Differentiation status determines the effects of IFNγ on the expression of PD-L1 and immunomodulatory genes in melanoma

Background Melanoma cells frequently dedifferentiate in response to inflammation which can increase responses to certain cytokines. Interferon-γ (IFNγ) is an integral part of the anti-tumour immune response and can directly induce both differentiational changes and expression of immunosuppressive proteins in melanoma cells. How the differentiation status of melanoma cells affects IFNγ responses remains unclear. Methods Dedifferentiation of melanoma cells was induced via either siRNA or shRNA mediated MITF knockdown and the cells were subsequently treated with IFNγ. Effects of MITF knockdown and IFNγ treatment on gene expression were evaluated via qPCR and RNA sequencing. A Luminex assay was used to analyse the effects of dedifferentiation and IFNγ treatment on cytokine secretion. Effects on PD-L1 protein expression were analysed via flow cytometry and western blotting. Inhibition of the JAK kinases, NF-κB and STAT3 with small molecule inhibitors, and siRNA mediated knockdown of STAT1 and IRF1 was applied to investigate the molecular mechanism behind IFNγ induced PD-L1 expression in dedifferentiated melanoma cells. The effects of inhibitor treatments and siRNA mediated knockdowns were evaluated via qPCR and western blotting. Bioinformatic analyses of publicly available RNA sequencing data, consisting of 45 patient derived melanoma cell lines, with or without IFNγ treatment, was conducted to assess the generalisability of the in vitro results. Results Dedifferentiation renders 624Mel melanoma cells hypersensitive to IFNγ stimulation in a context-dependent manner, resulting in non-additive upregulation of IFNγ-induced genes, increased PD-L1 protein expression and amplified secretion of CCL2, CXCL10 and IL-10. Furthermore, the intensified PD-L1 protein expression occurs through the JAK-STAT1-IRF1 axis. Lastly, dedifferentiated patient derived melanoma cell lines showed enhanced inflammatory signalling in response to IFNγ compared to differentiated cells, and tended to have higher PD-L1 expression, associated with increased IRF1 expression and activity. Conclusions Together, these findings indicate the existence of a molecular context linking dedifferentiation and IFNγ signalling in melanoma which may lead to immune evasion. Furthermore, these results imply that modulating melanoma differentiation may help shape IFNγ responsiveness.