**4. Oncogene-Induced Silencing of Immune Genes by DNA Methylation**

Methylation of cytosines within CpG dinucleotides is a highly abundant epigenetic modification of mammalian genomes [141]. Methylation patterns, which regulate gene expression, are dynamically regulated via the opposing activities of enzymes that introduce or remove this modification, known as 'writers' and 'erasers', respectively. This regulatory apparatus is complemented by chromatin 'readers', i.e., protein modules that recognize histone and DNA modifications [142]. In accord with the deregulation of methylation in cancer development, DNA methyl transferases (DNMTs, 1, 3A and 3B) are overexpressed in many tumors [143–146]. A connection between tumorigenic features of cancer cells, epigenetic silencing and defects in antiviral responses is already observed upon spontaneous immortalization of fibroblasts which results in epigenetic silencing of ISGs [147]. Numerous studies reported on promoter methylation and down regulation of different IRFs (e.g., different combinations of IRF4, IRF5, IRF6, IRF7) in cancers, including fibrosarcoma [148], melanoma [149], lung cancer [150], and gastric cancer [151]. Similarly, the promoter of IFN-γ was shown to be methylated in cervical cancer [104]. Moreover, our analysis of the TCGA skin cutaneous melanoma (SKCM) database revealed significantly higher methylation of promoters of genes presenting highly-correlated expression with STAT1 (a gene group that is enriched for cell autonomous immunity genes), as compared to randomly selected genes [152]. In accord with its tumor-promoting functions, RAS was termed as "silent assassin", due to its gene silencing abilities in cancer cells [153]. In this context, DNMT1 expression is transcriptionally regulated by RAS-induced signaling pathways [143,154,155]. RAS-mediated transformation also modulates the function of DNA-methylation readers such as MBD2 [156]. Furthermore, the expression of enzymes that revert DNA methylation (ten-eleven translocation (TET) methylcytosine dioxygenases) is also regulated by oncogenic signaling in general, and RAS signaling in particular; and ERK-mediated suppression of TET1 is required for K-RAS-induced cellular transformation and hypermethylation of DNA [157]. In accord with a functional interaction between RAS and DNMTs in mediating pro-tumorigenic features, a genome-wide RNA interference (RNAi) screen in K-RAS-transformed NIH-3T3 cells identified DNMT1 and members of the RAS/MEK pathway (ERK2 and MAP3K9) as required for the silencing of the pro-apoptotic FAS gene [158]. The role of such functional interaction in mediating the suppression of

immune responses is observed in the downregulation of the RAS-effector MYC and the upregulation of ISGs in lung cancer, following DNMT inhibition [159]. Interestingly, promoter methylation of IRF7 and enhancement of viral infection was observed in nasal epithelial cells exposed to cigarette smoke [160], suggesting that exposure to carcinogens may already set the stage for the silencing of immune genes observed in malignant cells.
