**1. Introduction**

Cutaneous melanoma (CM) is an aggressive neoplasm that evolves from the malignant transformation of neural crest stem cell-derived melanocytes [1]. The etiology of melanoma is multifactorial, and the most prominent factors include genetic predisposition, light skin color, multiple naevi, and excessive exposure to UV [2]. Although less prevalent than other skin malignancies, CM accounts for more than 70% of skin cancer-related deaths. Its incidence, however, is steadily increasing in fair-skinned populations worldwide [3]. Detected in the early stages, CM is generally curable, with a 5-year overall survival (OS) rate for the localized disease of 95%. In contrast, stage IV patients carry a grim prognosis, and the 5-year OS rate drops to 25%. CM mortality is usually related to delayed diagnosis or tumor refractoriness to conventional therapies, all of which contribute to metastatic disease [4]. The remarkable progress made in recent years in deciphering CM biology has resulted in the development of several targeted therapies and immune-checkpoint inhibitors (ICIs), such as anti-programmed death (PD-1) and anti-cytotoxic T lymphocyte antigen (CTLA-4), that have truly revolutionized metastatic CM treatment. For instance, therapies targeting critical nodes in the mitogen-activated protein kinase (MAPK) pathway have significantly improved patient OS [5,6]. Furthermore, immune therapies with ICIs led to more durable results and even pathological complete response (pCR) in several patients [7,8]. However, a significant proportion of CM patients fail to respond to these therapies due to the quick emergence of resistance, suggesting that it is critical to gain a deeper understanding of this disease's biology in order to improve its clinical management [9]. At present, CM

**Citation:** Dobre, E.-G.; Constantin, C.; Costache, M.; Neagu, M. Interrogating Epigenome toward Personalized Approach in Cutaneous Melanoma. *J. Pers. Med.* **2021**, *11*, 901. https://doi.org/10.3390/ jpm11090901

Academic Editor: Jun Fang

Received: 11 August 2021 Accepted: 6 September 2021 Published: 9 September 2021

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diagnosis and prognosis rely on assessing clinicopathological variables that do not consider CM genomic and immune heterogeneity [10,11]. In CM, blood biomarkers are very few; S100 and melanoma inhibitory activity (MIA) are the only generally accepted biomarkers that can predict CM evolution and that can indicate therapy efficacy [12] Nevertheless, in tumor-free patients these biomarkers lack utility and additional biomarkers like those related to immune response can be used to monitor the disease dynamics [13]. Although still used in oncology as a general biomarker, serum lactate dehydrogenase (LDH) lacks specificity. An increase in serum LDH would indicate in late stages a high tumor burden, without providing any information on how to guide metastatic patient treatment [14]. Given the poor prognosis of advanced-stage CM, novel biomarkers are needed to assist in early diagnosis and prognosis and stratifying patients into different risk groups to optimize their therapeutic protocols [15].

The idealized model for CM development and progression is the Clark model. This model progressively describes the histopathological changes accompanying normal melanocytes' linear progression to metastatic melanoma via a benign naevus [16] (Figure 1). Nevi are growth-arrested, clonal neoplasms of melanocytes, triggered by certain specific mutations in the MAPK pathway, usually BRAF V600E mutations [17]. Subsequently, the benign nevus evolves into a dysplastic nevus, which progresses through the primary tumor's radial and vertical growth phases (RGP and VGP) and ultimately invades the dermis and regional lymph nodes, from which it metastasizes to distal sites [18]. However, current literature highlights that more than two-thirds of melanomas arise de novo in normal skin without requiring a nevus precursor [19,20]. Given that the annual risk of an individual nevus progressing into melanoma is estimated to be far less than one in 33,000 and the majority of CM patients lack atypical nevi, de novo genesis may be the main route of CM development [21,22]. Although nevus-associated melanomas correlate with favorable prognostic factors, the assessment of nevus as a biomarker in melanoma is still controversial due to certain unconventional models suggested for CM progression [21,23]. Finally, there is also an epigenetic progenitor model, which suggests that a polyclonal epigenetic disruption of tissue-specific (non-cancerous) stem/progenitor cells might act as a driving force of carcinogenesis. The subsequent accumulation of genetic and epigenetic alterations further enhances the tumorigenesis process [24,25] (Figure 1). Regardless of the controversy posed by CM progression, melanomagenesis is fueled by a pro-inflammatory environment. Multiple environmental agents can induce inflammation; however, UV radiation remains the most prominent aggressor, resulting in significant DNA damage and reactive oxygen species (ROS) generation [26]. Inflammation has acute and chronic stages that can shift the physiological balance towards skin regeneration or tumorigenesis, depending on their intensities [18,27]. If the inflammation is persistent and acquires chronic attributes, it may lead to a pre-cancerous lesion in the form of a dysplastic nevus. If detected by a fully functional immune system, this pre-cancerous lesion subsides to healing, and the melanocyte regains its physiological functions. However, if the entire process develops as a sustained chronic inflammation, it triggers a plethora of molecular and cellular networks shaping an immunosuppressive milieu that sustains skin tumorigenesis and further metastasis [18,28] (Figure 1).

Epigenetic alterations have emerged as essential contributors in the pathogenesis of various human diseases, including CM. Epigenetics is another layer of instructions apart from the genetic code that controls how genes are read and expressed, involving a change in the cell phenotype without changes in the genotype [29]. Epigenetic regulation is an umbrella term that encompasses several mechanisms such as DNA methylation, histone post-translational modifications (PTMs), nucleosome remodeling, histone variants, and RNA-mediated post-transcriptional regulation [30]. Influenced by lifestyle and environmental factors, epigenetic changes are highly dynamic and reversible and thus easy to regulate [31]. Given that these epigenetic alterations occur before the clinical diagnosis of CM, these molecular defects may serve as a foundation for developing novel diagnostic tools for early detection [32]. Furthermore, the discovery that distinct epigenetic signatures

may associate with different disease subtypes highlights that these changes may also have prognostic applications in CM management [33,34]. Additionally, the study of epigenetic enzymes facilitates the design of novel treatment strategies that may help in delaying or reversing drug resistance, either as monotherapy or in combinations [35,36]. Of particular importance, observations that epigenetic regulators are often mutated in CM [37] and that mutant genes differ significantly between patients [38] suggest not only that the signaling pathways in these tumors are induced in a patient-dependent manner but also the need to implement personalized medicine in the clinical management of CM. Accordingly, once a certain abnormality is identified, it must be targeted with a specific treatment to reduce the side effects and maximize the therapeutic benefit of the patients [39]. Therefore, a better understanding of CM epigenetics will guide the precision medicine initiative in the field towards the identification of more specific diagnostic, prognostic, and predictive biomarkers, as well as more potent epigenetic inhibitors for the treatment of specific subtypes of CM.

— — — — — — — — — — — — — — **Figure 1.** Schematic representation of CM development from healthy melanocyte to metastatic disease (the Clark model) under a pro-inflammatory milieu. Abbreviations: PRRs—pattern recognition receptors, Tc—cytotoxic T cells, Th1-T helper type 1 cells, Tregs—regulatory T cells, Pax3—paired box gene 3, Sox10—SRY-box transcription factor 10, Endrb endothelin receptor type B, c-Kit—human receptor tyrosine kinase c-kit, Mitf—microphthalmia-associated transcription factor, CDKN2A—cyclin dependent kinase inhibitor 2A, CDK4—cyclin dependent kinase 4, CCND1—cyclin D1, APC adenomatous polyposis coli, PTEN—phosphatase and tensin homolog, hTERT—human telomerase reverse transcriptase, MMP2—matrix metalloproteinase 2, SWI/SNF—switch/sucrose non-fermentable chromatin remodeling complex, PI3K phosphoinositide 3-kinase, HIF-1α—hypoxia-inducible factor-1α, PRC2—polycomb repressive complex 2.

> — — — — 1α— 1α, PRC2— We present herein both old and new evidence regarding the roles of DNA methylation and chromatin modifications in melanoma pathogenesis and discuss recent advances in investigating their translational potential as biomarkers and therapeutic targets.

#### **2. Epigenetics: Another Layer of Information in Gene Expression Regulation**

The epigenetics field focuses on the study of heritable alterations in gene expression with no underlying changes in the DNA sequence. Epigenetic regulation has been extensively reviewed in association with various human biological processes, such as embryogenesis, cell differentiation, X chromosome inactivation, and pathologies such as cancer [40,41]. The best-characterized epigenetic mechanisms are DNA methylation and histone modifications; however, the epigenetic scenario is much more complicated with new

players and new mechanisms including non-coding RNA (ncRNA)-mediated regulation, histone variants, and ATP-dependent chromatin remodeling [42–44]. All these discrete but reversible modifications may orchestrate extensive changes in chromatin structure and conformation, interfering with the transcriptional machinery's ability to access its target genes and promoters [45,46].

The epigenetic machinery carefully modifies the homeostatic balance between euchromatin and heterochromatin, which is essential to genomic stability [31]. The epigenetic players involved are divided into three classes: writers (generally enzymes that induce chemical modifications in histones and DNA), erasers (entities that erase these chemical signatures), and readers (enzymes that recognize and interpret various chemical modifications) [47]. Within the review, we will focus mainly on DNA methylation and histone modifications. These two types of epigenetic changes appear to influence each other in the deposition during mammalian development and carcinogenesis; histone methylation appears to direct DNA methylation patterns, while DNA methylation may serve as a model for establishing certain histone changes [32]. Moreover, in CM, DNA methylation is gaining increased importance in PD-related immune therapy [48], while histone alterations are associated with BRAF-targeted therapy [49].

In mammals, DNA methylation is an epigenetic signature that occurs at the 5' position of the cytosine preceding a guanine nucleotide (denoted CpG, where *p* implies the phosphodiester bond between the two nucleosides) [50]. CpG-enriched regions, called 'CpG islands', are found in about 40% of mammalian gene promoters, making these particular regions prone to methylation [51]. Interestingly, methylation's biological effects can vary widely depending on genomic location; thus, methylation in the promoter regions of genes is usually associated with transcriptional repression, while methylation in the gene body promotes transcription [31]. The enzymes that perform this modification are DNA methyltransferases (DNMTs), using S-adenosylmethionine (SAM) as a donor of methyl groups [52]. The human genome encodes at least four DNMTs: DNMT1, DNMT3A, DNMT3B, and DNMT3L [53]. DNMT1 is responsible for maintaining DNA methylation, while DNMT3A and DNMT3B catalyze the de novo synthesis of 5-mC [52,54]. Conversely, 5-mC methylation marks can be deleted by ten-eleven translocation (TET) proteins [55]. DNMTs may play critical roles in embryonic development and the intergenerational propagation of specific methylation patterns [56]; thus, any disorder in the functionality of these enzymes can have important physiological consequences [53]. DNMTs were recently shown to be involved in immune therapy resistance in CM, as further detailed in [57].

Histones, especially their N-terminal tails (containing an average of 15–30 residues), can undergo a plethora of PTMs, such as acetylation, methylation, phosphorylation, and more rarely, ubiquitination, sumoylation, and glycosylation [58]. The acetylation (ac) of histone lysines (K), is associated with the transition to an "open" conformation; this weakens DNA–histone interactions, increases DNA accessibility, and facilitates transcription. Acetylated lysine residues may also serve as binding platforms for transcription factors and other histone-modifying enzymes, such as bromodomains [59]. Another type of histone modification is methylation (me) of lysine and arginine (R) residues. Histone methylation can be present in several forms, so that the lysine residues may be mono-, di- or trimethylated, while the arginine residues may be mono- or di-methylated. Methylated histone residues are recognized by several protein domains such as plant homeodomain zinc fingers, Tudor domains, or WD40 repeats. While histone acetylation is usually associated with transcriptional activation, histone methylation has various functions, depending on the type of histone, the type of amino acid, the degree of modification, and another histone's PTMs in the vicinity [60,61]. For example, in the promoter region, di- or tri-methylation of histone H3 at lysine 4 (H3K4me2, H3K4me3) is associated with transcriptional activation; in contrast, H3K27me3 and H3K9me3 are transcriptionally repressive marks [62]. Combinations of H3K4me1, H3K27me3, and H3K27ac marks can be used to distinguish active enhancers (H3K4me1 and H3K27ac positive) from inactive (H3K4me1 positive and H3K27ac negative) or poised (H3K4me1 and H3K27me3 positive) gene enhancers [63]. Interestingly, promoters of lineage-controlling developmental genes display a particular epigenetic signature that combines the activating H3K4me3 and the repressive H3K27me3 mark; these bivalent domains, which have also been reported in cancers, allow for rapid activation of developmental genes during embryogenesis, while maintaining repression in the absence of differentiation signals [64,65]. However, the balance between histone methylation and acetylation is tightly controlled by histone methyltransferases (HMTs)/demethylases (HDMs) and histone acetyltransferases (HATs)/histone deacetylases (HDACs), respectively [66]. Alterations in histone "writers" or "erasers" have been associated with many pathologies, including cancer [31,45,67]. Histone modifications can be associated in CM with therapy resistance [68] and/or with epithelial–mesenchymal transition (EMT) and metastasis [69]. κB oncog
