Abstract
Melanomas, which originate from melanocytic cells, mainly develop in the skin but can also arise at other body sites. The disease accounts for approximately 90% of deaths related to cutaneous tumors with late stage metastatic melanoma having a very poor prognosis of 6–9 month median survival for untreated patients. Research in the last decades resulted in ground-breaking discoveries of melanoma genetics and biology. High frequency mutations in genes like BRAF, NRAS and KIT, which lead to hyper-activation of the MAPK signaling pathway, drive melanoma progression. Targeting the MAPK signaling pathway has successfully been translated into effective therapies that significantly improve patient survival. Despite the unquestionable importance of such genetic events, the involvement of epigenetic alterations for melanoma development, and resistance to aforementioned therapies is becoming increasingly apparent. In this chapter, epigenetic alterations commonly found in melanoma are introduced, with a focus on histone and DNA modifications and their relevance for melanoma development, progression and therapy response. Detailed knowledge about this emerging aspect of melanoma research will help to understand the plastic nature of melanoma and set the foundation for novel treatment strategies that target aberrant gene regulation on genetic and epigenetic levels.
Keywords
- biomarker
- drug resistance
- histone modifications
- DNA methylation
- melanoma
- targeted therapy
1. Introduction
The grim prognosis for metastatic melanoma patients and the steadily increasing rates of melanoma incidents, that are projected to continuously rise within the next decades [1], represent a challenge for healthcare systems worldwide and highlight the importance of developing and optimizing prevention strategies, diagnostic approaches and treatment regimes. After many years of research with unsatisfying treatment options and poor clinical outcomes, last decade has seen major advances in the therapy of metastatic melanoma driven by the revolutionizing discoveries of driver mutations and immune escape mechanisms that contribute to the aggressive nature of this disease. Drugs, developed to specifically exploit these mechanisms, administered either alone or in combination, have been shown to be clinically effective treatment strategies significantly increasing survival rates of patients [2, 3, 4, 5]. Despite these recent ground-breaking advances in melanoma therapy, no currently available treatment options are curative in the majority of responding patients nor do all patients with
This chapter will briefly introduce the concept of epigenetics focusing on epigenetic alterations, especially changes in histone and DNA modifications during melanoma development and the emergence of therapy resistance. Detailed investigations into these changes will greatly contribute to our understanding of the heterogeneous and adaptive nature of melanoma. A thorough perception of how epigenetic drivers are modulating the genetic landscape will be the foundation for the development of new treatment strategies beyond pathway and immune checkpoint inhibitors.
2. Epigenetic changes
It has long been recognized that chromatin contains information beyond the primary DNA sequence. This information that is stored “on top of” the genetic information is highly dynamic and influences gene expression patterns and phenotypes without altering the nucleotide sequence while maintaining heritability to somatic daughter cells and in some cases even offspring
2.1. DNA methylation
The most well studied form of epigenetic information is stored by direct covalent chemical modification of the DNA itself. Cytosine residues in CpG dinucleotides are methylated at the fifth position generating 5-methyl cytosine (5-mC) (Figure 1) without affecting Watson-Crick base pairing and sequence information [7]. This modification is consistently found in most eukaryotic model systems [8]. Generally speaking, DNA methylation is associated with transcriptional repression [9] and established by DNA methyltransferases namely DNMT1, DNMT3A and DNMT3B. While DNMT1 is responsible for the maintenance of DNA methylation, DNMT3A and DNMT3B catalyze the
2.2. Histone modifications
Regulatory epigenetic information is also embedded in the basic structure of chromatin and the nucleosome. The nucleosome core particle comprises 147 bp of DNA that is wrapped around an octamer of histone proteins consisting of two copies of H2A, H2B, H3 and H4 (Figure 1). Histones, especially the N-terminal tails, are subject to a multitude of posttranslational modifications including acetylation, methylation, phosphorylation, sumoylation, ubiquitylation or O-GlcNAcetylation with new modifications continuously identified [13]. The genome can be classified in transcriptionally active “open” euchromatin and transcriptionally inactive “closed” heterochromatin. Histone lysine acetylation affects this “open” and “closed” states by converting the charge of the affected residue at the histone tail, which decreases the histone/DNA interactions, increases DNA accessibility and therefore facilitates transcription and replication [14]. Alternatively, histone modifications can act as binding motives for transcription factors and other histone-modifying enzymes. For example, bromodomains specifically recognize acetylated lysine residues and are an important part of many chromatin-associated proteins [14]. The second very prominently studied histone modification is methylation of lysine or arginine residues. In contrast to acetylation, methylation can be present in different forms. Lysine residues can be mono-, di- or tri-methylated, while arginine residues can be mono-methylated or symmetrically or asymmetrically di-methylated, neither of which affects the charge of the amino acid side chain (Figure 1) [15]. Instead, methylated histone residues are recognized by a plethora of protein domains including plant homeodomain (PHD) zinc fingers, chromodomains, Tudor domains or WD40 repeats [16]. While histone acetylation is generally associated with active transcription, histone methylation has more diverse functions depending on the location of the modification. For example, H3K4me3 or H3K36me3 are usually found in active gene promoters whereas H3K9me3 or H3K27me3 are linked to transcriptional repression [13]. Histone modifications are generally reversible and dysregulation of either ‘writers’ (e.g. histone acetyltransferase or histone methyltransferases) or ‘erasers’ (e.g. histone deacetylase or histone demethylases) are attributed to the pathogenesis of human diseases [17].
3. Epigenetics in melanoma initiation and development
High-throughput DNA sequencing enabled detailed investigations into the genetic makeup of cancer and revealed hundreds of genes that are frequently mutated in melanoma [18]. Among these, a set of driver mutations has been identified that allows melanocytes to proliferate excessively, to overcome senescence and to divide indefinitely, resulting in their transformation into melanoma [19]. Despite the undeniable importance of genetic events, detailed knowledge of the molecular mechanisms of tumor initiation is still absent. This is due to the fact that such events, like epigenetic changes, are challenging to observe because models that represent individual stages of melanomagenesis are required. Nevertheless, the importance of epigenetic dysregulation in melanoma development becomes increasingly apparent, which is emphasized by the high frequency of mutations found in epigenetic regulators [20].
3.1. DNA methylation in melanoma development
One model used to investigate epigenetic alterations during melanoma development utilizes sequential cycles of anchorage blockade to transform mouse melanocytes resulting in cell lines that show different degrees of aggressiveness and
While it appears that global DNA methylation levels are decreased during melanocyte transformation, many gene-specific CpG islands are hypermethylated. Comparing 24 primary cutaneous melanomas and 5 benign nevi using the Infinium BeadChip technology covering 27,578 CpG loci in the promoter regions of 14,495 genes identified 106 hypermethylated and 44 hypomethylated CpG islands. Among the 106 hypermethylated genes,
Differences in DNA methylation between melanocytes and melanoma can also be attributed to mutant BRAF, the most frequently mutated gene in melanoma [32]. Knockdown of BRAF in BRAF-mutant melanoma cell lines resulted in profound alterations of the methylation landscape with changes in gene expression affecting proliferation and invasion. Furthermore knockdown of BRAF significantly decreased DNMT1 and EZH2 expression suggesting that BRAFV600E-mediated pathway activation has a profound influence on the epigenetic landscape [33]. Analyzing BRAFV600E and BRAFWT samples from The Cancer Genome Atlas (TCGA) revealed that BRAFV600E correlates with global DNA hypomethylation. Primary melanoma samples showed a significantly decreased expression of
3.2. Histone modifications during melanoma development
Remarkable insights into the importance of histone modifications for melanoma development have been revealed using a zebrafish model in which the human
Several histone-modifying enzymes have been shown to function aberrantly and contribute to melanoma progression. The H3K9me3-specific histone methyltransferase SET domain bifurcated 1 (SETDB1) is recurrently amplified within a region of chromosome 1 and shows a high expression in melanoma compared to nevi or normal skin [39]. Using the same zebrafish model as described above (
Another deregulated histone-modifying enzyme during melanoma development is the H3K27me3-specific histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). EZH2 and H3K27me3 have been found to be elevated in aggressive melanoma cell lines and metastatic tumor samples. The expression of tumor suppressors RUNX3 and E-cadherin was found to be suppressed by EZH2 dependent H3K27me3 [40]. Accordingly, EZH2 is a major factor for melanoma initiation and progression. Knockout of EZH2 in a genetically engineered
A more specific example how histone modifications promote melanoma progression is found in the case of the telomerase reverse transcriptase (TERT). Recently, activating
A systematic overview of the epigenomic landscape of two phenotypically distinct melanocyte cell models that are characterized by low or high tumorigenicity showed distinct chromatin states associated with melanomagenesis. Specifically, chromatin state transitions characterized by loss of histone acetylation marks like H3K27Ac, H2BK5Ac and H4K5Ac and di-/trimethylation of H3K4 in regulatory domains associated with signaling pathways important for melanoma including phosphatidylinositol 3-kinase (PI3K), interferon (IFN) γ-, LKB1-, TRAIL- and platelet-derived growth factor (PDGF)-mediated signaling was observed, again emphasizing the link between epigenetic changes and melanoma development and progression [52].
Especially, loss of H3K4 methylation seems to be a key factor for melanoma growth and the highly problematic intratumor heterogeneity frequently observed in melanoma [53]. The histone 3 K4 demethylases jumonji/ARID1 (JARID1/KDM5B/PLU-1/RBP2-H1) defines a subpopulation of slow cycling melanoma cells, which is important for continuous growth of melanoma tumors. Interestingly, this subpopulation was found to be highly dynamic, as isolated KDM5B-positive and negative melanoma cells give rise to a heterogenous population consisting of both subpopulations [54] which highlights the variable nature of the epigenetic landscape in melanoma.
3.3. Epigenetic modifications as biomarkers and prognostic factors in melanoma
Because of the profound differences in DNA methylation patterns between melanocytic nevi and melanoma, several studies have investigated the suitability of DNA methylation as a predictive biomarker in melanoma. Unsupervised hierarchical clustering of 27 common benign nevi and 22 primary invasive melanomas resulted in separation of the two sample cohorts. Specifically, 22 genes were identified that significantly distinguished melanomas from nevi whereas 14 of these genes were validated in a separate set of 25 melanomas and 29 nevi [29] suggesting that analysis of differential DNA methylation patterns could be used as melanoma biomarkers. Later on Gao, et al. investigated the methylation differences of common nevi, dysplastic nevi, primary melanomas and metastatic melanomas and established a diagnostic algorithm based on promoter methylation patterns of
Besides changes in DNA methylation (5-mC), genome wide loss of the DNA demethylation intermediate 5-hydroxymethylcytosine (5-hmC) has recently been found to be a hallmark of melanoma [61]. Specifically, it has been shown that 5-hmC levels are progressively lost in melanoma compared to benign nevi, which was accompanied by decreased expression of TET family members and IDH2. Re-establishing 5-hmC by overexpression of TET2 reduced tumor growth and invasion suggesting an important function for 5-hmC in melanoma pathology. Accordingly, high levels of 5-hmC were found to negatively correlate with Breslow depth and predict better survival [61]. These findings were confirmed later on and suggest that 5-hmC analysis by immunohistochemistry could be a promising candidate as a prognostic biomarker in melanoma [62].
Presumed correlations between histone modifications and melanoma progression with prognosis have not been investigated compared to DNA methylation. This is in part because of technical challenges eminent by direct assessment of histone modifications [63]. Martinez, et al. performed immunohistochemical analyses of 10 benign nevi, 25 primary cutaneous melanomas without metastases, 19 primary cutaneous melanomas with metastases and 33 metastatic melanomas using an antibody specifically detecting H3K79 trimethylation and H3T80 phosphorylation (H3K79me3T80ph). They found a significant increase of H3K79me3T80ph in melanoma compared to nevi seemingly identifying a subset of primary melanomas with metastatic potential [64]. Another strategy to utilize histone modifications as biomarkers and prognostic factors that avoids the technical difficulties of direct assessment of histone modifications is to investigate the expression levels of histone-modifying enzymes. Along this line, it has been reported that the expression of the H3K27-specific histone methyltransferase EZH2 is increased during melanoma progression. However, only metastatic melanomas showed a significant increase compared to nevi [65]. Accordingly, analyses of EZH2 expression of TCGA melanoma samples showed a significantly shorter survival of patients with high EZH2 expression. Additionally, EZH2 high patients developed distant metastases faster, suggesting a role for EZH2 in metastasis formation [41]. In contrast to EZH2, KDM5B has been found to be significantly downregulated during melanoma development. About 70% of the investigated nevi samples showed a KDM5B expression compared to 10 and 30% in primary and metastatic melanoma samples, respectively [66].
To our knowledge and despite the wealth of epigenetic changes that differentiate melanocytes and melanoma, no epigenetic biomarkers are used in the clinic to date.
4. Impact of epigenetic modifications on melanoma therapy
4.1. Acquired drug resistance, an obvious problem in melanoma therapy
Despite tremendous advances in developing innovative cancer therapies within the last few years, mechanisms for treatment failure are still not fully understood. Targeted inhibition of oncogenic
Even though immunotherapies like IL-2, adoptive T-cell transfer or antibodies that block CTLA-4 or PD-1 have shown long-term responses in some patients [77, 78, 79, 80], many patients eventually relapse as melanoma cells escape immune surveillance. Genetic mechanisms like loss or mutation of specific antigens or parts of the major histocompatibility complexes that are involved in antigen presentation, have been attributed to immune evasion [81]. More recently, loss of function mutations in interferon-receptor signaling and in antigen presentation have been linked to resistance to PD-1 inhibition in three of four investigated patients [82]. Beside these genetic alterations that cause immunotherapy resistance, the expression of several melanoma antigens is linked to the dynamically regulated expression of NGFR [83] or can be reversibly lost in response to inflammation [84]. Another study found a correlation between a mesenchymal transcription signature, including WNT5A and ROR2, with resistance to anti-PD-1 therapy in metastatic melanoma [85] suggesting the involvement of epithelial-mesenchymal transition in immunotherapy failure.
In the following paragraphs, the current knowledge about epigenetic mechanisms contributing to drug resistance in melanoma is summarized.
4.2. Epigenetic alterations and targeted therapy
One of the most clinically relevant observations that point towards non-genetically regulated drug resistance is the concept of drug holidays, which describes the phenomenon of intermittent treatment schedules or treatment interruption. This delays the emergence of resistance. One of the first reports describing the benefit of treatment interruption was a case study of a patient diagnosed with an adenocarcinoma of the lungs. After initial chemotherapy, the patient enrolled in a phase I study of the orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib. After 18 month of drug response, the disease eventually progressed and was treated with a different combination of chemotherapy. One year after discontinuation of the initial treatment, gefitinib re-treatment resulted in a significant response [86]. Similar observations were further reported for patients treated with BRAF or BRAF/MEK inhibitors in which re-treatment with BRAF inhibitors resulted in a significant response after disease progression during an earlier treatment with BRAF or BRAF/MEK inhibitors [87]. A multi-institutional retrospective study later on found that 43% of patients that received re-treatment with BRAF inhibitors after disease progression and treatment interruption showed a clinically significant response [88]. Studies using vemurafenib-naive, primary human-patient-derived melanoma xenograft mouse models showed that vemurafenib resistance could be delayed by intermittent dosing schedules compared to continuous treatment [89].
The reversibility of drug resistance observed in clinical settings matches well with findings of slow cycling subpopulations that have been found to allow for reversible drug tolerance
Multiple studies proposed strategies to target the slow cycling drug-tolerant phenotype. Sharma, et al. showed that the KDM5Ahigh subpopulation that emerged after exposure to very high drug concentrations was susceptible to histone deacetylase (HDAC) inhibitors [74] because KDM5A is associated with histone decatylases during removal of histone modification marking active transcription [93]. HDAC inhibitors induced apoptosis in this subpopulation and combination of HDAC inhibitors with other drugs prevented the emergence of acquired resistance. Interestingly, HDAC inhibitors have to be present during the cytotoxic treatment as pre-treatment with histone deacetylase inhibitors followed by exposure to cytotoxic drugs alone was not sufficient to block acquired resistance [74]. This is important as it suggests that drug resistance is not mediated by a pre-existing subpopulation that carries intrinsic resistance mechanisms like additional mutations that can be eradicated, but by a dynamically regulated adaptive response that allows cancer cells to withstand unfavorable and toxic conditions. Roesch, et al. found that the KDM5Bhigh population enriched upon drug treatment in melanoma is dependent on oxidative phosphorylation as several members of the electron transport chain, including NADH dehydrogenase, ubiquinol cytochrome c reductase, cytochrome c oxidase and ATP synthase are highly expressed in these cells [90]. They further described that inhibition of the mitochondrial respiratory chain using oligomycin, rotenone or phenformin blocked endogenous KDM5B expression and decreased the drug-induced enrichment of KDM5Bhigh cells. Furthermore, combination of orally available NADH dehydrogenase inhibitor phenformin with BRAF inhibitor vemurafenib increased the tumor suppressive effects
The IDTC phenotype described by us is characterized by elevated expression of several histone-modifying enzymes showing no specific susceptibility to combinations of BRAF inhibitors with HDAC inhibitors, AKT inhibitors or oligomycin [91]. In accordance with previous studies, knockdown of KDM5B-sensitized melanoma cells to BRAF inhibition, but the surviving cells again displayed the IDTC phenotype. Exposure of established IDTCs to different drugs including MEK, AKT and HDAC inhibitors showed that these compounds effectively suppressed their target pathways within 3 days of treatment. However, slow cycling melanoma cells were able to adapt to this additional stressor and re-activated the respective pathways within 12 days of drug exposure. In the case of HDAC inhibitors, methylation patterns of histone 3 lysine 4 and 9, which have been shown to be co-regulated with histone acetylation via transcriptional regulation of histone methyltransferases and histone demethylases [95, 96] were re-established to resemble the H3K4me3low/H3K9me3high pattern seen in the slow cycling multidrug-tolerant cells [91]. A possible explanation for the discrepancy between the discussed studies in regards to the different strategies to target heterogenous slow cycling populations could be that the KDM5Ahigh or KDM5Bhigh cells are stringently selected subtypes of the slow cycling phenotype whereas IDTCs are characterized by multiple epigenetic modifiers, most likely including multiple subtypes that contribute to the same phenomenon. The dynamic signaling rewiring observed in the IDTC phenotype is reminiscent of the diverse drug resistance mechanisms that have been reported to emerge from slow cycling EGFR inhibitor addicted lung cancer cells [75], which suggests that an adaptive response as described for IDTCs in melanoma might be present in multiple cancer types. One key feature of all slow cycling drug-tolerant cell populations that emerge after 3–12 days of drug exposure is the reversibility upon drug withdrawal. However long-term exposure (90 days) of melanoma cells to BRAF inhibitors resulted in loss of the IDTC markers NGFR as well as KDM5B [91]. Interestingly, these cells displayed no multidrug resistance but maintained resistance to BRAF inhibitors despite drug withdrawal, suggesting the emergence of permanent resistance [91].
4.3. Epigenetic alterations and immunotherapy
Epigenetic regulation is a key mechanism for maintaining immune cell identity and differentiation. For example, CD8 positive cytotoxic T lymphocytes undergo dynamic changes of DNA methylation and histone modification patterns following infection that are important for regulation and maintenance of their differentiation states [97]. Therefore, it is important to consider that epigenetic targeting drugs will not only affect tumor cells but also influence immune cells and other cells of the tumor microenvironment. Herein, the effects of epigenetic alterations within cancer cells, specifically melanoma, and how these changes affect the therapeutic effect of immunotherapy will be discussed.
The most promising immunotherapies currently in clinical use are anti-PD-1 and PD-L1 therapies [98]. Analyses of 52 immunotherapy-naïve stage III melanomas specimens in regard to the PD-L1 expression suggested that PD-L1 negative status is associated with worse prognosis and a poor immune response gene signature. PD-L1 positive melanomas showed a significant association with the TCGA hypomethylation cluster suggesting that upregulation of immune checkpoint inhibitors is found in cancer cells with altered gene expression. Another study showed that treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine activates a viral defense pathway. Expression levels of these viral defense genes grouped different cancers including melanoma into separate categories where high expression was associated with the TCGA immune reactive (IMR) tumors with a good prognosis [99]. Melanoma patients with high levels of the viral defense signature correlated with response to anti-CTLA-4 for more than 6 month and combined treatment of 5-aza-2′-deoxycytidine and anti-CTLA4 immune checkpoint therapy in a B16-F10 mouse melanoma model enhanced tumor responses [99]. Another important factor for the successful immunotherapy response is the expression of tumor-associated antigens [100]. Along this line, it has been shown that the expression of high molecular weight-melanoma associated antigen (HMW-MAA) is regulated by DNA methylation as its expression correlates with promoter methylation. As such it is induced by treatment with 5-aza-2′-deoxycytidine [101].
Multiple studies reported the importance of histone modifications for the regulation of immunogenic factors. For example, H3K4me3 dependent PD-L1 expression has been observed in pancreatic cancer [102] or H3K27me3 and DNA methylation-mediated silencing of Th1-type chemokines CXCL9 and CXCL10 in ovarian cancer cells [103], suggesting an important role for histone modifications in the regulation of immunomodulatory factors across different cancer types. Further evidence of epigenetically regulated PD-L1 expression is provided by studies using HDAC inhibitors in melanoma cell lines. Specifically, treatment with class I HDAC inhibitors resulted in increased acetylation of histone 3 in PD-L1 and PD-L2 promoter regions, which resulted in increased PD-L1 expression
5. Conclusion
Keeping in mind the wealth of data describing epigenetic alterations during melanoma development and also in relation to the therapeutic response targeting or co-targeting these epigenetic events appears to be a very promising strategy for improving melanoma management. This is especially true in light of the highly heterogeneous and adaptive nature of melanoma which cannot be explained only by stable genetic events. While epigenetic biomarkers have not yet been put to clinical use, there is an overwhelming number of clinical trials utilizing and testing epigenetic drugs in different cancer types. These trials investigate the use of general epigenetic inhibitors targeting histone deacetylases, bromodomain and extra-terminal (BET) proteins (histone acetylation binding proteins) and more specific inhibitors targeting DNMT1, IDH1 and IDH2 (affect TET enzyme function), EZH2, DOT1L (histone H3K79 methyltransferase) or KDM1A [105].
Additionally, epigenetic drugs are tested in combination with already established chemo-, targeted- and immunotherapies. Besides synergistic effects of these drugs, this approach could also result in prevention or reversion of drug resistance, a concept that has already been shown
While these current clinical trials hold great promise, improved understanding of detailed epigenetic mechanisms, identification of new key players in epigenetic remodeling and the subsequent development of specific inhibitors, which modulate and target epigenetics have the potential to shape the future of melanoma therapy.
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