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Poster display session: Basic science, Endocrine tumours, Gastrointestinal tumours - colorectal & non-colorectal, Head and neck cancer (excluding thyroid), Melanoma and other skin tumours, Neuroendocrine tumours, Thyroid cancer, Tumour biology & pathology

4568 - BRAF V600E mutation in melanoma sustains IFN-gamma inducible PD-L1 expression by coactivating STAT1 and increasing protein translation

Date

21 Oct 2018

Session

Poster display session: Basic science, Endocrine tumours, Gastrointestinal tumours - colorectal & non-colorectal, Head and neck cancer (excluding thyroid), Melanoma and other skin tumours, Neuroendocrine tumours, Thyroid cancer, Tumour biology & pathology

Topics

Tumour Immunology;  Targeted Therapy

Tumour Site

Melanoma

Presenters

Maja Wasylecka-Juszczyńska

Citation

Annals of Oncology (2018) 29 (suppl_8): viii442-viii466. 10.1093/annonc/mdy289

Authors

M.M. Wasylecka-Juszczyńska, P. Górniak, M. Szydłowski, A. Polak, P. Juszczyński

Author affiliations

  • Dept. Of Experimental Hematology, Institute of Hematology and Transfusion Medicine, 02-776 - Warsaw/PL
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Resources

Abstract 4568

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Clinical trial identification

Legal entity responsible for the study

Institute of Hematology and Transfusion Medicine.

Funding

Polish National Science Center.

Editorial Acknowledgement

Disclosure

All authors have declared no conflicts of interest.

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