**6. Endogenous Retroviruses, Viral Mimicry That Elicits Anti-Tumor Immunity**

Tumor cells often show enhanced DNA methylation at CpG-rich sites, located in endogenous retroelements (reviewed in [168,169]). These elements, which make up more than 40% of the human genome, consist of repetitive sequences that belong to three major classes: endogenous retroviruses (ERVs), short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs). Endogenous retroelements have originated from ancient infections by exogenous retroviruses, which integrated their genomes into the genome of germ cells of the host. This allowed for the vertical transmission of these elements to the offspring of the infected host. During evolution, the majority of such elements have accumulated excessive DNA mutations that inactivated their genes. However, a minority (thousands) retained some of their protein coding potential. Importantly, peptides that are derived from human endogenous retroviruses (hERVs) can be recognized by immune cells. This is exemplified by the infiltration of T cells with receptors specific for hERVs-derived epitopes, into hERVs-expressing clear cell renal cell carcinoma tumors [170]. Furthermore, endogenous retroelements may express additional immunostimulators since transcription of these elements may generate dsRNA molecules (by bidirectional transcription, as well as by sense–antisense pairing); and if reverse transcription follows, complementary DNA (cDNA) and double-stranded DNA (dsDNA) may be created too. These products, which mimic viral infection, may then be sensed by endosomal TLR3, 7, 8 or 9, and/or by cytoplasmic PRRs, including RIG- I, MDA5, cGAS [168,169]. Sensing this 'viral mimicry',

activates antiviral signaling cascades, including an IFN response (see [171] and additional examples below). ERVs are repressed by variety of mechanisms, including epigenetic silencing through DNA methylation and histone modifications (reviewed in [172–175]).

Given the potential immunogenicity of endogenous retroelements and their epigenetic suppression, reactivation of these elements by epigenetic modifiers in cancer cells may results in the abovementioned viral mimicry, leading to an anti-cancerous state. For example, treatment of colorectal or ovarian cancer cells with DNMT inhibitors (DNMTis) results in induction of transcription from otherwise suppressed ERVs, the subsequent formation of dsRNA from specific ERV elements, recognition of these dsRNA molecules by MDA5/TLR3 sensors, activation of the mitochondrial antiviral-signaling protein (MAVS)-IRF7 axis and induction of IFN. Together, these result in enhanced anti-proliferative/apoptotic responses [176,177].

The complex interactions among oncogenic signaling, epigenetics and viral mimicry can be further demonstrated by the effects of the cyclin-dependent kinases 4 and 6 (CDK4/6) on cancer immunity [178]. CDK4/6, which interact with D-type cyclins, are central drivers of the cell cycle at the G1-S transition, transduce variety of mitogenic signals and their activity is associated with oncogenesis of several types of cancer (recently reviewed in [179]). Upon the induction of mitogenic signal, cyclin D-CDK4/6 complex promotes retinoblastoma (Rb) phosphorylation, leading to the release of transcription factor E2F from the Rb-E2F complex, and entry into S phase and DNA replication. One of the many targets of E2F is the *Dnmt1* gene [178,180]. Accordingly, CDK4/6 inhibition reduces DNMT1 activity, which leads to activation of ERVs expression, formation of ERVs dsRNA and IFN responses to this viral mimicry. Overall, this increases tumor antigen presentation and, together with additional effects of the CDK4/6 inhibitors, leading to cytotoxic T-lymphocytes (CTL) mediated clearance of the tumor cells in mouse models [178]. Thus, mitogenic signals suppress ERVs expression via DNA methylation, mediated by the CDK4/6-Cyclin D-Rb-DNMT1 axis, and inhibition of this axis results in ERVs activation followed by enhanced anti-tumor immunity.
