BRAFV600E is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAFV600E by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAFV600E inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAFV600E inhibitors remain to be determined. To address this question, we developed a metastatic BRAFV600E-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAFV600E-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAFV600E lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAFV600E inhibitor therapy to promote more durable responses in melanoma. Cancer Res; 74(24); 7298–308. ©2014 AACR.

Metastatic melanoma is a skin cancer with increasing incidence rate and poor prognosis. The mean 5-year relative survival rate for metastatic melanoma is only 16% (1). Until recently, the therapeutic options for patients with advanced-stage metastatic melanoma were very limited and remained largely ineffective in improving patient's survival (1). The identification of activating point mutations of the BRAF gene has been a major breakthrough in the management of metastatic melanoma (2). Valine to glutamic acid substitution at codon 600 (BRAFV600E) is the most common mutation present in BRAF gene and is approximately found in 50% of all melanoma cases (3). BRAFV600E has been shown to trigger constitutive activation of the MAPK pathway, resulting in increased cell proliferation and invasiveness (2). The large proportion of patients bearing BRAFV600E mutations provided a strong rationale for the development of small-molecule–based BRAFV600E-selective inhibitors. Vemurafenib (PLX4032), and its research analogue PLX4720, are ATP-competitive inhibitors for BRAFV600E shown to reduce the kinase activity of this protein, consequently inhibiting the MAPK pathway and cell proliferation of BRAFV600E-mutated melanoma (4, 5). Vemurafenib demonstrated improved overall and progression-free survival rates in most patients with previously untreated BRAFV600E melanoma (3, 6) and was approved in 2011 by the U.S. FDA in the treatment of late-stage or unresectable melanoma (FDA Reference ID: 3001518). However, complete and durable remissions were rarely observed and progression-free survival did not exceed 5 to 7 months upon treatment with BRAF inhibitors (7). Drug resistance remains to date a major factor that limits BRAFV600E inhibitor clinical efficacy and the discovery of strategies overcoming adaptive resistance may have a huge impact on a patient's clinical outcome (8, 9).

BRAFV600E mutation contributes to melanoma immune escape (10) and accumulating evidence indicates that the efficacy of BRAF inhibitors relies on the activation of immune components against cancer cells (11–14). BRAF inhibition was associated with a decreased production of immunosuppressive soluble factors such as IL10, VEGF, and IL6 (12, 14, 15). An enhanced expression of melanoma-associated antigens together with an increased infiltration of CD8+ T lymphocytes was also observed in metastases of patients receiving BRAF inhibitors (12–14, 16). Using resistant variants of BrafV600E-driven mouse melanoma and melanoma-prone mice, our group recently demonstrated that combination therapy between PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies had significant antitumor activity, suggesting that immune-based therapy represents a promising strategy to overcome BRAF inhibitor drug resistance (11, 17).

Tumor microenvironment differs greatly between visceral metastases and primary cutaneous melanoma and may directly affect antitumor immune reactions and the efficacy of BRAFV600E inhibitors (18, 19). A better understanding of the antimetastatic effect of BRAF inhibitors is therefore required. In this study, using the first described mouse model for metastatic BRAFV600E melanoma, we establish that natural killer (NK) cells are critical for the therapeutic effect of PLX4720 through a perforin-dependent pathway. We show that PLX4720 treatment, in the context of IL2, directly enhances mouse NK cell ERK1/2 phosphorylation, proliferation, and CD69 expression and human NK cell ERK1/2 phosphorylation, CD69 expression and IFNγ release post NKp30 ligation. Finally, we demonstrate that treatment with a low dose of IL2 improves the antimetastatic efficacy of PLX4720 providing a strong rationale for combining NK cell stimulatory agents and BRAF inhibitors in metastatic melanoma.

Cell lines

The BRAFV600E SM1WT1 melanoma cell line has already been described (11). The LWT1 cell line was derived from SM1WT1 by the intravenous injection of 5 × 105 SM1WT1 into C57BL/6 wild type (WT) mice (Fig. 1). Both SM1WT1 and LWT1 cell lines were maintained in complete RPMI-1640 with 10% heat-inactivated FCS, 2 mmol/L glutamax, 100 U/mL penicillin, and 100 μg/mL streptomycin. The genotyping for BRAFV600E was achieved as previously described (20). B16F10 cells were sourced from ATCC, cultivated in complete DMEM supplemented as above. The original B16F10 cells were short tandem repeat DNA profiled, grown to bulk, and were never passaged for more than 2 weeks. They were routinely tested for mycoplasma by MycoAlert mycoplasma detection kit (Lonza, catalog number LT07-318).

Figure 1.

LWT1 is a model of metastatic BRAFV600E melanoma sensitive to PLX4720 inhibition. A, metastatic BRAFV600E-mutant cell line LWT1 was generated by intravenous injection of 5 × 105 SM1WT1 cells into C57BL/6 WT mice. After 30 days, the lungs with tumor nodules were meshed through a 70-μm cell strainer and cultured as described in Materials and Methods. B and C, the indicated doses of LWT1 or SM1WT1 parental cell lines were injected intravenously in WT mice. After 14 days, lungs were harvested, perfused with India ink, and the number of metastases was counted under a microscope. B, representative pictures of lungs from each group of mice. C, graph representing the mean ± SEM number of lung metastases in the indicated group of mice. Each symbol represents one individual mouse. ND, not detected. D and E, LWT1 or B16F10 melanoma cells were treated in vitro with the indicated doses of PLX4720 or equivalent amount of DMSO. D, graph showing the mean ± SEM proliferation (Alamar Blue assay) of the LWT1 and B16F10 cell lines after 48 hours of culture with the indicated dose of PLX4720. Data are representative of three independent experiments. E, representative Western blot analysis with the indicated antibodies. Data are representative of three independent experiments. Fold increase in pERK1/2 relative to no PLX 4720 control is recorded above the lane. F and G, 5 × 105 LWT1 (F) or 2 × 105 B16F10 (G) were injected i.v. in C57BL/6 WT mice. Mice were treated with 20 mg/kg or equivalent amount of DMSO daily from day 1 to 7. After 14 days, lungs were harvested and representative picture of lungs from each group of mice and graph representing the mean ± SEM number of lung metastases in the indicated groups of mice are shown. Pooled data from two independent experiments are shown. Each symbol representing one individual mouse. ***, P < 0.001 Mann–Whitney test.

Figure 1.

LWT1 is a model of metastatic BRAFV600E melanoma sensitive to PLX4720 inhibition. A, metastatic BRAFV600E-mutant cell line LWT1 was generated by intravenous injection of 5 × 105 SM1WT1 cells into C57BL/6 WT mice. After 30 days, the lungs with tumor nodules were meshed through a 70-μm cell strainer and cultured as described in Materials and Methods. B and C, the indicated doses of LWT1 or SM1WT1 parental cell lines were injected intravenously in WT mice. After 14 days, lungs were harvested, perfused with India ink, and the number of metastases was counted under a microscope. B, representative pictures of lungs from each group of mice. C, graph representing the mean ± SEM number of lung metastases in the indicated group of mice. Each symbol represents one individual mouse. ND, not detected. D and E, LWT1 or B16F10 melanoma cells were treated in vitro with the indicated doses of PLX4720 or equivalent amount of DMSO. D, graph showing the mean ± SEM proliferation (Alamar Blue assay) of the LWT1 and B16F10 cell lines after 48 hours of culture with the indicated dose of PLX4720. Data are representative of three independent experiments. E, representative Western blot analysis with the indicated antibodies. Data are representative of three independent experiments. Fold increase in pERK1/2 relative to no PLX 4720 control is recorded above the lane. F and G, 5 × 105 LWT1 (F) or 2 × 105 B16F10 (G) were injected i.v. in C57BL/6 WT mice. Mice were treated with 20 mg/kg or equivalent amount of DMSO daily from day 1 to 7. After 14 days, lungs were harvested and representative picture of lungs from each group of mice and graph representing the mean ± SEM number of lung metastases in the indicated groups of mice are shown. Pooled data from two independent experiments are shown. Each symbol representing one individual mouse. ***, P < 0.001 Mann–Whitney test.

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Mice

C57BL/6 WT mice were purchased either from Animal Resources Centre or Walter and Eliza Hall Institute of Medical Research (Parkville, Australia) and maintained at QIMR Berghofer Medical Research Institute (QIMR, Herston, Australia). C57BL/6 Cd226−/−, Ifng−/−, and Pfp−/− mice have been previously described (11) and were bred, genotyped, and maintained at the QIMR. Seven- to 12-week-old male mice were used according to the QIMR animal ethics committee.

In vitro treatment of LWT1 and B16F10

The IC50 of the cell lines were determined by Alamar Blue (Invitrogen, catalog number DAL1025) assay according to the manufacturer's instructions. MHC class I, Rae-1, and CD155 expression were analyzed on LWT1 or B16F10 cells after 24 hours in the presence of PLX4720 (10 μmol/L) or an equivalent amount of DMSO. Cells were then incubated with Fc blocking buffer (2.4G2 antibody) and the following fluorescence conjugated mAbs: anti-mouse- CD155 (4.42.3), Rae-1 (186107), and relevant IgG isotype controls, all from Biolegend or R&D Systems. MHC class I was detected by incubation with biotinylated anti-mouse H2Kb (AF6-88.5) or H2Db (28-14-8), with subsequent incubation with Allophycocyanin-conjugated streptavidin, all from BD Biosciences (catalog number 554067). All antibodies were diluted in 0.5% FCS 2 mmol/L EDTA PBS. Samples were acquired on a LSR IV Fortessa Flow Cytometer (BD Biosciences). Data were analyzed on FlowJo V10 (Treestar).

Western blotting

LWT1 or B16F10 cells were treated with 2.5, 5.0, or 10.0 μmol/L of PLX4720 or equivalent amount of DMSO. Purified human and mouse NK cells were cultivated with 1 μmol/L of PLX4720 or DMSO in complete RPMI supplemented with 300 U/mL IL2. After 24 hours, cells were harvested and resuspended in RIPA lysis buffer with protease inhibitors (Roche Diagnostics GmbH), 1 mmol/L sodium orthovanadate (Sigma), and 10 mmol/L sodium fluoride. Cells were then lysed by passing them through a 25-gauge needle 10 times and centrifuged at 13,200 rpm/4°C for 20 minutes to remove cell debris. Protein concentration was determined using the bicinchoninic acid protein assay reagent (Pierce, Thermo Fisher Scientific). Each protein sample (30 μg) was resolved on SDS-polyacrylamide gels and transferred to a PROTRAN BA 83 nitrocellulose membrane (Whatman Schleicher & Schuell; Sigma). Immunodetections were performed using anti-pERK1/2Thr202/204 (D13.14.4, #4370) and ERK1/2 (137F5,#4695, Cell Signaling Technology), anti-c-MYC (Y69, #1472-1, Abcam) rabbit polyclonal antibodies were used in conjunction with an horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham, GE Healthcare). Equal loading was assessed using β-actin (Sigma) mouse monoclonal primary antibodies. The Super Signal chemiluminescent system (Pierce, Thermo Fisher Scientific) or ECL-plus (Amersham) was used for detection. Quantification of protein-band intensities by densitometric analysis was performed using NIH ImageJ software (NIH, Bethesda, MD).

Transplant model and treatments

Pulmonary metastasis assays were performed by tail vein injection of 5 × 105 LWT1 cells or 2 × 105 B16F10 cells. PLX4720 (20 mg/kg) or equivalent amount of DMSO was given daily from day 1 to day 7 relative to tumor cell inoculation by i.p. injections. To examine IL2 immunotherapy of metastases, some groups of mice received either PBS or recombinant IL2 (10,000 or 100,000 U, Chiron Corporation) from day 1 to 5. Antibody depletions were performed by i.p. injections of 100 μg of control rat IgG (HRPN – BioExcell), anti-CD4 (GK1.5 - BioExcell), anti-CD8β (53.5.8 - BioExcell), anti-NK1.1 (PK136 – BioExcell), anti-IFNγ (H22 – prepared in house), anti-NKG2D (C7 – prepared in house), or anti-asialo(as)GM1 (Wako Chemicals) on days −1, 0, and 7, relative to tumor challenge. The in vivo immune modulatory effects of PLX4720 on spleen NK cells were tested by daily treatment with 20 mg/kg of PLX4720 or an equivalent amount of DMSO for 3 days followed by euthanasia and analysis of NK cells from spleens by flow cytometry. To visualize the LWT1 metastasis, lungs were intratracheally perfused with 30% India Ink PBS, followed by PBS wash and 24-hour incubation with Fekete's Solution. Lung photographs and metastasis counts were both performed with a Nikon SMZ745T microscope and NIS-Elements (F.4) software.

Cells preparation and flow cytometry

Spleens were meshed, filtered at 70 μm, and washed in PBS. Red blood cells were lysed by ACK buffer incubation for 1 minute. Single-cell suspensions were incubated for 15 minutes in Fc blocking buffer (2.4G2 antibody) and stained with the following fluorescence-conjugated mAbs, all diluted at 0.5% FCS 2 mmol/L EDTA PBS: anti-mouse- CD3ϵ (145-2C11), TCRβ (H57-597), and NK1.1 (PK136). All mAbs were purchased from Biologend or eBioscience. Samples were acquired on a LSR IV Fortessa Flow Cytometer (BD Biosciences). Data were analyzed on FlowJo V10 (Treestar).

In vitro activation of mouse and human NK cells

C57BL/6 spleen NK cells were purified by flow cytometry (Beckman Coulter MoFlo High Speed Cell Sorting) after staining with anti-CD3ϵ (145-2C11) and NK1.1 (PK136). NK cells were stained with 1 μmol/L Cell Trace Violet (Invitrogen) for 10 minutes at 37°C followed by FCS and PBS wash. Human peripheral blood mononuclear cells (PBMC) were prepared on a Ficoll–Paque density gradient (Amersham Biosciences AB) by centrifugation (800 × g, 30 minutes at room temperature) and human CD3CD56+ NK cells were negatively selected by magnetically activated cell sorting (NK cell isolation kit II, Miltenyi Biotec) according to the manufacturer's instructions.

Mouse or human NK cells were then cultured with the indicated concentrations of PLX4720 or an equivalent amount of DMSO in complete RPMI-1640 supplemented with 300 U/mL of human recombinant IL2. The analysis of ERK phosphorylation was performed after 24 hours in both species. Mouse NK cell activation and proliferation were assessed by flow cytometry by CD69 staining (H1.2F3) and Cell Trace Violet dilution after 3 days of cultures. Human NK cell activation was analyzed by CD69 expression after 6 days of culture.

In some experiments, purified NK cells (from 9 different donors) were cultured overnight (20 hours) in complete medium (RPMI 10% plus Human AB serum, 1% penicillin/streptomycin, 2 mmol/L glutamine, 2 mmol/L sodium pyruvate) with DMSO, PLX (0.3–3.0 μmol/L) with or without IL2 (300 U/mL). NK cells were recovered, counted, and plated (5 × 104/well) in a 96-well MAXISORB cross-linking plate (Nunc) precoated with 2.5 μg/mL anti-NKp30 (clone 210847, R&D Systems) or the IgG2a isotype control. NK cells were incubated at 37°C for an additional 20 hours and the supernatants were harvested and IFNγ measured by ELISA (BD Biosciences).

51Cr release cytotoxicity assay

NK cells were stimulated with 10 ng/mL of IL15/IL15R complex (eBioscience; #14-8152-80) for 48 hours in complete RPMI and used as effector cells. LWT1 or B16F10 cells were treated with 10 μmol/L of PLX4720 or equivalent amount of DMSO for 24 hours and used as target cells. The target cells were incubated with 51Cr for 60 minutes, washed in PBS, and 1,000 cells dispensed in 96-well V bottom plates. Effector cells were plated at an effector:target ratio of 1:1, 5:1, and 10:1, and incubated at 37°C for 4 hours. When indicated rat IgG (HRPN – BioExcell, 10 μg/mL), or anti-CD226 (480.1 – BioExcell, 10 μg/mL) was used in the assay. 51Cr release in the supernatant was determined by reading on a Wallac 1470 WIZARD Gamma Counter and the % of killing calculated by the following equation: % specific killing = (sample cpm − spontaneous release)/(maximum release − spontaneous release) × 100.

Statistical analysis

Statistical analysis was achieved using GraphPad Prism 6 software. Unpaired Mann–Whitney test or Student t test was used for comparison between groups with statistical significance when P values were below or equal to 0.05 (*), 0.01 (**), or 0.001 (***).

LWT1 is a model of metastatic BRAFV600E melanoma sensitive to PLX4720 inhibition

In the absence of immune-competent BRAFV600E melanoma models, the mechanisms involved in antimetastatic efficacy of BRAFV600E-specific inhibitor PLX4720 remain unknown. We therefore derived an experimental metastatic cell line from the previously described BRAFV600E SM1WT1 melanoma (11, 20) by intravenous passage through the lungs of a wild-type (WT) mouse (Fig. 1A). This metastatic melanoma cell line, termed LWT1, induced in 2 weeks the consistent formation of metastatic colonies restricted to the lungs in a dose-dependent manner, whereas the injection of an equal number of parental cell line SM1WT1 did not (Fig. 1B and C). Consistent with previous results obtained with the parental cell line SM1WT1 (11), we found that PLX4720 had modest activity against LWT1 (somewhat PLX4720 resistant; IC50 = 15.5 μmol/L), whereas PLX4720 had little detectable effect on BRAFwt melanoma cell line B16F10 (IC50 = 69.2 μmol/L; Fig. 1D). ERK1/2 was constitutively phosphorylated in the LWT1 BRAFV600E cell line and treatment with PLX4720 for 24 hours produced a dose-dependent reduction in the phosphorylation of ERK1/2 (Fig. 1E). This was associated with a decrease in c-Myc expression, a downstream target of ERK1/2 involved in cell proliferation (4), confirming the inhibition of the MAPK pathway by PLX4720 (Fig. 1E). In contrast, the basal level of ERK1/2 phosphorylation was very low in BRAFwt B16F10 tumors but low concentrations of PLX4720 slightly increased the phosphorylation of ERK1/2 and had no impact on c-Myc expression (Fig. 1E). These data confirm the findings of previous reports showing that PLX4720 promotes the phosphorylation of ERK in BRAFWT melanoma cells (21, 22). We then tested the antimetastatic effect of PLX4720 in vivo and it was able to reduce the quantity of pulmonary metastatic foci of LWT1 by approximately 50% (Fig. 1F), while it did not affect the number of B16F10 metastases (Fig. 1G). PLX4720 was superior to either anti-CTLA-4 or anti-PD-1 treatment alone and enabled enhanced survival in this model (Supplementary Fig. S1). These results show in a metastatic context that PLX4720 has specific antimetastatic effects against BRAFV600E melanoma cell lines.

PLX4720 control of pulmonary LWT1 metastasis is NK cell dependent

Given the accumulating evidence showing the ability of BRAF inhibitors to modulate antimelanoma immune reactions, we analyzed the role of immune components in PLX4720 antimetastatic properties in vivo. PLX4720 greatly reduced the number of metastases in the lungs of Ig-treated mice as compared with DMSO-treated group (Fig. 2A and B). Similar reduction with PLX4720 was observed in mice depleted with both CD4 and CD8 mAbs (Fig. 2B). In contrast, we found that this drug had no detectable antimetastatic effect in NK cell-depleted mice (Fig. 2A and B). The role of NK cells in PLX4720 efficacy was subsequently validated using another antibody to deplete NK cells (anti-NK1.1; PK136; Fig. 2B). The antimetastatic effect of PLX4720 was lost in mice depleted of NK cells even at a lower dose of LWT1 tumor cells (Supplementary Fig. S2). These results demonstrated that the antimetastatic effects of PLX4720 relied not only on the intrinsic inhibition of melanoma cell proliferation, but also required the action of host NK cells.

Figure 2.

Antimetastatic effects of PLX4720 require NK cells. C57BL/6 WT mice were injected with the indicated depleting antibodies and were challenged intravenously with 5 × 105 LWT1 melanoma cells. Mice were subsequently treated with 20 mg/kg or equivalent amount of DMSO daily from day 1 to 7. After 14 days, lungs were harvested and perfused with India ink and lung metastases were counted under a microscope. A, representative pictures of lungs from the indicated group of mice. B, graph representing the mean ± SEM numbers of lung metastases in the indicated group of mice. Pooled data from two independent experiments are shown. Each symbol represents one individual mouse. NS, nonsignificant, P > 0.05; *, P < 0.05; ***, P < 0.001 Mann–Whitney test.

Figure 2.

Antimetastatic effects of PLX4720 require NK cells. C57BL/6 WT mice were injected with the indicated depleting antibodies and were challenged intravenously with 5 × 105 LWT1 melanoma cells. Mice were subsequently treated with 20 mg/kg or equivalent amount of DMSO daily from day 1 to 7. After 14 days, lungs were harvested and perfused with India ink and lung metastases were counted under a microscope. A, representative pictures of lungs from the indicated group of mice. B, graph representing the mean ± SEM numbers of lung metastases in the indicated group of mice. Pooled data from two independent experiments are shown. Each symbol represents one individual mouse. NS, nonsignificant, P > 0.05; *, P < 0.05; ***, P < 0.001 Mann–Whitney test.

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Antimetastatic effect of PLX4720 depends on NK cell-mediated cytotoxicity

We next evaluated the importance of effector pathways in PLX4720-driven NK cell-mediated control of LWT1 lung metastasis. We observed that PLX4720 was still effective in mice neutralized for IFNγ or in mice deficient for IFNγ (−/−), suggesting that the IFNγ pathway was not required for PLX4720 efficacy in vivo in this mouse tumor model (Fig. 3A). In contrast, mice deficient in perforin (Pfp−/−) alone or additionally neutralized for IFNγ were unable to control LWT1 metastasis when treated with PLX4720 (Fig. 3A). These results suggested that in the LWT1 tumor model, perforin played a major role in controlling the antimetastatic efficacy of PLX4720.

Figure 3.

PLX4720 requires perforin and CD226 for optimal antimetastatic activity. A and D, the indicated strains of mice were challenged intravenously with 5 × 105 LWT1 melanoma cells and subsequently treated with 20 mg/kg or equivalent amount of DMSO daily on days 1 to 7. Antibody neutralization was performed on days −1, 0, and 7 relative to tumor challenge. After 14 days, lungs were perfused with India ink and metastases were counted under a microscope. A and D, the mean ± SEM number of lung metastases in the indicated groups of mice are shown. Data are pooled from two independent experiments. Each symbol represents an individual mouse. B, LWT1 cells were stained with antibodies against the indicated extracellular proteins and analyzed by flow cytometry. The gray histograms represent isotype controls, whereas the black lines represent the test staining. C, LWT1 cells were 51Cr-labeled followed by 4 hours of coculture with activated NK cells in the presence of Ig control or anti-CD226 antibodies. Graph represents the mean ± SD of experimental replicates. Data are representative of three independent experiments. E and F, LWT1 and B16F10 cells were treated in vitro with 10 μmol/L of PLX4720 or equivalent amount of DMSO for 24 hours. Cells were either labeled 51Cr and used as target in a cytotoxicity assay (E) or analyzed by flow cytometry for Rae-1 and CD155 expression (F). E, NK cells were activated in vitro for 48 hours with media supplemented with 10 ng/mL IL15/IL15Rα followed by 4 hours of incubation with PLX4720 or DMSO-treated B16F10 or LWT1 cells. Data are representative of three independent experiments. Graphs show mean ± SD of three experimental replicates. NS P > 0.05; *, P < 0.05; **, P < 0.01; Mann–Whitney test.

Figure 3.

PLX4720 requires perforin and CD226 for optimal antimetastatic activity. A and D, the indicated strains of mice were challenged intravenously with 5 × 105 LWT1 melanoma cells and subsequently treated with 20 mg/kg or equivalent amount of DMSO daily on days 1 to 7. Antibody neutralization was performed on days −1, 0, and 7 relative to tumor challenge. After 14 days, lungs were perfused with India ink and metastases were counted under a microscope. A and D, the mean ± SEM number of lung metastases in the indicated groups of mice are shown. Data are pooled from two independent experiments. Each symbol represents an individual mouse. B, LWT1 cells were stained with antibodies against the indicated extracellular proteins and analyzed by flow cytometry. The gray histograms represent isotype controls, whereas the black lines represent the test staining. C, LWT1 cells were 51Cr-labeled followed by 4 hours of coculture with activated NK cells in the presence of Ig control or anti-CD226 antibodies. Graph represents the mean ± SD of experimental replicates. Data are representative of three independent experiments. E and F, LWT1 and B16F10 cells were treated in vitro with 10 μmol/L of PLX4720 or equivalent amount of DMSO for 24 hours. Cells were either labeled 51Cr and used as target in a cytotoxicity assay (E) or analyzed by flow cytometry for Rae-1 and CD155 expression (F). E, NK cells were activated in vitro for 48 hours with media supplemented with 10 ng/mL IL15/IL15Rα followed by 4 hours of incubation with PLX4720 or DMSO-treated B16F10 or LWT1 cells. Data are representative of three independent experiments. Graphs show mean ± SD of three experimental replicates. NS P > 0.05; *, P < 0.05; **, P < 0.01; Mann–Whitney test.

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NK cell release of cytotoxic granules is controlled by the integration of signals received by a wide set of activation and inhibitory receptors (23, 24). We could not detect Rae-1 family ligands while CD155 was highly expressed on LWT1 cells (Fig. 3B). In contrast, the expression of MHC-I molecules H2-Kb and H2Db was low at the cell surface of LWT1 cells (Fig. 3B). In accordance with the low density of MHC-I and the presence of CD226 ligands, we found that LWT1 was well killed by activated NK cells in 4-hour classical 51Cr assays (Fig. 3C). Interestingly, the presence of anti-CD226 antibodies in the assay was able to limit LWT1 killing, suggesting that the CD226 interaction with CD155 is important for NK cell recognition of LWT1 (Fig. 3C).

Therefore, using gene-targeted mice for CD226 (Cd226−/−), we tested the role of these receptors in PLX4720-driven NK cell-mediated control of LWT1 lung metastasis. We found that the effect of PLX4720 was partially compromised in Cd226−/− mice (Fig. 3D). Additional neutralization of NKG2D did not further abrogate the activity of PLX4720 (Fig. 3D). Altogether, our results show that the antimetastatic effects of PLX4720 require both perforin-mediated cytotoxicity and in part, tumor recognition via CD226.

To test whether PLX treatment of tumor cells increased LWT1 sensitivity to NK cell-mediated killing, LWT1 or B16F10 was cultured in vitro in the presence of 10 μmol/L of PLX4720 or DMSO for 24 hours. We found that PLX4720 pretreatment of these target cells neither affected the NK cell-mediated killing of BRAFV600E nor BRAFwt tumor cells (Fig. 3E). Consistent with these findings, the expression levels of Rae-1, CD155, and MHC class I molecules were not affected by BRAF inhibition (Fig. 3F).

PLX4720 directly impacts mouse NK cells

We next tested freshly purified NK cells activated with IL2 in the presence of increasing concentrations of PLX4720. Supplementation with PLX4720 significantly increased NK cell CD69 (Fig. 4A) and NK cell proliferation (Fig. 4B). In contrast, we did not observe any impact of PLX4720 on IFNγ production by NK cells in response to IL12 and IL18 cytokine combinations (Supplementary Fig. S3) or PLX4720 on NK cell-mediated cytotoxicity induced by IL15 (Supplementary Fig. S4). PLX4720 has been shown to increase ERK1/2 phosphorylation in BRAFwt cells (21, 22). We observed an increase in ERK1/2 phosphorylation after 24-hour PLX4720 treatment and an increase in total ERK1/2 expression after PLX4720 treatment, while β-actin levels were not modulated (Fig. 4C). PLX4720 also displayed NK cell modulatory functions in vivo because C57BL/6 mice bearing LWT1 lung metastases and treated with PLX4720 (20 mg/kg daily) for 24 hours had an increased NK cell frequency in the lungs compared with DMSO-treated mice or mice that had not been inoculated intravenously with LWT1 melanoma (Fig. 4D).

Figure 4.

PLX4720 enhances NK cell activation and proliferation. A and B, Cell Trace Violet-labeled purified NK cells were cultured with the indicated doses of PLX4720 in media supplemented with 300 U/mL IL2. Analysis of CD69 and proliferation was performed by flow cytometry. A, the mean ± SD CD69 expression of triplicates (left) and representative histograms showing CD69 expression (right) are shown. B, the mean ± SD percentage of divided NK cells (left) and representative histograms showing Cell Trace Violet dilution (right) after 3 days in culture are shown. A and B, data are representative of three independent experiments. Statistical analysis performed by unpaired Student t test, *, P < 0.05. C, 2 × 106 purified NK cells were cultured for 24 hours in the presence of PLX4720 or DMSO in media supplemented with 300 U/mL IL2. The phosphorylation of ERK1/2 and the total amount of ERK1/2 and β-actin were measured in cell lysate by Western blot analysis. Fold increase relative to no PLX4720 control is recorded above the lane. Representative images of three independent experiments are shown. D, naïve C57BL/6 WT mice (control) or C57BL/6 WT mice injected i.v. with 5 × 105 LWT1 melanoma cells (LWT1) were treated i.p. with 20 mg/kg PLX4720 or an equivalent amount of DMSO (vehicle) for 24 hours. Twenty-four hours after this treatment, mice were sacrificed and frequencies of NK cells in the lungs, peripheral blood, and spleens were determined by flow cytometry. The mean ± SEM percentages of NK cells are shown. Data pooled from two independent experiments are shown. *, P < 0.05 Mann–Whitney test.

Figure 4.

PLX4720 enhances NK cell activation and proliferation. A and B, Cell Trace Violet-labeled purified NK cells were cultured with the indicated doses of PLX4720 in media supplemented with 300 U/mL IL2. Analysis of CD69 and proliferation was performed by flow cytometry. A, the mean ± SD CD69 expression of triplicates (left) and representative histograms showing CD69 expression (right) are shown. B, the mean ± SD percentage of divided NK cells (left) and representative histograms showing Cell Trace Violet dilution (right) after 3 days in culture are shown. A and B, data are representative of three independent experiments. Statistical analysis performed by unpaired Student t test, *, P < 0.05. C, 2 × 106 purified NK cells were cultured for 24 hours in the presence of PLX4720 or DMSO in media supplemented with 300 U/mL IL2. The phosphorylation of ERK1/2 and the total amount of ERK1/2 and β-actin were measured in cell lysate by Western blot analysis. Fold increase relative to no PLX4720 control is recorded above the lane. Representative images of three independent experiments are shown. D, naïve C57BL/6 WT mice (control) or C57BL/6 WT mice injected i.v. with 5 × 105 LWT1 melanoma cells (LWT1) were treated i.p. with 20 mg/kg PLX4720 or an equivalent amount of DMSO (vehicle) for 24 hours. Twenty-four hours after this treatment, mice were sacrificed and frequencies of NK cells in the lungs, peripheral blood, and spleens were determined by flow cytometry. The mean ± SEM percentages of NK cells are shown. Data pooled from two independent experiments are shown. *, P < 0.05 Mann–Whitney test.

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PLX4720 and IL2 combination therapy suppresses melanoma metastasis

Given the demonstrated effects of PLX4720 and IL2 on NK cell proliferation ex vivo and the important role of NK cells in PLX4720 mechanism of action in vivo, we next assessed whether a combination of PLX4720 and IL2 could combine to suppress LWT1 metastasis in mice. We have previously shown the antitumor activity of IL2 in other models of tumor metastasis in mice (25) and herein used a similar dose/regimen of daily IL2 for 5 days concurrent with PLX4720 treatment. Notably, mice receiving a combination of PLX4720 and IL2 displayed significantly lower levels of LWT1 pulmonary metastasis compared with mice treated with either agent alone or DMSO/PBS (Fig. 5). Because IL2 has been standardly used in the treatment of advanced human malignant melanoma (26), these data suggest that this combination should now be further explored in this preclinical model.

Figure 5.

IL2 enhances the antimetastatic activity of PLX4720. A total of 5 × 105 LWT1 melanoma cells were injected i.v. into C57BL/6 WT mice and mice were subsequently treated with 20 mg/kg or equivalent amount of DMSO daily on days 1 to 7 and/or PBS or the indicated amount of IL2 (10,000 U or 100,000 U i.p.) given daily on days 1 to 5. Mice were sacrificed on day 14 and lungs harvested and fixed as described in Materials and Methods. Data are from two pooled experiments, with graphs representing the mean ± SEM pulmonary colonies of each group of five mice. Statistical analysis by Mann–Whitney test (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001).

Figure 5.

IL2 enhances the antimetastatic activity of PLX4720. A total of 5 × 105 LWT1 melanoma cells were injected i.v. into C57BL/6 WT mice and mice were subsequently treated with 20 mg/kg or equivalent amount of DMSO daily on days 1 to 7 and/or PBS or the indicated amount of IL2 (10,000 U or 100,000 U i.p.) given daily on days 1 to 5. Mice were sacrificed on day 14 and lungs harvested and fixed as described in Materials and Methods. Data are from two pooled experiments, with graphs representing the mean ± SEM pulmonary colonies of each group of five mice. Statistical analysis by Mann–Whitney test (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001).

Close modal

PLX4720 and IL2 increase human NK cell functions

Finally, to determine whether a similar mechanism may occur in PLX4720-treated cancer patients, we investigated whether PLX4720 directly impacted human NK cells. We observed a clear increase in ERK1/2 phosphorylation after PLX4720 treatment for 24 hours (Fig. 6A). Interestingly, like in mouse NK cells, an increase in total ERK1/2 was associated with PLX4720 treatment, whereas β-actin expression remained comparable between the two groups of NK cells (Fig. 6A). We next cultured NK cells for 6 days with IL2 and increasing concentrations of PLX4720. We observed that PLX4720 induced a dose-dependent increase in CD69 expression on NK cells, demonstrating that PLX4720 potentiates human NK cell activation induced by IL2 (Fig. 6B). Finally, human NK cells secrete IFNγ when ligated via NKp30 in the context of IL2 activation (27). We assessed the effect of PLX4720 on NKp30/IL2-induced IFNγ secretion by purified NK cells from 9 different human donors. We observed that increasing concentrations of PLX (0.3 to 3 μmol/L) significantly enhanced the level of IFNγ above IL2 or NKp30 triggering alone (Fig. 6C). In contrast, PLX was unable to enhance the IFNγ secreted from human NK cells exposed to IL12/IL18 (data not shown).

Figure 6.

PLX4720 enhances human NK cell activation. A, 2 × 106 human NK cells purified from PBMC of healthy donors were cultured for 24 hours in the presence of PLX4720 or DMSO in media supplemented with 300 U/mL IL2. The phosphorylation of ERK1/2, the total amount of ERK1/2 and β-actin were measured in cell lysate by Western blot analysis. Fold increase relative to no PLX4720 control is recorded above the lane. Representative images of two independent experiments are shown. B, purified human NK cells were cultured with the indicated doses of PLX4720 in media supplemented with 300 U/mL IL2. The representative histograms showing CD69 expression (left) and the mean ± SD CD69 expression of experimental replicates (right) are shown. Data are representative of four independent healthy donors. Statistical analysis was performed by unpaired Student t test, **, P < 0.01. C, purified human NK cells from nine donors were cultured in DMSO or PLX4720 (0.3–3.0 μmol/L as indicated) with or without IL2 (300 U/mL) for 20 hours. NK cells were then plated (5 × 104/well) in wells coated with anti-NKp30 (±IL2 1,000 U/mL) and incubated for an additional 20 hours. Supernatants were harvested and IFNγ released in the supernatant measured by ELISA. Statistical analysis performed by Mann–Whitney test as indicated (*, P < 0.05).

Figure 6.

PLX4720 enhances human NK cell activation. A, 2 × 106 human NK cells purified from PBMC of healthy donors were cultured for 24 hours in the presence of PLX4720 or DMSO in media supplemented with 300 U/mL IL2. The phosphorylation of ERK1/2, the total amount of ERK1/2 and β-actin were measured in cell lysate by Western blot analysis. Fold increase relative to no PLX4720 control is recorded above the lane. Representative images of two independent experiments are shown. B, purified human NK cells were cultured with the indicated doses of PLX4720 in media supplemented with 300 U/mL IL2. The representative histograms showing CD69 expression (left) and the mean ± SD CD69 expression of experimental replicates (right) are shown. Data are representative of four independent healthy donors. Statistical analysis was performed by unpaired Student t test, **, P < 0.01. C, purified human NK cells from nine donors were cultured in DMSO or PLX4720 (0.3–3.0 μmol/L as indicated) with or without IL2 (300 U/mL) for 20 hours. NK cells were then plated (5 × 104/well) in wells coated with anti-NKp30 (±IL2 1,000 U/mL) and incubated for an additional 20 hours. Supernatants were harvested and IFNγ released in the supernatant measured by ELISA. Statistical analysis performed by Mann–Whitney test as indicated (*, P < 0.05).

Close modal

Because of the lack of a mouse model, the mechanisms involved in the control of metastatic melanoma by BRAFV600E inhibitor has remained until now, unknown. In this study using a syngeneic BRAFV600E metastatic melanoma mouse model in immune-competent mice, we demonstrated that the antimetastatic effects of a selective BRAFV600E inhibitor PLX4720 require the action of host NK cells and perforin, and in part, the recognition/adhesion molecule, CD226. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAFV600E lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30/IL2 stimulation. Finally, we demonstrated that therapy with a low or high dose of IL2 combined with PLX4720 was able to limit metastatic burden in mice. These findings revealed the importance of NK cells in treating BRAFV600E metastatic melanoma and now provide the basis for additionally exploring the curative potential of BRAFV600E inhibitors in combination with immunotherapies that engage NK cells.

Many reports have highlighted the importance of NK cells for immunotherapy, such as dendritic cell-based immunotherapy (28), IFNα and IL2 (29, 30). Now we can consider that BRAFV600E inhibitors may exert a large part of their antimetastatic activity via NK cells. Furthermore, the safety and efficacy of vemurafenib combined with IFN α-2b is currently under investigation in a new clinical trial (Clinical Trials.gov identifier NCT01943422), although the study is reportedly not recruiting yet. IFNα2b is an adjuvant treatment for patients with resected stage III melanoma, known to upregulate the expression of MHC class I on tumor cells, therefore increasing tumor antigenicity for recognition by CD8+ T cells (31). Although engaging CD8+ T cells is generally a positive approach to therapy, the recognition of MHC class I by NK cells triggers inhibitory signaling to reduce NK cell-mediated cytotoxicity and IFNγ production (23). Because here we found that the antimetastatic effects of PLX4720 require NK cells, it is possible that the upregulation of MHC class I on BRAFV600E-mutant melanoma by IFNα2b might also impair the NK cell-related antimetastatic effects of the BRAF inhibitor, vemurafenib. The enhanced antimetastatic activity of low-dose IL2 and PLX4720 in this model of BRAFV600E-mutant melanoma encourages us to explore other means to improve the NK cell-mediated control of metastasis in humans with BRAFV600E-mutant melanoma. IL21 has shown promise in the treatment of malignant melanoma (32) and has antimetastatic activity and can terminally differentiate NK cells (33). Antibodies reactive with KIR, that relieve MHC class I inhibition, have also been shown to promote NK cell antitumor activity (34). Combinations of these agents with BRAF inhibitors in the treatment of melanoma are now worthy of further exploration.

Although cutaneous melanoma can be treated by surgical excision with great effectiveness when diagnosed early, metastasis of such tumors remains incurable (35). Therefore, investigation of strategies that eliminate remaining or resistant tumor cells is urgently needed. Once it was clear that NK cells were critical in controlling pulmonary metastasis of LWT1, we then tested whether NK cells were necessary for the antimetastatic activity of PLX4720. This was of important, because BRAFV600E inhibitors have mostly been examined in immune-deficient SCID mice that lack T and B cells, but do have NK cells (4). Here, we showed that the depletion of NK cells completely abolished the antimetastatic effects of PLX4720 treatment. We directly demonstrated ex vivo the enhancement of mouse NK cell pERK and CD69 expression, and proliferation by the BRAFV600E inhibitor in the context of IL2 culture. Human NK cell cultures revealed similar pERK1/2 and CD69 upregulation and an increase in IFNγ release by NK cells triggered via the activation receptor, NKp30, in the context of IL2. PLX4720 possibly increases ERK1/2 phosphorylation in BRAF WT cells (like NK cells) by a previously described mechanism (21, 22), whereas ATP-competitive inhibitors increase pERK1/2 levels by inhibiting CRAF inhibitory autophosphorylation, thereby activating the RAF and MAPK pathways.

Follow-up studies should now be performed on peripheral blood and other samples from human cancer patients receiving BRAF inhibitors to determine whether NK cell proliferation and effector functions are being regulated by treatment and how durable or transient these changes might be. It is possible that BRAF inhibitors may have a slightly different spectrum of activities on human NK cells to those observed in mouse NK cells and caution must be taken in translating observations from the mouse to humans. Furthermore, patients with melanoma are now receiving BRAF inhibitor and MEK inhibitor in combination and we might predict that this combination might abrogate any activation of BRAF WT NK cells. Thus far, the clinical focus has been to try and improve the curative potential of BRAFV600E inhibitors by combination with T-cell checkpoint inhibitors, such as anti-CTLA-4 or anti-PD-1. These combination approaches have surprisingly revealed some new toxicities (36). In contrast, therapies that attempt to improve the antimetastatic activities of BRAFV600E inhibitors by promoting NK cell function have not been rationally explored in the clinic. These approaches may improve overall survival in patients receiving BRAF inhibitors.

No potential conflicts of interest were disclosed.

Conception and design: L. Ferrari de Andrade, S.F. Ngiow, L. Martinet, M.J. Smyth

Development of methodology: L. Ferrari de Andrade, L. Martinet

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Ferrari de Andrade, S.F. Ngiow, K. Stannard, S. Rusakiewicz, M. Kalimutho, S.-K. Tey, L. Zitvogel, L. Martinet, M.J. Smyth

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Ferrari de Andrade, S.F. Ngiow, S. Rusakiewicz, M.J. Smyth

Writing, review, and/or revision of the manuscript: L. Ferrari de Andrade, S.F. Ngiow, K.K. Khanna, L. Martinet, M.J. Smyth

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Takeda

Study supervision: S.F. Ngiow, K.K. Khanna, M.J. Smyth

The authors thank Kate Elder and Liam Town for the care, maintenance, and genotyping of the mouse colonies.

L. Ferrari de Andrade was supported by a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) scholarship and M.J. Smyth was supported by a National Health and Medical Research Council of Australia (NH&MRC) Australia Fellowship and NH&MRC Program Grant.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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