title: Inducible but not constitutive expression of PD-L1 in human melanoma cells is dependent on activation of NF-κB.
abstract: Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity.
authors.affiliation: Melanoma Research, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, Sydney, New South Wales, Australia.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Gowrishankar
authors.affiliation: Melanoma Research, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, Sydney, New South Wales, Australia.
authors.forename: Dilini
authors.initials: D
authors.lastname: Gunatilake
authors.affiliation: Melanoma Research, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, Sydney, New South Wales, Australia.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Gallagher
authors.affiliation: Melanoma Research, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, Sydney, New South Wales, Australia.
authors.forename: Jessamy
authors.initials: J
authors.lastname: Tiffen
authors.affiliation: Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Helen
authors.initials: H
authors.lastname: Rizos
authors.affiliation: Melanoma Research, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, Sydney, New South Wales, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Hersey
mesh_terms: D060890:B7-H1 Antigen / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology*; D008545:Melanoma / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology*; D013997:Time Factors; D000077484:Vemurafenib
chemical_list: C471109:4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; D060890:B7-H1 Antigen; C423236:CD274 protein, human; D007093:Imidazoles; D007211:Indoles; D016328:NF-kappa B; D011810:Quinoxalines; D013449:Sulfonamides; D000077484:Vemurafenib; D007371:Interferon-gamma
references.citation: J Biol Chem. 2003 Jan 17;278(3):1450-6
references.pmid: 12403772
references.citation: N Engl J Med. 2013 Jul 11;369(2):122-33
references.pmid: 23724867
references.citation: J Immunol. 2001 May 1;166(9):5337-45
references.pmid: 11313369
references.citation: Genes Dev. 2012 Feb 1;26(3):203-34
references.pmid: 22302935
references.citation: J Invest Dermatol. 2014 Nov;134(11):2795-805
references.pmid: 24906137
references.citation: Clin Cancer Res. 2014 Jul 1;20(13):3446-57
references.pmid: 24812408
references.citation: J Dermatol Sci. 2005 Nov;40(2):95-103
references.pmid: 16085391
references.citation: Cancer Discov. 2013 Dec;3(12):1355-63
references.pmid: 24078774
references.citation: PLoS One. 2013;8(4):e61602
references.pmid: 23585913
references.citation: N Engl J Med. 2012 Jun 28;366(26):2443-54
references.pmid: 22658127
references.citation: Ann Oncol. 2014 Oct;25(10):1935-40
references.pmid: 25009014
references.citation: Immunol Rev. 2012 Mar;246(1):193-204
references.pmid: 22435556
references.citation: Pigment Cell Melanoma Res. 2014 Nov;27(6):1126-37
references.pmid: 24924589
references.citation: Clin Cancer Res. 2013 Feb 1;19(3):598-609
references.pmid: 23095323
references.citation: Expert Opin Biol Ther. 2013 Jun;13(6):847-61
references.pmid: 23421934
references.citation: J Invest Dermatol. 2012 Jul;132(7):1850-9
references.pmid: 22437314
references.citation: FEBS Lett. 2006 Feb 6;580(3):755-62
references.pmid: 16413538
references.citation: J Leukoc Biol. 2004 Feb;75(2):163-89
references.pmid: 14525967
references.citation: Acta Pharmacol Sin. 2005 Apr;26(4):462-8
references.pmid: 15780196
references.citation: Nat Med. 2007 Jan;13(1):84-8
references.pmid: 17159987
references.citation: Cytokine Growth Factor Rev. 2010 Feb;21(1):11-9
references.pmid: 20018552
references.citation: N Engl J Med. 2013 Jul 11;369(2):134-44
references.pmid: 23724846
references.citation: Nature. 2010 Dec 23;468(7327):1119-23
references.pmid: 21068722
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20852-7
references.pmid: 19088198
references.citation: Cancer Immunol Immunother. 2012 Jan;61(1):101-8
references.pmid: 21853301
references.citation: Sci Transl Med. 2013 Aug 28;5(200):200ra116
references.pmid: 23986400
references.citation: Nat Rev Cancer. 2012 Jul;12(7):465-77
references.pmid: 22722403
references.citation: J Clin Oncol. 2014 Apr 1;32(10):1020-30
references.pmid: 24590637
references.citation: Annu Rev Immunol. 2008;26:677-704
references.pmid: 18173375
references.citation: Melanoma Res. 2012 Jun;22(3):244-51
references.pmid: 22516966
references.citation: Pigment Cell Res. 2006 Apr;19(2):112-24
references.pmid: 16524427
references.citation: Curr Opin Immunol. 2013 Jun;25(3):381-8
references.pmid: 23582509
references.citation: Clin Cancer Res. 2014 Oct 1;20(19):5064-74
references.pmid: 24714771
references.citation: Nat Rev Cancer. 2012 Feb;12(2):121-32
references.pmid: 22257950
references.citation: Sci Transl Med. 2012 Mar 28;4(127):127ra37
references.pmid: 22461641
references.citation: Clin Cancer Res. 2006 Feb 1;12(3 Pt 1):950-60
references.pmid: 16467110
references.citation: Nat Med. 2002 Aug;8(8):793-800
references.pmid: 12091876
references.citation: Cell Cycle. 2010 Feb 15;9(4):829-39
references.pmid: 20107316
references.citation: Cancer Cell. 2011 Apr 12;19(4):429-31
references.pmid: 21481782
references.citation: Cancer Res. 1999 Jun 1;59(11):2747-53
references.pmid: 10364001
references.citation: Immunol Rev. 2012 Mar;246(1):379-400
references.pmid: 22435567