Anne Fröhlich1, Judith Sirokay1, Simon Fietz2, Timo J Vogt3, Jörn Dietrich3, Romina Zarbl3, Mike Florin2, Pia Kuster1, Gonzalo Saavedra2, Susana Ramírez Valladolid2, Friederike Hoffmann1, Lukas Flatz4, Sandra S Ring5, Carsten Golletz6, Torsten Pietsch7, Sebastian Strieth3, Peter Brossart8, Gerrit H Gielen7, Glen Kristiansen6, Friedrich Bootz3, Jennifer Landsberg1, Dimo Dietrich9. 1. Department of Dermatology and Allergy, University of Bonn, Bonn, Germany. 2. Department of Dermatology and Allergy, University of Bonn, Bonn, Germany; Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. 3. Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. 4. Institute of Immunobiology, Kantonsspital St Gallen, St Gallen, Switzerland.; Department of Oncology and Haematology, Kantonsspital St Gallen, St Gallen, Switzerland; Department of Dermatology, University Hospital Zurich, Zurich, Switzerland; Department of Dermatology and Allergology, Kantonsspital St Gallen, St Gallen, Switzerland. 5. Institute of Immunobiology, Kantonsspital St Gallen, St Gallen, Switzerland.; Microbiology and Immunology PhD Program, University of Zurich, Zurich, Switzerland. 6. Institute of Pathology, University Hospital Bonn, Bonn, Germany. 7. Institute of Neuropathology, University Hospital Bonn, Bonn, Germany. 8. Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany. 9. Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Electronic address: dimo.dietrich@gmail.com.
Abstract
BACKGROUND: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. METHODS: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). FINDINGS: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. INTERPRETATION: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.
BACKGROUND: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. METHODS: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanomapatients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). FINDINGS: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. INTERPRETATION: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.
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