**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 *BRAFV600E* melanomas became the poster child of exciting initial therapeutic responses unfortunately followed by long-term resistance. Development of therapy resistance is the major obstacle for the successful use of targeted therapies, where almost all patients, who respond initially, are relapsing, irrespectively of single or combined inhibition of the MAPK pathway [67]. Furthermore, 15–20% of mutant *BRAF* tumors do not respond to targeted therapy in the clinical setting [68], suggesting the presence of pre-existing resistance mechanisms. Resistance to MAPK pathway inhibition has been shown to involve emergence of genetic mutations in *RAS* or *MEK*, amplification of mutant *BRAF* or alternative BRAF splicing [69, 70]. However, such genetic resistance mechanisms are absent in approximately 40% of patient samples, indicating the involvement of other mechanisms contributing to therapy failure [67]. Among these mechanisms, the upregulation of CRAF [71] or the SOX10-mediated activation of TGF-β that results in increased EGFR and PDGFRβ expression [72] that have been reported to mediate non-genetic resistance. Elevated EGFR and PDGFRβ levels have been shown to be reversible after discontinuing BRAF and MEK inhibitor treatment, while expression of EGFR or treatment with TGF-β resulted in a slow cycling drug-resistant phenotype [72]. This observation reflects findings by our group [73] and others [74, 75] of reversible multidrug-tolerant slow-cycling state following stressors like drug treatment. Beside failure of BRAF inhibition, a recent study found that dynamic and recurrent non-genomic alterations following chronic BRAF inhibitor treatment also affect tumor immunity possibly resulting in cross resistance to anti PD-1 therapy [76].

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 [77–80], 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**

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

To our knowledge and despite the wealth of epigenetic changes that differentiate melanocytes

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 *BRAFV600E* melanomas became the poster child of exciting initial therapeutic responses unfortunately followed by long-term resistance. Development of therapy resistance is the major obstacle for the successful use of targeted therapies, where almost all patients, who respond initially, are relapsing, irrespectively of single or combined inhibition of the MAPK pathway [67]. Furthermore, 15–20% of mutant *BRAF* tumors do not respond to targeted therapy in the clinical setting [68], suggesting the presence of pre-existing resistance mechanisms. Resistance to MAPK pathway inhibition has been shown to involve emergence of genetic mutations in *RAS* or *MEK*, amplification of mutant *BRAF* or alternative BRAF splicing [69, 70]. However, such genetic resistance mechanisms are absent in approximately 40% of patient samples, indicating the involvement of other mechanisms contributing to therapy failure [67]. Among these mechanisms, the upregulation of CRAF [71] or the SOX10-mediated activation of TGF-β that results in increased EGFR and PDGFRβ expression [72] that have been reported to mediate non-genetic resistance. Elevated EGFR and PDGFRβ levels have been shown to be reversible after discontinuing BRAF and MEK inhibitor treatment, while expression of EGFR or treatment with TGF-β resulted in a slow cycling drug-resistant phenotype [72]. This observation reflects findings by our group [73] and others [74, 75] of reversible multidrug-tolerant slow-cycling state following stressors like drug treatment. Beside failure of BRAF inhibition, a recent study found that dynamic and recurrent non-genomic alterations following chronic BRAF inhibitor treatment also affect tumor immunity possibly resulting in

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 [77–80], 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

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**

metastatic melanoma samples, respectively [66].

12 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

cross resistance to anti PD-1 therapy [76].

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 *in vitro.* One of the first reports of such a drug-tolerant subpopulation showed that a very small fraction of cancer cells including melanoma survives treatment with drug concentrations 100 fold higher than the IC50 [74]. These surviving cells were found to be mainly quiescent and in G1 arrest, they eventually continued growth in the presence of the drug. Importantly, drug withdrawal re-sensitized these drug-tolerant cells and re-established the same cellular heterogeneity as found in the initial sensitive population. Mechanistically, the surviving drug-tolerant cells exerted an altered chromatin state with increased expression levels of the histone demethylase KDM5A (JARID1A/RBP2) and concomitantly reduced levels of H3K4me2/3. RNAi-mediated knockdown of KDM5A confirmed that this histone demethylase is important for the establishment of the reversible drug-tolerant state [74]. This observation of an epigenetically regulated mainly G1 arrested state surviving exposure to high drug concentrations is similar to the previously mentioned slow cycling KDM5Bhigh subpopulation that is important for continuous melanoma growth [54]. KDM5Bhigh cells have been found to be enriched upon drug treatment and resemble a slow cycling drug-tolerant state in melanoma as shRNA-mediated knockdown of KDM5B increased sensitivity to different drugs [90]. In accordance with the dynamic nature of KDM5A and KDM5B positive subpopulations, we have observed that chronic exposure to external stressors, rather than specific drug treatment, initiates an innate cellular response whereupon cells adopt a slow cycling, multidrug-tolerant phenotype [91]. Continuous exposure of melanoma cells to sub lethal BRAF inhibitor concentrations for 12 days initiated a cellular transformation and not the selection of a pre-existing subpopulation, which resulted in a slow cycling, mainly G1 arrested phenotype. These so called induced drug-tolerant cells (IDTCs) were unresponsive to 20-fold higher BRAF inhibitor concentrations as well as multiple other drugs including the MEK inhibitor GSK1120212 or cisplatin. As demonstrated for the KDM5Ahigh subpopulation [74], IDTCs re-gained drug sensitivity upon 7 days of drug withdrawal. On the molecular level IDTCs displayed elevated expression of drug efflux genes including *ABCB5*, *ABCA5*, *ABCB8* and *ABCB4*, as well as melanoma stem cell markers *NGFR*, *SOX10*, *CD44*, *SOX2* and *SOX4,* suggesting the transition into an undifferentiated state [91]. These molecular changes were accompanied by a profound decrease of histone marks H3K4me3, H3K27me3 that were decreased and H3K9me3, which was increased. Accordingly, expression of several histone-modifying enzymes including the H3K27-specific demethylases, KDM6A, KDM6B and the H3K4-specific demethylases, KDM1B, KDM5A and KDM5B was increased at the IDTC state [91]. Interestingly, a similar transition into an H3K4me3low/H3K27me3low/H3K9me3high state was triggered by hypoxia and nutrient starvation and IDTCs generated by these stressors exhibited tolerance to BRAF inhibitors or cisplatin treatment, suggesting an epigenetically regulated drug-independent generic stress response that allows cells to cope with difficult environmental conditions [91]. Similar to our proposed IDTCs, a slow cycling, reversible NGFRhigh state that displays features of de-differentiation has also been described, which has been shown to be susceptible to inhibition of epigenetic modifiers as bromodomain inhibitors, that block recognition of acetylated histones, suppressed the slowly cycling NGFRhigh state [92].

rotenone or phenformin blocked endogenous KDM5B expression and decreased the druginduced enrichment of KDM5Bhigh cells. Furthermore, combination of orally available NADH dehydrogenase inhibitor phenformin with BRAF inhibitor vemurafenib increased the tumor suppressive effects *in vivo* [90]. In the same year, Yuan, et al. showed AMPK-dependent synergistic cytotoxicity of combining BRAF inhibitors and phenformin which also suppressed the

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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].

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 thera-

**4.3. Epigenetic alterations and immunotherapy**

peutic effect of immunotherapy will be discussed.

emergence of a drug-resistant phenotype [94].

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 druginduced enrichment of KDM5Bhigh cells. Furthermore, combination of orally available NADH dehydrogenase inhibitor phenformin with BRAF inhibitor vemurafenib increased the tumor suppressive effects *in vivo* [90]. In the same year, Yuan, et al. showed AMPK-dependent synergistic cytotoxicity of combining BRAF inhibitors and phenformin which also suppressed the emergence of a drug-resistant phenotype [94].

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**

as shRNA-mediated knockdown of KDM5B increased sensitivity to different drugs [90]. In accordance with the dynamic nature of KDM5A and KDM5B positive subpopulations, we have observed that chronic exposure to external stressors, rather than specific drug treatment, initiates an innate cellular response whereupon cells adopt a slow cycling, multidrug-tolerant phenotype [91]. Continuous exposure of melanoma cells to sub lethal BRAF inhibitor concentrations for 12 days initiated a cellular transformation and not the selection of a pre-existing subpopulation, which resulted in a slow cycling, mainly G1 arrested phenotype. These so called induced drug-tolerant cells (IDTCs) were unresponsive to 20-fold higher BRAF inhibitor concentrations as well as multiple other drugs including the MEK inhibitor GSK1120212 or cisplatin. As demonstrated for the KDM5Ahigh subpopulation [74], IDTCs re-gained drug sensitivity upon 7 days of drug withdrawal. On the molecular level IDTCs displayed elevated expression of drug efflux genes including *ABCB5*, *ABCA5*, *ABCB8* and *ABCB4*, as well as melanoma stem cell markers *NGFR*, *SOX10*, *CD44*, *SOX2* and *SOX4,* suggesting the transition into an undifferentiated state [91]. These molecular changes were accompanied by a profound decrease of histone marks H3K4me3, H3K27me3 that were decreased and H3K9me3, which was increased. Accordingly, expression of several histone-modifying enzymes including the H3K27-specific demethylases, KDM6A, KDM6B and the H3K4-specific demethylases, KDM1B, KDM5A and KDM5B was increased at the IDTC state [91]. Interestingly, a similar transition into an H3K4me3low/H3K27me3low/H3K9me3high state was triggered by hypoxia and nutrient starvation and IDTCs generated by these stressors exhibited tolerance to BRAF inhibitors or cisplatin treatment, suggesting an epigenetically regulated drug-independent generic stress response that allows cells to cope with difficult environmental conditions [91]. Similar to our proposed IDTCs, a slow cycling, reversible NGFRhigh state that displays features of de-differentiation has also been described, which has been shown to be susceptible to inhibition of epigenetic modifiers as bromodomain inhibitors, that block recognition of acetylated

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,

histones, suppressed the slowly cycling NGFRhigh state [92].

14 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

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].

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 *in vitro* more than 15 years ago [106]. In melanoma, one clinical trial is currently investigating the combination of the BRAF/MEK inhibitors vemurafenib and cobimetinib with the DNA hypomethylating agent decitabine (NCT01876641). However, the main focus in the field appears to be the combination of epigenetic drugs, especially DNA methyltransferase and histone deacetylase inhibitors with immunotherapy, which is currently tested in numerous clinical trials [107] and the outcome of these promising approaches is highly anticipated.

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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

Dermatology Research Centre, The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Brisbane, Australia

[1] Whiteman DC, Green AC, Olsen CM. The growing burden of invasive melanoma: Projections of incidence rates and numbers of new cases in six susceptible populations

[2] Sosman JA, et al. Survival in BRAF V600-mutant advanced melanoma treated with

[3] Long GV, et al. Overall survival and durable responses in patients with BRAF V600 mutant metastatic melanoma receiving Dabrafenib combined with Trametinib. Journal

[4] Hodi FS, et al. Improved survival with ipilimumab in patients with metastatic mela-

[5] Larkin J, et al. Combined nivolumab and lpilimumab or monotherapy in untreated mel-

[6] Allis CD, Jenuwein T. The molecular hallmarks of epigenetic control. Nature Reviews.

through 2031. The Journal of Investigative Dermatology. 2016;**136**(6):1161-1171

vemurafenib. The New England Journal of Medicine. 2012;**366**(8):707-714

noma. The New England Journal of Medicine. 2010;**363**(8):711-723

anoma. The New England Journal of Medicine. 2015;**373**(1):23-34

the potential to shape the future of melanoma therapy.

\*Address all correspondence to: h.schaider@uq.edu.au

of Clinical Oncology. 2016;**34**(8):871-878

Genetics. 2016;**17**(8):487-500

Heinz Hammerlindl and Helmut Schaider\*

**Author details**

**References**

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 *in vitro* and *in vivo* [104].

#### **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 *in vitro* more than 15 years ago [106]. In melanoma, one clinical trial is currently investigating the combination of the BRAF/MEK inhibitors vemurafenib and cobimetinib with the DNA hypomethylating agent decitabine (NCT01876641). However, the main focus in the field appears to be the combination of epigenetic drugs, especially DNA methyltransferase and histone deacetylase inhibitors with immunotherapy, which is currently tested in numerous clinical trials [107] and the outcome of these promising approaches is highly anticipated.

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.
