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.

Methylation of DNA at CpG sites is the most common and stable of epigenetic changes in cancer. Hypermethylation acts to limit immune checkpoint blockade immunotherapy by inhibiting endogenous interferon responses needed for recognition of cancer cells. By contrast, global hypomethylation results in the expression of programmed death ligand 1 (PD-L1) and inhibitory cytokines, accompanied by epithelial-mesenchymal changes that can contribute to immunosuppression. The drivers of these contrasting methylation states are not well understood. DNA methylation also plays a key role in cytotoxic T cell 'exhaustion' associated with tumor progression. We present an updated exploratory analysis of how DNA methylation may define patient subgroups and can be targeted to develop tailored treatment combinations to help improve patient outcomes. DNA Methylation, ICB, and Cancer DNA methylation can have major effects on gene expression and is the most commonly studied type of epigenetic modification (see Glossary). It comprises the covalent modification of the nucleotide cytosine at the 5ʹ position at sites preceding guanine (CpG) [1]. During mammalian cell division, it is replicated by the maintenance enzyme DNA methyltransferase 1 (DNMT1) on the daughter strand cytosine at the complementary CpG, usually resulting in gene silencing. New sites of DNA methylation, known as de novo methylation, can be introduced by DNMT3a or DNMT3b (Box 1). Conversely, methyl groups can be erased by ten-eleven translocation (TET) family proteins followed by glycosylation and replacement with an unmethylated cytosine. DNMT1 recruitment to replicating chromatin is facilitated by the 'ubiquitin-like with PHD and ring-finger domains' (UHRF) E3 ubiquitin ligase UHRF1 [2,3]. Methylation at CpG sites can also be recognized by methyl CpG binding 'reader' proteins such as methyl-CpG binding domain protein 1 (MBD1) and methyl-CpG binding protein 2 (MeCP2) harboring transcriptionally repressive activity. The latter may bind histone deacetylases (HDACs), which can contribute to the repression of certain genes [4]. Highlights Genome-wide DNA methylation is a relatively stable epigenetic characteristic of cells, which can be dysregulated in cancer cells by oncogenic signals.

The canonical Wnt signalling pathway induces the β-catenin/LEF transcription factors. It is activated in various cancers, most characteristically carcinomas, in which it promotes metastatic spread by increasing migration and/or invasion. The Wnt/β-catenin signalling pathway is frequently activated in melanoma, but the presence of β-catenin in the nucleus does not seem to be a sign of aggressiveness in these tumours. We found that, unlike its positive role in stimulating migration and invasion of carcinoma cells, β-catenin signalling decreased the migration of melanocytes and melanoma cell lines. In vivo, β-catenin signalling in melanoblasts reduced the migration of these cells, causing a white belly-spot phenotype. The inhibition, by β-catenin of migration was dependent on MITF-M, a key transcription factor of the melanocyte lineage, and CSK, a Src-inhibitor. Despite reducing migration, β-catenin signalling promoted lung metastasis in the Nras-driven melanoma murine model. Thus, β-catenin may play conflicting roles in the metastatic spread of melanoma, repressing migration while promoting metastasis. These results highlight that metastasis formation requires a series of successful cellular processes, any one of which may not be optimally efficient.