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. 2013 Jan 10;62(4):811–822. doi: 10.1007/s00262-012-1389-z

Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties

Patrick A Ott 1, Trevor Henry 1, Sonja Jimenez Baranda 1, Davor Frleta 1, Olivier Manches 1, Dusan Bogunovic 1, Nina Bhardwaj 1,✉
PMCID: PMC11028975  PMID: 23306863

Abstract

Purpose

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAFV600 mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAFV600 mutant melanoma.

Methods

We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.

Results

Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAFV600E mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.

Conclusions

BRAFV600E mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-012-1389-z) contains supplementary material, which is available to authorized users.

Keywords: Dendritic cell, Melanoma, BRAF, MEK, Immune suppression, Tumor microenvironment

Introduction

Metastatic melanoma is an aggressive skin cancer that is largely resistant to treatment with systemic chemotherapy. Recent advances with targeted agents that block driver oncogenic mutations such as BRAFV600E/K have shown impressive clinical efficacy in patients with advanced melanoma [1–5], whereas immunotherapy using negative co-stimulatory molecule blockade with monoclonal antibodies such as anti-CTLA-4, PD-1, or PD-L1 can lead to durable responses in melanoma patients [6–9].

T-cell-mediated immunity has been shown to control and eliminate melanoma burden in humans [10]. However, clinical responses of patients with metastatic melanoma are still difficult to achieve with most immune-based interventions. The immunosuppressive milieu within the tumor is known to be one explanation for the failure of many immunotherapies. Several mechanisms that negatively impact the melanoma-specific T-cell response in the tumor microenvironment have been identified and validated in vivo [11, 12]. They include suppression by CD4+CD25+FoxP3+ T regulatory cells [13, 14], inhibition by negative regulatory surface molecules such as PD-1 and PD-L1 [15, 16], and the production of immunosuppressive mediators such as IL-10, IL-6, TGF-beta, and VEGF. The upregulation of cell signaling pathways involved in the survival and proliferation of tumor cells has been implicated in promoting these suppressive immune networks in the tumor environment [17, 18]. In melanoma cells, the mitogen-activated protein kinase (MAPK) signaling pathway, which is critical for tumor cell growth, is constitutively activated through a mutation of the oncogene BRAF at codon 600 (V600E and V600K) in 50 % of patients and has emerged as a key therapeutic target. Pharmacological inhibition of the BRAFV600E mutation leads to objective tumor responses in approximately 50 % of advanced melanoma patients carrying the mutation and resulted in improved overall survival compared to standard chemotherapy [1].

In humans, the impact of MAPK pathway upregulation and the effect of its inhibition are beginning to be explored in the context of a melanoma-specific immune response [19]. Recent studies suggest that MAPK pathway inhibition with small molecule inhibitors in melanoma patients can have a favorable effect on melanoma-specific immune responses [20]. Since oncogenic pathway inhibition and immunotherapy with immune-checkpoint blockade are now important strategies in the treatment for melanoma, knowledge about the interplay of the MAPK pathway and the immune system may prove critical for the development of novel therapeutic intervention approaches in melanoma. Dendritic cells (DCs) are potent antigen-presenting cells (APC) that are crucial for effective priming and boosting of tumor-specific T cells [21]. In this study, we asked whether the MAPK pathway in BRAFV600E mutant melanoma cells contributes to compromised function of DCs in the melanoma microenvironment by studying co-cultures of melanoma cell lines and DCs. Furthermore, we examined the direct effect of MAPK pathway inhibition with MEK and BRAF inhibitors on DC viability and function, as incorporating MAPK inhibition into DC-based vaccination approaches for patients with advanced or high-risk melanoma is being considered clinically.

Materials and methods

Human monocyte-derived DCs

Buffy coats, which served as sources for peripheral blood mononuclear cells (PBMCs), were purchased from the New York Blood Center. Human monocyte-derived DCs were differentiated from monocyte fractions of PBMCs as described previously [22]. Briefly, monocytes that attached to the tissue culture plates following plating of PBMCs for 1 h were cultured at 37 °C in RPMI with 5 % PHS containing 300 IU/mL recombinant human (rHu) IL-4 (Immunex) and 116 IU/mL rHu granulocyte macrophage colony-stimulating factor (GM-CSF; R&D). On days 2 and 4 of culture, additional IL-4 and GM-CSF were added. Immature DCs were harvested on day 5 for use in experiments. The purity of DC was at least 95 % based on CD11c expression assessed by FACS.

Melanoma cell lines

GMEL, 888-mel, M44 (all BRAFV600E), and FM29 (BRAF wild-type (WT)) were obtained from Dr. K. Palucka (Baylor Institute for Immunological Research). SK19 (BRAFV600E), SK197 (BRAF WT), and SK173 (BRAF WT) were obtained under a materials transfer agreement (MTA) from Dr. Alan Houghton (Memorial Sloan Kettering Cancer Center, NY). Melanoma lines were cultured in RPMI containing 10 % fetal bovine serum, 1 % Hepes buffer and gentamycin (0.2 %) at 37 °C in 5 % CO2 unless specified otherwise. BRAF mutational analysis was performed using conventional sequencing as described previously [23].

Reagents

All antibodies (CD80, CD83, CD86, MHC-I, CD40, and CD11c) for staining of human cells were purchased from BD Pharmingen and used following the manufacturer’s instructions. Vemurafenib (also known as plx4032 or RG7204) was obtained under an MTA with Roche and provided by Dr. Fei Su. Vemurafenib was dissolved in dimethyl sulfoxide (DMSO; Fisher Scientific) to a stock concentration of 10 mM and used as previously described at 1 μM unless stated otherwise [24]. The MEK 1/2 inhibitor U0126 and its inactive analogue U0124 were obtained from Calbiochem and used at a concentration of 12.5 μmol/L unless specified otherwise. The dose of 12.5 μmol/L was chosen based on the minimal concentration required for reliable downstream pERK inhibition in different melanoma cell lines [25].

Quantification of chemokine/cytokine secretion and flow cytometry

Melanoma or DC culture supernatants were collected and tested for the presence of the following cytokines: IL-12p70, TNF-α, IL-10, -6, -1β and -8 by flow cytometry using a cytometric bead array (CBA) (BD Biosciences). Levels of free, bioactive TGF-β1 were tested in an ELISA assay (Invitrogen). A panel of 23 factors (Eotaxin, GM-CSF, IFN-γ, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-15, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP10, MCP1, MIP1α, RANTES, VEGF, and TNF-α) was measured in melanoma supernatants by using the Luminex platform and a kit from Millipore. Flow cytometry was conducted in a FACS Calibur (BD Biosciences) flow cytometer, and data analysis was performed with FlowJo software (Tree Star).

Melanoma cell and DC co-culture

On day one, 40,000–50,000 melanoma cells were plated in triplicate in 96-U-bottom wells and treated with U0126, vemurafenib, U0124, or DMSO. After 24 h, supernatants were removed and 5-day (immature) DCs were added at 80,000 cells/well. On day 3 of melanoma culture (24 h of co-culture), DCs were stimulated with polyI:C (Amersham) at 2 μg/mL. Twenty-four hours after stimulation, supernatants and cells were removed and cytokine and surface marker analysis was performed by CBA and FACS, respectively.

Allogeneic T-cell reaction

Naïve CD4 cells were isolated from healthy donor PBMC by using EasySep® Human CD4+ T Cell Enrichment Kit (STEMCELL) and loaded with carboxyfluorescein succinimidyl ester (CFSE). Immature DCs were treated with U0126, vemurafenib, U0124, or DMSO for 24 h, stimulated with polyI:C for 24 h, and incubated in 96-well round-bottom tissue culture plates at a ratio of 1:10 with allogeneic naïve CD4 cells for 5–7 days in RPMI media. CFSE dilution over time was analyzed by FACS as a measure of T-cell proliferation. Data analysis was performed with FlowJo software (Tree Star).

Assessment of apoptosis and cell viability assays of DCs

Dendritic cells were treated with U0126, vemurafenib, U0124, or DMSO as vehicle control at different concentrations. After 24 h, DCs were stimulated with polyI:C and cells/supernatant harvested after 18 h of stimulation. DCs were assessed for apoptosis by staining with Annexin V–FITC and 7-Amino-Actinomycin (7-AAD) (BD Biosciences), as previously described. At least 5,000 events per sample were collected by flow cytometry. For viability measurements, DCs were plated in triplicate and treated as above in 96-well opaque-walled plates; CellTiter-Glo reagent (Promega Corporation) was used according to the manufacturer’s instructions. Luminescence was recorded with a Synergy HT Multi-Detection Microplate Reader (Biotek Instruments, Inc.). Each experiment was done in triplicate and repeated at least twice.

Statistical analysis

The GraphPad Prism (GraphPad Software) package program and Microsoft Excel were used for data presentation. All statistical analyses were conducted using paired t test and Mann–Whitney test.

Results

Cytokine production is variable across different human melanoma cell lines and can be inhibited with MAPK inhibition

Cytokine production after 48 h of culture was analyzed in the melanoma cell lines M44, GMEL, SK19, and 888-mel (all BRAFV600E) and in the melanoma cell lines FM29, SK173, and SK197 (all BRAF WT). IL-8 was secreted by all cell lines, whereas modest amounts of IL-6 and IL-10 were produced (Supplemental Fig. 1a–c). IL-8 production was heterogeneous among the cell lines and higher in 3 of 4 BRAFV600E mutant cell lines compared to WT. No other cytokines or chemokines, including a panel of 23 soluble factors (as delineated in the Methods) measured by Luminex assay as well as TGF-β1 measured by ELISA, were detected consistently after 24–48 h of culture (not shown). MAPK pathway inhibition with U0126 led to suppression of IL-8 production in BRAFV600E mutant and WT cell lines (Supplemental Fig. 1d).

MAPK blockade reverses BRAFV600E mutant melanoma-induced suppressed cytokine secretion and surface marker expression of poly:IC-matured DCs

To assess whether MAPK pathway upregulation in BRAFV600E mutant cell lines impacts the function of DCs, BRAFV600E mutant and BRAF WT melanoma lines were co-cultured with immature DCs generated from healthy donors. Prior to adding DCs, melanoma cell lines were treated for 24 h with U0126 versus its inactive analogue U0124 and supernatants were removed. After 24 h of co-culture with melanoma cell lines, DCs were matured with polyI:C and cytokine production was measured in the supernatants. Exposure of DCs from 11 healthy donors to the BRAFV600E mutant cell line GMEL treated with U0124 resulted in decreased TNF-α and IL-12 production (Fig. 1a, b). In contrast, when GMEL cells were treated with U0126 prior to the exposure to DCs, no decrease in TNF-α or IL-12 secretion was observed. Similar results were seen with DCs from 11 healthy donors exposed to the BRAFV600E mutant line 888-mel (Fig. 1c, d). To assess whether the diminished suppressive capacity of melanoma cell lines upon MEK inhibition is specific to BRAF mutant melanoma cell lines, DCs from 19 healthy donors were co-cultured with the BRAF WT cell line FM29 pretreated with U0126 for 24 h versus U0124. Both TNF-α and IL-12 secretion were suppressed when FM29 cells were treated with inactive analogue. In contrast to the BRAF mutant cell lines 888-mel and GMEL, pretreatment with U0126 did not mitigate the suppression of either TNF-α or IL-12 by FM29 (Fig. 1e, f). Similarly, co-culture of DCs from 11 healthy donors and the WT cell line SK173 resulted in profound inhibition of both TNF-α and IL-12 production, which was not reversed after pretreatment of SK173 with U0126 (Supplemental Fig. S2a, b). These data indicate that MEK inhibition in BRAFV600E mutant cell lines leads to a partial reversal of their ability to suppress TNF-α and IL-12 production by DCs. This effect appears to be mediated by inhibition of an upregulated MAPK pathway since it was not observed with MEK inhibition in WT cell lines.

Fig. 1.

Fig. 1

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Suppressed cytokine production of poly:IC-matured DCs co-cultured with melanoma cell lines is restored by MEK inhibition in BRAFV600E mutant, but not BRAF WT melanoma cell lines. Human melanoma cell lines GMEL (a, b) and 888-mel (c, d), both BRAFV600E and FM29 (e, f) (BRAF WT) were treated with the MEK inhibitor U0126 or the inactive analogue U0124. After 24 h, supernatants were removed and immature DCs were added. On day 3 of melanoma culture (24 h of co-culture), DCs were stimulated with polyI:C. Using CBA, supernatants of the melanoma/DC co-cultures were tested for the production of TNF-α (a, c, e) and IL-12 (b, d, f). DCs from 11 to 19 healthy donors were analyzed in triplicate in at least 5 independent experiments. Mel melanoma cells, NS not significant

We next investigated whether inhibition of BRAF activity with vemurafenib resulted in a similar partial reversal of TNF-α and IL-12 suppression mediated by BRAFV600E mutant cell lines. Co-culture of polyI:C-matured DCs from 11 healthy donors and GMEL (BRAFV600E) resulted in profound inhibition of TNF-α production when melanoma cells were pretreated with control vehicle (DMSO) (Fig. 2a). However, when GMEL cells were pretreated with the BRAF inhibitor vemurafenib, no suppression of TNF-α production in DCs was detected. IL-12 production of DCs was only modestly suppressed upon co-culture with GMEL cells, and no significant difference was seen between vemurafenib and control (Fig. 2b). Co-culture of DCs and 888-mel cells (BRAFV600E) lead to marked inhibition of TNF-α and IL-12 in 13 healthy donors, whereas no inhibition of TNF-α and IL-12 secretion was detected with vemurafenib pretreatment of 888-mel (Fig. 2c, d). Exposure of DCs from 15 donors to FM29 cells (BRAF WT) lead to inhibition of TNF-α production. In contrast to the BRAFV600E mutant cell lines, pretreatment of FM29 with vemurafenib did not prevent the inhibition of TNF-α secretion by DCs (Fig. 2e). Furthermore, no inhibition of IL-12 production and no differences between vemurafenib and control were seen with FM29 (Fig. 2f). Similar results were seen with another BRAF WT cell line, SK197 (Supplemental Fig. S2c, d). These data indicate that BRAF inhibition in BRAFV600E mutant melanoma cells completely or partially reverses the suppression of TNF-α and IL-12 production by DCs in co-culture and corroborate, in an independent set of healthy donors, the observations made with the MEK inhibitor U0126.

Fig. 2.

Fig. 2

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Suppressed cytokine production of poly:IC-matured DCs co-cultured with melanoma cell lines is restored by BRAF inhibition in BRAFV600E mutant, but not BRAF WT melanoma cell lines. Experiments were performed as described for Fig. 1. Melanoma cell lines were treated with the BRAF inhibitor vemurafenib versus vehicle control (DMSO). Supernatants of the melanoma/DC co-cultures were tested for the production of TNF-α (a, c, e) and IL-12 (b, d, f). DCs from 11 to 15 healthy donors were analyzed in triplicate in at least 3 independent experiments. Mel melanoma cells, vemu vemurafenib, NS not significant

No difference in the decrease in IL-12 and TNF-α production by DCs exposed to 888-mel, and the reversal with vemurafenib pretreatment (or the lack thereof with FM29) was seen between direct co-culture (Fig. 3a, c) and separation of melanoma cells and DCs by a membrane in trans-well plates (Fig. 3b, d). This suggests that the suppressive effect on cytokine secretion is not cell-contact dependent.

Fig. 3.

Fig. 3

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Reversal of suppressed cytokine production mediated by BRAF inhibition is not cell-contact-dependent; downregulation of expression of co-stimulatory molecules on poly:IC-matured DCs co-cultured with melanoma cell lines is partially reversed by BRAF inhibition. Melanoma cell lines 888-mel (BRAFV600E) and FM29 (BRAF WT) were cultured and treated with vemurafenib or control vehicle (DMSO). After 24 h, supernatants were removed and immature DCs from healthy donors added either directly in the well (a, c) or in trans-wells (b, d). On day 3 of melanoma culture (24 h of co-culture), DCs were stimulated with polyI:C. Using CBA, supernatants of the melanoma/DC co-cultures were tested for the production of IL-12 (a, b) and TNF-α (c, d). The experiment was repeated twice with similar results. 888-mel and GMEL were treated with vemurafenib or DMSO and co-cultured with DCs from healthy donors as described above (e). Expression of CD83, CD80, and CD86 on DCs was analyzed by FACS. Triplicates are shown. The asterisks indicate p values: *p < 0.05; **p < 0.005

Furthermore, the expression of CD80, CD83, and CD86 on DCs was decreased upon co-culture with 888-mel and GMEL. Inhibition of the MAPK pathway in the melanoma cells with both U0126 and vemurafenib prior to DC exposure markedly reduced this inhibitory effect (results for vemurafenib are shown in Fig. 3e).

MAPK pathway inhibition with a MEK inhibitor, but not with a mutation-specific BRAF inhibitor, suppresses cytokine production of polyI:C-matured DCs

There is a strong clinical interest in combination strategies using immune-check point blockade (for example with anti-CTLA-4 blocking antibodies) and either BRAF or MEK inhibitors. A phase I trial exploring the combination of ipilimumab and vemurafenib in advanced melanoma patients is currently ongoing (Clinicaltrials.org: NCT01400451). Recent in vitro studies have shown that BRAF inhibition, even at high concentrations, does not compromise T-cell function, and there is emerging data showing that low doses of RAF inhibition might even enhance T-cell activation [26, 27].

We asked whether the function of polyI:C-matured DCs is affected by direct BRAF or MEK inhibition. DCs from healthy donors were treated with U0126 and vemurafenib, respectively, and the production of different cytokines after maturation with polyI:C was measured. In a group of 15 healthy donors, U0126 at a dose of 12.5 μmol/L significantly inhibited IL-12 production and TNF-α secretion of DCs (Fig. 4a, b). In contrast, BRAF inhibition with vemurafenib had no effect on IL-12 or TNF-α production (Fig. 4c, d).

Fig. 4.

Fig. 4

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Cytokine production of polyI:C-matured DCs is altered by MEK, but not BRAF inhibition. IL-12 and TNF-α production of polyI:C-matured DCs are not affected by vemurafenib over a wide range of concentrations. Immature DCs from 15 healthy donors were treated with U0126 at 12.5 umol/L, vemurafenib at 1 umol/L, and controls (DMSO or U0124). After 24 h, DCs were stimulated with polyI:C. Using CBA, supernatants were tested for the production of IL-12 (a, c) and TNF-α (b, d). Individual data points represent means of triplicates. Immature DCs were treated in triplicate with different concentrations of vemurafenib (e, f), U0126 (g, h), or vemurafenib at different concentrations in addition to U0126 at a fixed dose of 12.5 umol/L (i, k). After 24 h, DCs were stimulated with polyI:C. Using CBA, supernatants were tested for the production of IL-12 (e, g, i) and TNF-α (f, h, k). One representative donor out of 6 tested in 3 independent replicate experiments is shown. Error bars indicate standard deviation (SD) of triplicates. The asterisks indicate p values: *p < 0.05; **p < 0.005

Dose titration experiments revealed that secretion of IL-12 and TNF-α by DCs is not directly affected by vemurafenib over a wide range of concentrations (Fig. 4e, f). Only at a dose level of 50 μmol/L, a level that is at least 50 times higher than the IC50 for most BRAF mutant melanoma cell lines, did vemurafenib lead to inhibition of both IL-12 and TNF-α production. In contrast, MEK inhibition with U0126 results in dose-dependent suppression of cytokine production in polyI:C-stimulated DCs from healthy donors (Fig. 4g, h). No additive effects on IL-12 or TNF-α production were observed when BRAF inhibition was added to MEK inhibition with U0126 at the standard dose of 12.5 μmol/L (Fig. 4i, k).

MEK, but not BRAF inhibition leads to downregulation of co-stimulatory molecules and reduces the T-cell priming capacity of polyI:C-matured DCs

The effect of MEK inhibition on the expression of a panel of co-stimulatory and activation markers was assessed by FACS analysis. The expression of CD40, CD80, CD83, and MHCI of DCs stimulated with polyI:C was completely or almost completely inhibited by U0126, whereas it was not affected by vemurafenib (Fig. 5a). Furthermore, naïve T cells stimulated with allogeneic DCs treated with U0126 prior to maturation with polyI:C proliferated less compared to DCs treated with inactive analogue, whereas no effect on T-cell stimulation capacity as measured by proliferation was observed upon exposure to vemurafenib (Fig. 5b).

Fig. 5.

Fig. 5

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Surface marker expression and T-cell stimulatory capacity of polyI:C-matured DCs are altered by MEK, but not BRAF-specific inhibition. a Immature DCs from healthy donors were treated with U0126 at 12.5 μmol/L, vemurafenib at 1 μmol/L, and control (DMSO). After 24 h, DCs were stimulated with polyI:C. Expression of CD40, CD80, CD83, and MHC-I was measured. One representative donor out of 4 tested in 3 independent replicate experiments is shown. Error bars indicate SD of triplicates. The asterisks indicate p values: *p < 0.05. b Proliferation of naïve T cells stimulated with DCs treated with vemurafenib at 1 and 10 μmol/L and with U0126 at 12.5 μmol/L prior to maturation with polyI:C was analyzed in triplicate. Bars indicate the percentage of T-cell proliferation compared to control (DMSO for vemurafenib; U0124 for U0126), where control is 100 %. Vemu vemurafenib

Cell viability and apoptosis of DCs are affected by exposure to U0126, but not vemurafenib

Apoptosis in response to U0126 and vemurafenib exposure was examined at different concentrations using FACS-based analysis of annexin V and 7-AAD. Markedly increased apoptosis of polyI:C-matured DCs treated with U0126 compared to control was noted, even at the standard dose of 12.5 μmol/L (Supplemental Fig. S3a). In contrast, no apoptosis was observed with vemurafenib at doses of 10 μmol/L, which constitutes 10× the IC50 for most BRAFV600 mutant melanoma cell lines (Supplemental Fig. S3b). To corroborate these findings, DC cell viability after treatment with U0126 and vemurafenib was also assessed using an ATP-based assay. No effect on cell viability was seen when DCs were treated with vemurafenib at doses of up to 50 μmol/L, whereas U0126 resulted in markedly decreased cell viability (Supplemental Fig. S3c).

Discussion

Immune-checkpoint blockade with the anti-CTLA-4 antibody ipilimumab leads to superior overall survival over standard chemotherapy with dacarbazine and vaccine treatment with the peptide gp100 [6, 7]. In patients with BRAFV600 mutant metastatic melanoma, MAPK pathway inhibition with the BRAF inhibitor vemurafenib or the MEK inhibitor trametinib leads to response rates of approximately 50 and 20 %, respectively; both agents demonstrated improved overall survival compared to chemotherapy in phase 3 trials [1, 28]. Despite the impact on survival and encouraging response rates, treatment with BRAF or MEK inhibition is limited by the relative short duration of responses in most melanoma patients. Tumor response achieved by BRAF or MEK inhibition is largely thought to be a direct effect of the drug on melanoma cells, mediating apoptotic cell death. However, it has been shown that activation of the MAPK pathway by the oncogenic BRAF mutation V600E may lead to immune evasion through secretion of the immunosuppressive cytokines IL-10, IL-6, and VEGF [29]. Indeed, recent preclinical and clinical studies have shown an enhanced T-cell response upon MAPK pathway inhibition, suggesting that MAPK pathway blockade may have beneficial effects on immune cells in the melanoma microenvironment in addition to direct cytotoxic effects on melanoma cells. Specifically, inhibition of both MEK and BRAF leads to increased tumor antigen presentation in melanoma cell lines, resulting in improved antigen recognition (gp100 and MART-1) by T cells [19]. In advanced melanoma patients treated with the BRAF inhibitors dabrafenib or vemurafenib, post-treatment tumor biopsies showed increased infiltration with CD4 and CD8 lymphocytes and a correlation of CD8 lymphocyte infiltration with tumor response [30].

In this study, we asked whether there is a molecular link between upregulated signaling through the MAPK pathway in BRAFV600E mutant melanoma cells and the function of DCs. We demonstrate that suppression of IL-12 and TNF-α production in DCs mediated by melanoma cells can be restored, partially or completely, by inhibition of the MAPK pathway with both MEK and BRAF inhibition. Furthermore, melanoma-induced downregulation of the co-stimulatory molecules and activation markers CD80, CD83, and CD86 in polyI:C-matured DCs was reversed upon MAPK blockade in melanoma cells co-cultured with DCs. Importantly, this reversal of suppressed cytokine production and activation marker expression was not seen when BRAF WT melanoma cell lines were co-cultured with DCs. Trans-well experiments showed that the suppression of DC function by melanoma cells is most likely mediated by a soluble factor. Nevertheless, despite extensive analysis, no significant amounts of candidate factors, including IL-10, TGF-β, and VEGF, were found to be secreted by any of the melanoma cell lines in our study. IL-8, which was detected in higher amounts in some (but not all) of the BRAFV600E mutant cell lines and was recently shown to be important for immunogenicity of DCs, seems an unlikely mediator of the melanoma-induced suppression of DC function seen in our studies [31]. It has been shown previously that TLR-4-dependent IL-12 production by DCs can be suppressed by melanoma lysates [32]. This effect could be reversed by MEK inhibition with U0126. Our studies confirm the observation of suppressed IL-12 production induced by melanoma cells in polyI:C-stimulated DCs. Importantly, in the present study, we investigate, in co-culture experiments, the impact of MAPK inhibition in melanoma cells on the function of DCs that are exposed to melanoma cells, rather than direct MEK inhibition of DCs exposed to melanoma cells.

PolyI:C induces the secretion of proinflammatory cytokines in the absence of IL-10, and polyI:C has been shown as one of the most potent TLR agonists [33]. Moreover, it has been widely used as cancer vaccine adjuvant in clinical trials, including melanoma, and was therefore chosen as the DC maturation stimulus in our study. Our findings in DCs corroborate the recent evidence for beneficial effects of MEK and BRAF inhibitors on the T-cell response, addressing a cell type that is critical for the induction of strong tumor-specific T-cell responses. Furthermore, it is conceivable that the advantageous effect on antigen presentation by improving DC function seen in our study would be synergistic with the upregulation of melanoma differentiation antigens observed with MEK and BRAF inhibition in previous studies for the induction of a melanoma-specific T-cell response [19]. Our findings indicate that constitutive upregulation of the MAPK pathway in melanoma cells by a BRAFV600E mutation is connected to compromised DC function. Consistent with previous observations [34, 35], no or minimal apoptosis was seen in BRAFV600E mutant and WT melanoma cell lines (888-mel, GMEL, FM29, and SK197) after 48 h of MEK or BRAF inhibition (Supplemental Fig. S3e). Since in the co-culture experiments DCs were exposed to melanoma cells 24 h after treatment with MEK or BRAF inhibition (with the exposure lasting only 24 h prior to TLR stimulation), it is unlikely that the reversal of compromised DC function mediated by melanoma cells could be explained by the mere death of melanoma cells after MAPK pathway inhibition. In trans-well experiments, we observed that the suppression of DC function as measured by TNF-α and IL-12 production is not contact-dependent. Either no cytokine production or small amounts of IL-6 and IL-10 (< 100–150 pg/ml) were detected in the supernatants of 7 cell lines after 48 h of culture. In a previous study, it was demonstrated that the addition of supernatant from the A375mel line to DC cultures prior to maturation by LPS led to suppression of IL-12 and TNF-α [29]. This effect was mediated by IL-6, IL-10, and VEGF and could be partially reversed by pretreatment of the melanoma cells with BRAFV600E RNAi. Our observation that the suppressive effect of melanoma cells on IL-12 and TNF-α production by DCs is not contact-dependent in the co-cultures also suggests that the inhibition is mediated by a soluble factor such as a cytokine or chemokine. However, in our study, we did not find significant inhibition of IL-12 or TNF-α after addition of supernatants from melanoma cell lines to immature DCs and subsequent maturation with polyI:C. This may be explained by the small amounts (if any) of IL-6, IL-10, and VEGF detected after 24–48 h of melanoma cell culture detected in our experiments. Another explanation is the time of exposure of DCs to the melanoma supernatant, which in our study was added to fully differentiated immature DCs on day 5 as opposed to day one of the monocyte culture in the previous study [29]. It is possible that continuous local production of soluble mediators by melanoma cells in close proximity to DCs accounts for the inhibitory effect observed in the melanoma cell/DC co-culture experiments in our study.

Our studies and recent emerging data [20] indicate that immunotherapeutic strategies may be synergistic with MAPK pathway inhibition in BRAF mutant melanoma. Specifically, DC-based vaccination could be potentially combined with a BRAF and/or a MEK inhibitor for the treatment of advanced melanoma patients, provided that these agents do not have unfavorable effects on DCs. Furthermore, co-targeting of BRAF and MEK leads to superior response rates and response duration in patients with metastatic melanoma, suggesting that combined BRAF/MEK inhibition may be superior to monotherapy with either of these agents [4].

Recent in vitro studies have shown that lymphocyte function is not compromised by BRAF inhibition, while MEK inhibition negatively affects viability, numbers, and IFN-γ production of MART-1 and gp100-specific cytotoxic T lymphocytes (CTLs) [19, 27]. Moreover, frequencies of T cells, B cells, NK cells, DCs, monocytes, and regulatory T cells in peripheral blood from patients with advanced melanoma were not affected by treatment with the BRAF inhibitor dabrafenib [36].

There is some controversy in the literature as it relates to the role of the MAPK pathway in DC maturation. MEK inhibition was shown to enhance LPS- and TNF-α-mediated functional and phenotypic DC maturation (increased expression of co-stimulation molecules, increased IL-12 production, and increased T-cell stimulatory capacity [37]), while constitutive activation of ERK leads to immune suppression, mediated by TGF-β and Treg cells [38]. Other studies have demonstrated no effect or only a minimal impact of MEK inhibition on DC function [39–42]. Differences in the maturation stimuli are one important factor that might account for some of the inconsistencies.

Recent studies have shown paradoxical activation of the MAPK pathway upon BRAF and MEK inhibition in WT tumor cells [43, 44], suggesting that signaling through the pathway can be impacted in cells not carrying a BRAF mutation even with highly specific inhibitors that have a high avidity for mutated BRAF in tumor cells, highlighting the importance of careful examination of this new generation of kinase inhibitors with regard to their effects on immune cells.

Specifically, with the goal of combining MAPK blockade and DC vaccination in melanoma patients, we investigated viability, apoptosis, and function of polyI:C-matured DCs upon treatment with the BRAF inhibitor vemurafenib and/or the MEK inhibitor U0126. We find that U0126 at standard doses within the range of IC50 of many melanoma cell lines [25] consistently leads to inhibition of IL-12 and TNF-α, decreased expression of co-stimulation and activation markers such as CD80 and CD83, and induction of apoptosis. In contrast, BRAF inhibition with vemurafenib has no impact on these parameters even at doses that are at least ten times higher than the IC50 for most BRAF mutant melanoma cells. Therefore, these findings, in conjunction with our observation that compromised DC function can be restored with MAPK pathway blockade in melanoma cells, support the combinatorial use of BRAF inhibition and DC immunotherapy. In contrast, MEK inhibition, despite its effectiveness in reversing suppressed DC function, is not advisable as a combination strategy for a DC-based immunotherapeutic approach based on its direct effects on DCs. DC frequencies in the peripheral blood of melanoma patients do not seem to be affected by treatment with a BRAF inhibitor, supporting our findings [45]. Validation of phenotypic and functional analysis of DC properties in melanoma patients treated with BRAF and/or MEK inhibition would be desirable, ideally in pre- and post-treatment samples. This is of particular importance prior to conducting clinical investigations with these combinatorial approaches in the adjuvant setting.

Electronic supplementary material

Below is the link to the electronic supplementary material.

262_2012_1389_MOESM1_ESM.ppt (236KB, ppt)

Supplemental Figure S1: Cytokine production is variable across different human melanoma cell lines and can be inhibited with MAPK inhibition. BRAFV600E mutant cell lines (M44, GMEL, SK19, 888-mel) and BRAF WT lines (FM29, SK173, SK197) were cultured in 96-well plates at 250,000 cells/ml, and supernatants were harvested after 48 h. IL-6, IL-8, and IL-10 were measured using CBA (a-c). Mean cytokine levels are shown from at least 5 independent experiments. M44, SK19, 888-mel, and FM29 were cultured for 48 h with the inactive analogue U0124 versus addition of the MEK inhibitor U0126; IL-8 production was measured (d). Bars indicate the percentage of IL-8 production compared to control (U0124), where control is 100 %. Apoptosis was assessed by staining for Annexin V–FITC and 7-AAD in GMEL, 888-mel (both BRAFV600E mutant), FM29, and SK197 cells (both BRAF WT) treated in duplicates with U0126, vemurafenib, U0124, and DMSO (e). Vemu: vemurafenib. (PPT 236 kb)

262_2012_1389_MOESM2_ESM.pptx (210KB, pptx)

Supplemental Figure S2: Suppressed cytokine production of poly:IC-matured DCs co-cultured with melanoma cell lines is not restored by MEK or BRAF inhibition in BRAF WT melanoma cell lines. Melanoma cell line SK173 (BRAF WT) was treated with the MEK inhibitor U0126 or the inactive analogue U0124 and co-cultured with DCs as described in Fig. 1 (a, b). Melanoma cell line SK197 (BRAF WT) was treated with the BRAF inhibitor vemurafenib versus vehicle control (DMSO) and co-cultured with DCs as described in Fig. 1 (c, d). Using CBA, supernatants of the melanoma/DC co-cultures were tested for the production of TNF-α (a, c) and IL-12 (b, d). DCs from 11 donors were analyzed in triplicate in 5 independent experiments (a, b); DCs from 7 donors were analyzed in triplicate in 3 independent experiments (c, d). Mel: melanoma cells; vemu: vemurafenib; NS: not significant. (PPTX 210 kb)

262_2012_1389_MOESM3_ESM.ppt (161KB, ppt)

Supplemental Figure S3: Cell viability of polyI:C-matured DCs is altered by MEK, but not BRAF inhibition. Apoptosis was assessed by staining for Annexin V–FITC and 7-AAD in DCs treated in duplicates with U0126 (a) or vemurafenib (b) at different concentrations prior to polyI:C stimulation. Viability of DCs treated with U0126 or vemurafenib prior to polyI:C stimulation was examined using an ATP-based assay (c). One representative donor out of 6 tested in 3 independent experiments is shown. Bars indicate SD of triplicates. NT: not treated. (PPT 161 kb)

Acknowledgments

Melanoma Research Alliance (Young Investigator Award to PAO), Cancer Research Institute (NB).

Conflict of interest

The authors declare that they have no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

262_2012_1389_MOESM1_ESM.ppt (236KB, ppt)

Supplemental Figure S1: Cytokine production is variable across different human melanoma cell lines and can be inhibited with MAPK inhibition. BRAFV600E mutant cell lines (M44, GMEL, SK19, 888-mel) and BRAF WT lines (FM29, SK173, SK197) were cultured in 96-well plates at 250,000 cells/ml, and supernatants were harvested after 48 h. IL-6, IL-8, and IL-10 were measured using CBA (a-c). Mean cytokine levels are shown from at least 5 independent experiments. M44, SK19, 888-mel, and FM29 were cultured for 48 h with the inactive analogue U0124 versus addition of the MEK inhibitor U0126; IL-8 production was measured (d). Bars indicate the percentage of IL-8 production compared to control (U0124), where control is 100 %. Apoptosis was assessed by staining for Annexin V–FITC and 7-AAD in GMEL, 888-mel (both BRAFV600E mutant), FM29, and SK197 cells (both BRAF WT) treated in duplicates with U0126, vemurafenib, U0124, and DMSO (e). Vemu: vemurafenib. (PPT 236 kb)

262_2012_1389_MOESM2_ESM.pptx (210KB, pptx)

Supplemental Figure S2: Suppressed cytokine production of poly:IC-matured DCs co-cultured with melanoma cell lines is not restored by MEK or BRAF inhibition in BRAF WT melanoma cell lines. Melanoma cell line SK173 (BRAF WT) was treated with the MEK inhibitor U0126 or the inactive analogue U0124 and co-cultured with DCs as described in Fig. 1 (a, b). Melanoma cell line SK197 (BRAF WT) was treated with the BRAF inhibitor vemurafenib versus vehicle control (DMSO) and co-cultured with DCs as described in Fig. 1 (c, d). Using CBA, supernatants of the melanoma/DC co-cultures were tested for the production of TNF-α (a, c) and IL-12 (b, d). DCs from 11 donors were analyzed in triplicate in 5 independent experiments (a, b); DCs from 7 donors were analyzed in triplicate in 3 independent experiments (c, d). Mel: melanoma cells; vemu: vemurafenib; NS: not significant. (PPTX 210 kb)

262_2012_1389_MOESM3_ESM.ppt (161KB, ppt)

Supplemental Figure S3: Cell viability of polyI:C-matured DCs is altered by MEK, but not BRAF inhibition. Apoptosis was assessed by staining for Annexin V–FITC and 7-AAD in DCs treated in duplicates with U0126 (a) or vemurafenib (b) at different concentrations prior to polyI:C stimulation. Viability of DCs treated with U0126 or vemurafenib prior to polyI:C stimulation was examined using an ATP-based assay (c). One representative donor out of 6 tested in 3 independent experiments is shown. Bars indicate SD of triplicates. NT: not treated. (PPT 161 kb)

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