We have previously shown that melanoma cells were resistant to apoptosis induced by TNF family members Fas ligand (FasL), TNF-α, and CD40L. FasL also was not involved in CD4 T cell-mediated killing of melanoma cells. In the present study, we have tested melanoma cells for their susceptibility to apoptosis induced by human TNF-related apoptosis-inducing ligand (TRAIL) and the ability of a mAb against TRAIL to inhibit apoptosis and CD4 CTL-mediated killing of melanoma and Jurkat target cells. The results show that TRAIL-induced apoptosis in cells from 7 of 10 melanoma cell lines tested as well as in Jurkat T cells. Susceptibility to apoptosis was increased in some of the cell lines by treatment with cyclohexamide or actinomycin D. The melanoma cells were resistant to apoptosis induced by FasL, TNF-α, and CD40L. mAb M180 against TRAIL inhibited apoptosis induced by TRAIL. It was also found to inhibit CD4 CTL-mediated killing of Jurkat T cells as well as autologous and allogeneic melanoma cells. The degree of inhibition produced by the mAb varied between different clones of CTL and according to the susceptibility of the target cells to TRAIL-induced apoptosis. These results suggest that TRAIL is an important mediator of cell death induced by CTL and may have an important therapeutic role against human melanoma.

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.

To study the effects of intralesional Rose Bengal for chemoablation of metastatic melanoma. Twenty-six target lesions in 11 patients with locoregionally recurrent disease were injected with the agent PV-10 (10% w/v Rose Bengal in saline) at a dose of 0.5 ml/cc lesion volume. An additional 28 untreated lesions were observed for potential bystander effect. The treatment was well tolerated and an objective response was observed in 12 target lesions, with an additional seven lesions remaining static over at least 12 weeks of observation. In this dose-evaluation study, response rate was dose dependent, increasing to 69% after higher dose injections. Nontarget lesions exhibited a 27% objective response rate that increased to 44% in patients exhibiting a positive response of target lesions. These findings indicate that intralesional Rose Bengal is nontoxic and could benefit patients with metastatic melanoma.

Long-term safety of pembrolizumab in melanoma was analyzed in KEYNOTE-001, KEYNOTE-002, and KEYNOTE-006. Analysis involved patients who received ≥1 pembrolizumab dose. Lead-time bias was addressed via landmark analyses in patients who were progression-free before day 147. Adverse events (AEs) were analyzed for 1567 patients (median follow-up, 42.4 months). Most AEs were mild/moderate; grade 3/4 treatment-related AEs occurred in 17.7% of patients. Two pembrolizumab-related deaths occurred. Any-grade immune-mediated AEs (imAEs) occurred in 23.0%, most commonly hypothyroidism (9.1%), pneumonitis (3.3%), and hyperthyroidism (3.0%); grade 3/4 imAEs occurred in 6.9% of patients. Most imAEs occurred within 16 weeks of treatment. In landmark analysis, patients who did (n = 79) versus did not (n = 384) develop imAEs had similar objective response rates (ORRs) (64.6% versus 63.0%); median time to response (TTR), 5.6 months for both; median duration of response (DOR), 20.0 versus 25.3 months; median progression-free survival (PFS), 17.0 versus 17.7 months; median overall survival (OS), not reached (NR) versus 43 months (p = 0.1104). Patients who did (n = 17) versus did not (n = 62) receive systemic corticosteroids had similar ORRs (70.6% vs. 62.9%) and median TTR (6.4 vs. 5.6 months) but numerically shorter median PFS (9.9 vs. 17.0 months); median DOR, 14.2 months versus NR; median OS, NR for both. These results enhance the knowledge base for pembrolizumab in advanced melanoma, with no new toxicity signals after lengthy follow-up of a large population. In landmark analyses, pembrolizumab efficacy was similar regardless of imAEs or systemic corticosteroid use. NCT01295827, NCT01704287, NCT01866319.

Constitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy.