Approximately 40-60% of melanoma patients have activating BRAF mutations. Targeting BRAF inhibits proliferation of melanoma cells and increases their immunogenicity. PD-1 immune checkpoint blockade is another breakthrough in melanoma therapy, shown to effectively restore T cell function. These observations indicate that combinatorial use of BRAF inhibitors and immunotherapy might be a rational therapeutic strategy. Herein, we studied the role of BRAF V600E in regulating the expression of PD-L1 in melanoma cells.
CD274 gene transcript abundance and PD-L1 protein level was measured with qPCR, Western-blot and FACS. Identification of signaling pathways responsible for regulation of PD-L1 expression was performed using western blot, FACS and reporter assays. Azide-alkyne cycloaddition (“Click-it” chemistry) was used to analyze de novo protein synthesis.
BRAF-mutant melanoma cell lines exhibited low basal expression of PD-L1 that was markedly induced by IFN-γ, pointing to an adaptive mechanism of PD-L1 expression. BRAF inhibitor significantly reduced IFN-γ-induced PD-L1 levels. In cell lines treated with IFN-γ, vemurafenib decreased STAT1 S727 phosphorylation and expression of PD-L1, suggesting direct regulation of STAT1 by ERK. In A375 cells with constitutively active MEK kinase, vemurafenib had no effect on STAT1 phosphorylation and CD274 transcript level. These results indicate that RAS/RAF/MEK/ERK axis is crucial for maintaining IFN-γ-induced CD274 gene transcription. In addition, vemurafenib decreased activity of proteins responsible for translation regulation (pS6, p4E-BP1), suggesting that inhibition of protein synthesis could be another mechanisms leading to PD-L1 decrease. To test this hypothesis, we measured de novo PD-L1 synthesis following BRAF inhibition, and found markedly reduced PD-L1 translation. Importantly, we also noted decreased translation of other immunoregulatory proteins, such as galectin-1.
BRAF mutations influence PD-L1 expression by modulating its transcription and translation. BRAF inhibition has a potential immunomodulatory effect, at least in part by decreasing IFN-γ induced PD-L1 expression.
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Institute of Hematology and Transfusion Medicine.
Polish National Science Center.
All authors have declared no conflicts of interest.