PO-397 Role of IFNγ in adaptation and immunotherapy resistance in melanoma

Abstract

IntroductionIFNγ is a key component of our immune system, regulating a plethora of biological processes, including antitumor immunity. Its role in tumour immunology is however contradictory, exerting anti-tumour effects early in tumour development but promoting tumour immune evasion and tumour development at later stages. Its involvement in immunotherapy resistance is also extremely complex and is potentially mediated through multiple cellular processes. In this study, we aimed to generate further insights into the role of sustained IFNγ signalling in acquired resistance to immune selection and immunotherapy.Material and methodsA panel of established human and mouse melanoma cell lines were treated with increasing doses of IFNγ for 24 hour and the levels of IFNγ pathway activation were assessed by phospho-STAT1 immunoblotting. To assess the effects of chronic low-dose IFNγ treatment in gene expression of melanoma cell lines, RNA sequencing was performed in melanoma cell lines treated with INFγ for 2 weeks, followed by pathway analysis and gene set enrichment analysis of the differentially expressed genes. Finally, we assessed the DNA damage effects of chronic low-dose IFNγ treatment in melanoma cell lines by γH2AX immunofluorescent staining.Results and discussionsWe showed that established melanoma cell lines retain responsiveness to IFNγ, as reflected by the increased levels of phospho-STAT1 after treatment with increasing doses of IFNγ. Interestingly, RNAseq in melanoma cell lines chronically treated with low-dose IFNγ revealed oxidative stress response as well as DNA damage and repair pathways among the top upregulated pathways in IFNγ treated cells. In line with this, gene set enrichment analysis showed enrichment of DNA damage response and repair related signatures in genes upregulated by IFNγ treatment. Finally, we showed that chronic low-dose IFNγ treatment of melanoma cell lines induces DNA damage, as revealed by increased levels of γH2AX foci per cell and percentage of γH2AX positive cells.ConclusionIn vitro chronic treatment of melanoma cells with low-dose IFNγ has been previously shown to make cells acquire an immunotherapy resistance phenotype. In this study we showed that chronic low-dose treatment of human and mouse melanoma cell lines induces DNA damage, as revealed by γH2AX immunofluorescent staining and RNAseq followed by pathway analysis. The role of IFNγ-driven constitutive DNA damage response in immune evasion and immunotherapy resistance merits further evaluation as an actionable therapeutic target.