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

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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.

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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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Authors and Affiliations

  1. New York University Cancer Institute, New York University School of Medicine, 522 First Avenue, SRB 1303, New York, NY, 10016, USA

    Patrick A. Ott, Trevor Henry, Sonja Jimenez Baranda, Davor Frleta, Olivier Manches, Dusan Bogunovic & Nina Bhardwaj

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Correspondence to Nina Bhardwaj.

Electronic supplementary material

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262_2012_1389_MOESM1_ESM.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

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

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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Ott, P.A., Henry, T., Baranda, S.J. et al. Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties. Cancer Immunol Immunother 62, 811–822 (2013). https://doi.org/10.1007/s00262-012-1389-z

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