H3K9 Methyltransferases

Another histone-modifying enzyme involved in CM pathogenesis is bifurcated domain SUV39/SET 1 (SETDB1), which belongs to the SUV39 family of histone lysine methyltransferases [127]. This enzyme is involved in the trimethylation of H3K9, which is a specific signature of transcriptionally repressive chromatin [128].

In human melanoma samples, SETB1 often appears amplified in association with another histone methyltransferase, EHMT1 [129]. Subsequently, a positive association between SETB1 expression and several prognostic factors such as increased mitotic index, advanced Clark levels, and epidermal involvement in tissue biopsies of CM patients was also described [130]. A recent study, led by Orouji at al., revealed that the expression and amplification rate of SETDB1 may serve as an individual prognostic biomarker in CM, with increased levels of SETB1 protein being associated with metastasis and lower patient survival rates [131]. Compared to normal melanocytes, melanoma cells showed 8–13.9 times higher levels of SETDB1. Interestingly, they found that all those SETDB1 highly amplified human cell lines were BRAF V600E mutants [131]. Functional studies have shown that SETDB1 exerts its oncogenic effects in CM by modulating the expression of thrombospondin 1, a molecule known for its involvement in cell migration and invasiveness [131]. Surprisingly, it was found that SETDB1 regulates not only H3K9 methylation patterns but also H3K4me1 deposition, emphasizing that SETDB1's involvement in tumorigenesis is much broader than previously thought. Furthermore, treatment with

an H3K9me-specific histone methyltransferase inhibitor is highly effective in this context, leading to a considerable decrease in tumor cell viability. Interestingly, melanoma cells harboring low levels of SETDB1 were not affected by treatment with epigenetic inhibitors, underscoring SETDB1's role as a valuable therapeutic target in CM [131].

## 4.2.2. Histone Modifications "Readers"

Protein readers can recognize specific chromatin changes or combinations of PTMs and histone variants to further direct the transcriptional outcome [47]. Some of the beststudied families of chromatin readers are the bromodomain and extra-terminal domain (BET) family of adapter proteins (BRD2, BRD3, BRD4, and BRDT). BET proteins have an increased affinity for acetylated histone residues, enabling transcriptional activation by interaction with the transcriptional machinery [132]. Notably, the SWI/SNF complex, which uses energy from ATP hydrolysis to reshape the structure of chromatin, is also dependent on the presence of bromodomain-containing domains to be fully functional [133]. BET protein involvement in melanomagenesis was demonstrated by Segura at al. when it was shown that BRD2 and BRD4 are overexpressed in human melanoma cell lines and tissues, controlling the expression of certain genes involved in cell cycle progressions and survival [134]. Gene expression and IHC analysis of human tissue biopsies have confirmed higher levels of BRD2 and BRD4 in primary and metastatic tumors relative to melanocytes and nevi, suggesting that these BET proteins are involved in melanoma tumorigenesis [134]. Treatment with BET inhibitors (BETi) significantly reduced tumor growth and metastasis in both in vitro and in vivo models, and those effects were recapitulated by individual silencing of BRD4. Notably, the pharmacological capabilities of BETi have not been influenced by the mutational status of BRAF or NRAS, offering new promises for the treatment of patients that do not harbor actionable mutations [134]. Collectively, all these observations potentiate the pivotal role of the BRD4 protein in CM tumorigenesis, propelling it as a potential prognosis biomarker and therapeutic target in CM.
