018 Clinical Cancer Research3378



http://www.broadinstitute.org/cclehttps://portal.gdc.cancer.gov/http://clincancerres.aacrjournals.org/

Figure 1.

Melanoma cells express PD-1. A, Box plots of PD-1, PD-L1, and PD-L2 Affymetrix mRNA expression values of 61 melanoma cell lines included in theCCLE. B, Bar graph of PD-1 expression values compared with s100B in the same 61 melanoma cell lines. C, Box plots representing levels of PD-1 in the61 melanoma cell lines and in the 18 T-cell neoplasia cell lines included in CCLE. D, Flow chart of the TCGA data analysis. Samples with histologicimmune infiltrate were excluded from further analyses. E, Box plots of PD-1, PD-L1, and PD-L2 mRNA expression values of 214 melanomas without histologicevidence of immune infiltrate.

BRAF and MEK Inhibitors Increase PD-1–Positive Melanoma

www.aacrjournals.org Clin Cancer Res; 24(14) July 15, 2018 3379



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Sanlorenzo et al.





In vitro cell viability assayTo test the number of viable, metabolically active cells after

treatment with BRAF/MEKi alone or in combination with anti–PD-1 antibody we used a method based on the quantitation ofATP present (CellTiter-Glo Luminescent Cell Viability Assay,Promega Italia s.r.l) according to the manufacturer's protocol.

In vivoNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were pur-

chased from Charles River Laboratories Italia s.r.l. (Calco - Lecco,Italy). Mice were injected subcutaneously with 1,5 � 106 A375melanoma cells and randomly assigned to treatment groups.Treatments were started when tumors became palpable andcontinued for 40 days. Dabrafenib and trametinib were admin-istrated by oral gavage 5 consecutive days a week at a dose ofrespectively 600 and 4 mg. Anti–PD-1 antibody and respectiveisotype control mAb were injected intraperitoneally (200 mg perinjection) three times a week. Mice were sacrificed when themaintumor diameter reached 2 cm or massive ulceration occurred. Allprocedures were performed accordingly Institutional ReviewBoard–approved protocols.

Statistical analysesThe statistical analyses were performed using Stata 12.0 statis-

tical software (Stata), and Prism7 (GraphPad Software, Inc.). Allvariables were tested for normal distribution with the Shapiro–Wilk test, and none of them was found normally distributed.Comparisons between two independent non-normally distribut-ed groups were performed using the nonparametric Wilcoxonrank-sum test. Comparisons between matched groups were per-formed with Wilcoxon signed rank test. Correlations betweenvariables were tested with the Spearman's rank correlation test.Differences in tumor volumes were statistically assessed usingrepeating measurements two-way ANOVA followed by Sidakcorrection and with two-tails. P values less than 0.05 were con-sidered statistically significant.

ResultsMelanoma cells express low but consistent levels of PD-1

To investigate PD-1 expressiononmelanoma cells, we analyzedtwo datasets: the CCLE and TCGA.

All 61 CCLE melanoma cell lines expressed PD-1 withmRNA values comparable to those of PD-L1 and PD-L2 (Affy-metrix mRNA values: PD-1 4.20 (3.81–4.65), PD-L1 4.63(3.73–7.84), and PD-L2 3.73 (3.97–8.50; Fig. 1A). Averagemelanoma PD-1 values were about 40% of those of theestablished melanoma antigen s100B [Affymetrix mRNAvalues: 10.39 (3.28–13.80)], used as an internal control puttingthe mRNA values into perspective (Fig. 1B), and around 95% of

the average PD-1 expression found in 18 T-cell neoplasia celllines included in the CCLE [Affymetrix mRNA values: 4.39(3.98–5.34); Fig. 1C].

From the 470 TCGA patient-derived melanomas, we matchedgene-expression data with corresponding histologic reports andwe excluded all the samples with histologic evidence of immuneinfiltrate, as those would interfere with the assessment of mela-noma-intrinsic PD-1 expression (Fig. 1D). PD-1 was expressedin 99.5% of the samples, with a median expression comparablewith PD-L1 and PD-L2 (Fig. 1E). We did not find significantdifferences when we compared samples with (N ¼ 100)and without (N ¼ 114) stromal cells [median FPKM values32.5 (0–1282.8), and 64.7 (0.4–1461.3) respectively, P ¼0.0970]. Furthermore, we observed positive correlations betweenPD-1 andPD-L1 (r¼ 0.66,P 0.05; N ¼7; Fig. 2C; Supplementary Fig. S2B], we found a significantincrease of PD-L2þ melanoma cells during BRAF/MEKi treat-ment [8.5% (0.0–63.0) vs. 1.5% (0.2–43.3), P ¼ 0.0312; N ¼7; Fig. 2D; Supplementary Fig. S2C].

In BRAFV600 mutant cells, the combination of BRAF andMEK inhibitors led to the highest percentage of PD-1þ cells,

Figure 2.BRAF/MEK inhibitors increase the rates of PD-1þ and PD-L2þ tumor cells in BRAFV600 and NRASQ61 mutant melanomas. A, Representative flow cytometry plotsof a BRAFV600mutantmelanoma cell line (A375) treatedwith dabrafenib (1 mmol/L), trametinib (5 nmol/L), the combination of dabrafenibþ trametinib (1 mmol/Lþ5nmol/L), and fotemustine (50 mg/mL) for 96 hours. DAPI staining was used to identify viable cells. PD-1, PD-L1, and PD-L2 plots were performed considering onlyviable cells. Rates of (B) PD-1–, (C) PD-L1–, and (D) PD-L2–positive melanoma cells untreated and after treatment with BRAF/MEKi [dabrafenib þ trametinib(1 mmol/Lþ 5 nmol/L) in BRAFV600mutant cell lines and trametinib (5 nmol/L) in the NRASQ61mutant cell line for 96 hours;N¼ 7]; � , P

and showed the greatest anti-tumor effect, whereas in theNRASQ61 mutant cell line this was observed with MEK inhib-itor alone (Supplementary Fig. S3). Fotemustine efficientlykilled tumor cells, but did not significantly change levels ofPD-1þ cells (Fig. 2E).

Rates of PD-1þ melanoma cells increase in a time- and drug-dependent manner during BRAF/MEKi treatment

We explored whether the percentage of PD-1þ melanoma cellswas influenced by time or drug exposure. Increasing the time oftreatment with BRAF/MEKi, we observed a progressive increaseof the percentage of PD-1þ cells. After 8 days of treatment, viablePD-1þ melanoma cells increased to 31.8% on average (15.0%–50.3%;N¼ 3; Fig. 2F and G]. Following BRAF/MEKi withdrawal,the rate of PD-1þ melanoma cells returned back to the loworiginal value (Fig. 2H and I; N ¼ 3).

PD-1þ melanoma cells are more quiescent and presentstemness features

Since during BRAF/MEKi treatment PD-1þ cells reach signifi-cant percentages among the viable tumor cell population, we

compared their proliferative capabilities with PD-1� cells. Weused a carboxyfluorescein succinimidyl ester (CFSE) dye-basedassay, where the dye decrease corresponds to higher mitoticactivity and faster proliferation rate. Treatment with BRAF/MEKidecreased the overall proliferation (Fig. 3A) with PD-1þ cellsproliferating less than the PD-1� counterparts; on average16.1% less after 96 hours (N ¼ 3), and 65% less after 7 days(N ¼ 2; Fig. 3B; Supplementary Fig. S4).

PD-1 was reported to be preferentially expressed on putativemelanoma cancer stem cells (6). To test this, we used a lentiviralvector carrying eGFP under the transcriptional control of theOct4 stemness gene promoter (LV-Oct4.eGFP; Fig. 3C; ref. 15).This system visualizes putative cancer stem cells as eGFPþ,based on their selective ability to activate the Oct4 promoter. Inthree LV-Oct4.eGFP transduced patient-derived cell lines(mMel2-Oct4, mMel3-Oct4, mMel7-Oct4), BRAF/MEKi led tooverall eGFPþ cell enrichment (on average 2.4-fold), suggestinga lower sensitivity of eGFPþ melanoma cells to these drugs(Fig. 3D). Moreover, eGFPþ putative cancer stem cells wereenriched among PD-1þ cells compared with PD-1� cells (onaverage 1.6-fold; N ¼ 3; Fig. 3E).

Figure 3.

PD-1þmelanoma cells have reduced proliferative potential and show stemness features.A,Melanoma cell proliferation after 96 hours of treatment with BRAF/MEKi[dabrafenibþ trametinib (1 mmol/Lþ 5 nmol/L) in BRAFV600 mutant cell lines and trametinib (5 nmol/L) in the NRASQ61 mutant cell line], compared with untreatedcontrols measured with CFSE assay. N ¼ 3 (A375, SKMEL2, and SKMEL5). Average values in red. No significant differences were found. B, Proliferation ratesof PD-1þ melanoma cells compared with the PD-1� counterparts after 96 hours (N ¼ 3) and 7 days (N ¼ 2) of BRAF/MEKi treatment [dabrafenib þ trametinib(1 mmol/Lþ 5 nmol/L) in BRAFV600 mutant cell lines and trametinib (5 nmol/L) in the NRASQ61 mutant cell line]. No significant differences were found. C, Schematicrepresentation of the lentiviral vector used to transduce melanoma cells; the eGFP expression is controlled by the promoter regulatory element of the Oct4 gene(LV-Oct4. eGFP). D, Percentage of eGFPþ cells at baseline and after 96 hours of BRAF/MEKi treatment (dabrafenib þ trametinib; 1 mmol/L þ 5 nmol/L), inpatient-derived cell lines transduced with the LV-Oct4.eGFP vector (N ¼ 3). Average values and standard deviation. No significant differences were found.E, Percentage of eGFPþ/PD-1þ cells compared with eGFPþ/PD-1� cells in patient-derived cell lines transduced with the LV-Oct4.eGFP vector (N ¼ 3). Averagevalues and standard deviation. No significant differences were found.

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Anti–PD-1 antibody prolongs the antitumor response toBRAF/MEKi

Considering the hypothesis that PD-1 activation could lead tomelanoma proliferation (7), we tested whether PD-1-blockagecould have a direct anti-tumor effect.

In vitro, the sole use of anti–PD-1 antibody did not affect cellviability (Supplementary Fig. S5). When we combined anti–PD-1antibody with BRAF/MEKi, we observed only a trend toward abetter anti-tumor effect compared with BRAF/MEKi alone duringshort-term drug exposure (Supplementary Fig. S6). To test thehypothesis that the subset of PD-1þ melanoma cells, which arepreferentially endowed with stemness features, might contributeto the development of BRAF/MEKi resistance, we set up an in-vivolong-term experiment. We used non-obese diabetic/severe com-bined immunodeficient (NOD-SCID)/interleukin 2 receptor[IL2r]gnull (NSG) mice bearing palpable subcutaneous xenograftmelanoma. The treatmentwith anti–PD-1 antibody alone did nothave any beneficial effect on tumor growth (N ¼ 6; Fig. 4A). Onthe other side, when combined with BRAF/MEKi, anti–PD-1antibody (N¼ 6) significantly prolonged the antitumor responseand delayedmelanoma relapse compared to controls treated onlywith BRAF/MEKi (N ¼ 6; P ¼ 0.0006).

DiscussionBRAF/MEK inhibitors (BRAF/MEKi) and anti–PD-1 antibody

combinations might be a therapeutic strategy for metastaticmelanoma patients, and phase II and III clinical trials(NCT02910700, NCT02224781, NCT02130466, NCT02967692,NCT02858921) are currently recruiting. These trials are basedon preclinical models which explain the synergism by positiveBRAF/MEKi effects on T-cell recruitment, PD-L1 upregulation ontumor cells and consequent enhancement of anti–PD-1 antibody

antitumor effect (1, 2). Our results point to a novel, lymphocyte-independent, mechanism of action: BRAF/MEKi treatmentleads to higher rates of viable melanoma cells expressing PD-1and PD-L2, and therefore it could sensitize the tumor to a directinhibitory effect of anti–PD-1 antibody.

"Ectopic" melanoma-intrinsic PD-1 expression and its possiblerole in promoting tumor growth were proposed (7), but thebiological relevance of thesefindings is still not clear. Such subsetswere observed to have tumor-initiating properties; thus, theycould contribute to the development of drug resistance.

We first chose an in vitro platform to characterize melanoma-intrinsic PD-1 expression in normal conditions and duringBRAF/MEKi treatment. We confirmed that melanoma cells doexpress intrinsic PD-1, but at very low rates. Such low percent-age of PD-1þ cells is unlikely to account for large functionaleffects and indeed, treatment with anti–PD-1 antibody aloneaffected neither tumor cell viability in-vitro, nor tumor growthin immunodeficient xenograft models.

However, upon treatment with BRAF/MEKi, percentages ofPD-1þ cells increased to relevant numbers in all tested mela-noma cell lines, likely capable to enhance tumor proliferation ifactivated. Furthermore, we found that BRAF/MEKi also upre-gulated the PD-1-ligand PD-L2 on melanoma, therefore ajuxtacrine, pro-proliferative PD-1-activation on melanoma isfeasible and biologically plausible. This interaction wouldsupport melanoma proliferation and thus counteract desiredanti-tumor effects of BRAF/MEKi.

The increased percentage of PD-1þ melanoma cells duringtreatment with BRAF/MEKi can be the result of a molecularmodulation of PD-1 protein expression, but also of a selectiveprocess of PD-1þ melanoma cells less sensitive to target therapy.The linear kinetics of PD-1 expression during BRAF/MEKi treat-ment, and its rapid reversion upon drug withdrawal endorse the

Figure 4.

Anti–PD-1 antibody prolongs the antitumor response of BRAF/MEKi in immunodeficient mice. A, Kinetics (mean � SEM) of A375 xenograft growth in NSG micetreatedwith anti–PD-1 antibody (anti–PD-1mAb; 200mg three times aweek;N¼ 6) or isotype control antibody (200mg three times aweek;N¼6). Treatment start ismarked by the arrow.B,Kinetics (mean�SEM) ofA375 xenograft growth inNSGmice treatedwithBRAF/MEKi (dabrafenibþ trametinib; respectively, 600 and4mg,five consecutive days a week) and anti–PD-1 antibody (anti–PD-1 mAb; 200 mg three times a week; N ¼ 6) or BRAF/MEKi (dabrafenib and trametinib) alone(N ¼ 6). Treatment start is marked by the arrow. Statistical analysis was carried out using two-way ANOVA followed by Sidak correction and with two-tails;� , P < 0.05; ��, P < 0.001.






hypothesis of a dynamic modulation. On the other hand, ourfinding that the PD-1þ cell subsets are enriched with cells withstemness features supports the idea of a higher resistance of thosecells to target therapy treatment. The description of the exactmechanism leading to the increase of PD-1þ melanoma cellsduring treatment with BRAF/MEKi is beyond the scope of thiswork. Instead, we focused our efforts to investigate the biologicalrelevance of our findings.

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2018;24:3377-3385. Published OnlineFirst April 12, 2018.Clin Cancer Res Martina Sanlorenzo, Igor Vujic, Arianna Floris, et al.

PD-1 Antibody−Anti Leading to a Potential Lymphocyte-Independent Synergism with

BRAF and MEK Inhibitors Increase PD-1-Positive Melanoma Cells

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BRAF and MEK Inhibitors Increase PD-1-Positive Melanoma Cells … · Figure 1. Melanoma cells express PD-1. A, Box plots of PD-1, PD-L1, and PD-L2 Affymetrix mRNA expression values