Molecular, clinicopathological, and immune correlates of LAG3 promoter DNA methylation in melanoma
Anne Fröhlich,
Judith Sirokay,
Simon Fietz,
Timo J. Vogt,
Jörn Dietrich,
Romina Zarbl,
Mike Florin,
Pia Kuster,
Gonzalo Saavedra,
Susana Ramírez Valladolid,
Friederike Hoffmann,
Lukas Flatz,
Sandra S. Ring,
Carsten Golletz,
Torsten Pietsch,
Sebastian Strieth,
Peter Brossart,
Gerrit H. Gielen,
Glen Kristiansen,
Friedrich Bootz,
Jennifer Landsberg,
Dimo Dietrich
Affiliations
Anne Fröhlich
Department of Dermatology and Allergy, University of Bonn, Bonn, Germany
Judith Sirokay
Department of Dermatology and Allergy, University of Bonn, Bonn, Germany
Simon Fietz
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
Timo J. Vogt
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
Jörn Dietrich
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
Romina Zarbl
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
Mike Florin
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
Pia Kuster
Department of Dermatology and Allergy, University of Bonn, Bonn, Germany
Gonzalo Saavedra
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
Susana Ramírez Valladolid
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
Friederike Hoffmann
Department of Dermatology and Allergy, University of Bonn, Bonn, Germany
Lukas Flatz
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
Sandra S. Ring
Institute of Immunobiology, Kantonsspital St Gallen, St Gallen, Switzerland.; Microbiology and Immunology PhD Program, University of Zurich, Zurich, Switzerland
Carsten Golletz
Institute of Pathology, University Hospital Bonn, Bonn, Germany
Torsten Pietsch
Institute of Neuropathology, University Hospital Bonn, Bonn, Germany
Sebastian Strieth
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
Peter Brossart
Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany
Gerrit H. Gielen
Institute of Neuropathology, University Hospital Bonn, Bonn, Germany
Glen Kristiansen
Institute of Pathology, University Hospital Bonn, Bonn, Germany
Friedrich Bootz
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
Jennifer Landsberg
Department of Dermatology and Allergy, University of Bonn, Bonn, Germany
Dimo Dietrich
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany; Corresponding author.
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.
