*6.2. Combinatorial Therapies in CM*

Several lines of evidence support that, in addition to their potential use as single agents, epigenetic drugs may be extremely potent in sensitizing tumor cells to other anti-cancer therapies or may help in circumventing the major hurdle of acquired drug resistance [68,203,267]. Chemotherapy, targeted therapy, and immunotherapy are the major pillars of the anticancer treatment arsenal in CM. In this section, we review the current literature by which epi-drugs may regulate the sensitivity of cancer cells to targeted therapies and immunotherapies, two of the most used therapies in CM clinical management.

#### 6.2.1. Combinations with Targeted Therapy

Several research groups have shown that the administration of epigenetic inhibitors, such as HDACs, BETi, or DNMTi in combination with BRAFi and/or MEKi can reverse

acquired resistance to targeted therapy. For instance, the use of BRAFi encorafenib in combination with HDACi panobinostat decreases PI3K oncogenic pathway activity and increases the expression levels of pro-apoptotic proteins BIM and NADPH oxidase activator (NOXA) in CM cell lines with acquired BRAFi resistance [268]. In parallel, it has been reported that BETi JQ1 treatment can sensitize vemurafenib-resistant melanoma cells to BRAFi by modulating the histone acetylation patterns of the P-gp gene in the promoter region to prevent drug efflux [267]. Additionally, combining BET and HDAC inhibitors has strong synergistic effects in the induction of apoptosis and suppression of Akt and Yap signaling in melanoma cells, even in those with acquired resistance to BRAFi [172]. In addition, the combination of vemurafenib and decitabine has shown increased efficacy and tolerability in BRAF V600E metastatic tumors in a Phase I clinical trial (NCT01876641) [212]. Although this study was prematurely terminated due to loss of funding, 3/14 patients achieved a complete response, 3/14 had a partial response, and 5/14 had stable disease. The highest response rate was reported in clinical cohorts that utilized low-dose, long-term decitabine, emphasizing that future studies should focus on long-term use of decitabine at the lowest dose of 0.1 mg/kg [212]. At the moment, a phase II clinical trial is testing the efficacy and safety of tazemetostat in combination with dual BRAF/MEK inhibition in patients with BRAF-mutated metastatic melanoma who progressed on prior BRAF/MEKi therapy (NCT04557956).

## 6.2.2. Combinations with Immunotherapy

Several other reports highlighted that combining epigenetic therapies with immunotherapeutics may result in considerable clinical benefits in cancer patients. This is possible due to the ability of epigenetic therapies to modulate the immune response in tumors [269]. The mechanisms by which these epi-therapies exert their activity are not fully elucidated, but it has been suggested that they may involve reactivation of ERVs, upregulation of tumor-surface antigens, stimulation of antigen-presenting mechanisms, activation of IFN response pathways, transcriptomic reprogramming of TME cells, or induction of immune checkpoint blockade targets such as PD-1/PD-L1 on both tumor cells and lymphocytes alongside reversal of T cell exhaustion [79]. The mechanisms underlying these actions are depicted in Figure 3.

A bourgeoning body of evidence suggests that despite their reduced anti-cancer activities, DNMTis have the potential to increase the immunogenicity and immune recognition of CM cells [270]. Pioneering studies in this field have highlighted that DNMTi hypomethylating agent decitabine may orchestrate re-expression of HLA class I antigens on melanoma cells, which enables tumor cell recognition by MAGE-specific cytotoxic T lymphocytes and their further elimination [271]. Since the studied melanoma cell line was obtained from a metastatic lesion of a nonresponding patient undergoing MAGE-3.A1 T-cell-based peptide immunotherapy, the authors postulated that hypermethylation-induced loss of HLA class I expression may be the cause of the impaired response to vaccination; however, this study highlights for the first time that DNMTis should be tested for their efficiency on reversing the acquired resistance to immunotherapy in CM [271]. Other preclinical studies have shown that DNMTi may trigger the activation of ERVs, which are normally transcriptionally silenced, leading to the activation of a type I IFN response and cytotoxic T cell recruitment into the TME [196,272]. DNMTi treatments were also linked with CTA induction in melanoma cells, with important pharmacological applications in CM management, as the homogeneous expression of a therapeutic target in neoplastic cells is a prerequisite to effectively target tumors by vaccination-induced CTA-directed immune response [273]. MAGE-A3 and NYESO-1-based vaccines are currently being tested in clinical trials for the regression of CM (Li at al. 2020). Therefore, epigenetic regulation may be harnessed to broaden the eligibility and benefits of vaccine-based therapies in CM patients, but also to tailor specific immunotherapies for each patient [273].

— — — — — — — — — — — — — — — — **Figure 3.** The proposed mechanisms by which epigenetic agents may regulate the immune response in cancers. Abbreviations: IFNs—interferons; APC—antigen-presenting cell; MHCI major histocompatibility complex I; TCR—T-cell receptor; PD-1—programmed cell death protein 1; CTLA4—cytotoxic T-lymphocyte-associated protein 4; ICB—immune checkpoint blockade; PD-L1 programmed death-ligand 1; CD80—cluster of differentiation 80; TME—tumor microenvironment; TAAs—tumor-associated antigens; CTAs—cancer testis antigens; MAGE—human melanoma antigen; NY-ESO-1—New York esophageal squamous cell carcinoma 1; TLR3—Toll-like receptor; dsRNA double-stranded RNA.

Currently, two clinical trials evaluating the safety and efficiency of DNMTi combined with immunotherapy are underway in CM. Preliminary results of a phase II study of azacitidine in combination with pembrolizumab in patients with metastatic melanoma (NCT02816021) revealed that although the combination is relatively well tolerated, it is potent only in PD-1-naïve patients (55% ORR) [274]. However, these observations remain to be further confirmed in larger clinical cohorts and correlated with the molecular characteristics of tumor biopsies. Another phase I clinical trial evaluating guadecitabine with anti-CTLA-4 showed promising tumor immunomodulatory and anti-cancer activities in CM metastatic patients; briefly, the use of DNMTi led to the induction of HLA class I molecules and IFNGsignaling pathways and increased tumor infiltration by CD8 <sup>+</sup> T cells, rendering those tumors more sensitive to immunotherapy [214].

Recent studies have provided evidence that the efficiency of immunotherapy may also be potentiated in cancers when used in combination with HDACis. HDACis are known to increase MHC class I and II expression on the cell surface or regulate PD-L1 and PD-L2 expression on cancer cells, thereby rendering the tumor vulnerable to T-cell mediated immune responses [275,276]. Furthermore, it is well documented that HDACis may be involved in TME reprogramming to deactivate immunosuppressive cells such as MDSCs and increase cytotoxic T cell activity within the TME [277]. Interestingly, in a melanoma mouse model, HDAC6 inhibitor Nexturastat A given in conjunction with anti-PD-1 antibodies significantly impaired tumor growth by causing a decrease in the antiinflammatory phenotype of macrophages and increased infiltration of CD8 <sup>+</sup> T cells and NK cells into the TME [278]. Moreover, upon exposure to the HDACi entinostat in combination with azacytidine, the syngeneic mouse melanoma models displayed improved rates of response to both anti-PD-1 and anti-CTLA-4 immunotherapies, and the tumors were eradicated in 80% of the experimental animals [279]. Functional analysis revealed that the primary targets of the epigenetic drugs were the MDSCs, which exert immunosuppressive effects in tumors [279]. Similarly, mocetinostat decreases immunosuppressive immune

cell phenotypes while augmenting anti-tumor phenotypes in both preclinical and clinical settings, providing a rationale for the use of these agents in conjunction with ICIs in CM [225]. The promising results of treatment regimens incorporating immunotherapy and HDACi have led to the recent initiation of several clinical trials in CM. Preliminary results from a phase I clinical trial evaluating the combination of pembrolizumab and HDACi entinostat demonstrated a favorable response in patients with immune checkpoint inhibitorresistant CM and acceptable safety (NCT02437136) [224]. Notably, for one patient with a confirmed partial response, transition from a PD-L1 negative phenotype (pretreatment tumor biopsy) to a PD-L1 positive state (post-treatment biopsy) was reported, as a hallmark of increased sensitivity to immunotherapy [224]. In contrast, in another phase I clinical trial, panobinostat showed limited efficiency when added to standard ipilimumab therapy in CM metastatic patients (NCT02032810) [222]. Other drug combinations such as that of anti-PD-L1 antibody nivolumab with tinostamustine, an alkylating HDACi (NCT03903458) are now under clinical investigation in refractory, locally advanced, or metastatic CM patients (Table 1).

Finally, several studies that highlight the combination of EZH2i and MAPKi or immunotherapy show an enhanced apoptosis and increased tumor control in CM cell lines and mouse models [197,198]. There is also the possibility of using EZH2i in conjunction with DNMTi and/or HDAC inhibitors as these agents have demonstrated the ability to upregulate immune response pathways in many solid cancers, including CM [57,235].

#### **7. Discussion**

Notorious for its increased unpredictable behaviors, distant metastatic patterns, increased inflammatory status, and intrinsic resistance to therapy, CM is an unsolved clinical and social issue [280]. The remarkable progress made in recent years in deciphering CM biology has resulted in the development of several targeted therapies and immune checkpoint inhibitors that have truly revolutionized the treatment of metastatic CM. For instance, therapies targeting nodes in the MAPK pathway have greatly improved OS; furthermore, immune therapies with immune-checkpoint modulators have led to more durable results and even pCR in several patients [192]. Despite these promising results, targeted and immune therapies' effectiveness is often limited by the emergence of drug resistance. In CM, tumor refractoriness has been extensively linked with mutations in genes regulating drug efflux mechanisms, apoptotic machinery, DNA damage repair, cancer stemness, as well as many other biological processes [280–283]; however, for a significant proportion of CM, the cause of the resistance appears to be non-genetic. Peculiarities such as the rapid kinetics and the transient nature of refractory phenotypes suggest the existence of an epigenetic basis for drug resistance in CM, pointing out that epigenetic regulation is a fundamental feature of tumor development and adaptation to therapy [157]. Accordingly, epigenetic alterations are currently being investigated as potential biomarkers and are being envisioned as promising therapeutic targets for CM clinical management [284].

We revised how epigenetic mechanisms such as DNA methylation and histone modifications are fine-tuning gene expression programs in CM, thus influencing almost all the biological properties of these tumors. Distinct epigenetic signatures show promise for assisting in distinguishing between benign and malignant lesions, as well as between certain disease subtypes histologically classified or evaluated into blood circulation [34,103,285]. Moreover, altered epigenetic patterns have been shown to contribute to the acquisition of drug resistant phenotypes in CM and some of these modifications seem to have important prognostic and predictive applications [189,195]. Given the critical roles of epigenetic alterations in CM development and drug resistance, but also their reversible nature, targeting or co-targeting these epigenetic events appears to be a promising strategy for improving the clinical condition of CM patients. Although epigenetic biomarkers have not yet found their place in clinical practice, an impressive number of epigenetic drugs are constantly being developed and tested for their cytotoxicity and efficacy in clinical trials in CM [203].

There are several generations of epigenetic drugs. The first two generations of epitherapies include DNMTis and HDACis, some of which have already gained FDA approval for the treatment of blood cancers [202]. Notably, these earliest generations of epigenometargeted therapies were tailored according to the "one size fits all" principle, showing poor efficiency and selectivity and increased toxicity in CM patients [39]. However, clinical responses achieved with certain third-generation epigenetic agents designed according to precision medicine paradigms have provided new hope in the treatment of solid cancers [286,287]. Therefore, epi-drug development should follow a more personalized approach, with further identification of robust predictive biomarker selection and subsequent validation of this strategy in clinical studies [39]. Current literature highlights that, besides their use as monotherapy, epigenetic drugs may synergize with other anti-cancer compounds and reverse therapy resistance in both preclinical and clinical settings [236]. The administration of certain epigenetic drugs before chemotherapy or targeted therapy can be used for priming cancer cells to be more sensitive to these approaches, as epigenetic drugs can induce chromatin decompaction, making it more accessible to antineoplastic compounds [203]. A phase I clinical study (NCT01876641) has already shown that vemurafenib is more effective in CM patients when given in combination with decitabine in low doses. In line with these observations, the preclinical assessment reported activity of the combination and an increased potential in delaying the development of acquired resistance [212]. Moreover, co-administration of two epigenetic agents (such as DNMTi and HDACi) with different mechanisms of action may allow these compounds to increase each other's efficiency and overcome the issue of CM drug resistance [279]. Another rationale for using DNMTi in conjunction with HDACi is that such a combination may be extremely potent in stimulating the immune system to fight against tumors [39]. Finally, combinations of epigenetic agents with immunotherapy are currently tested in CM in clinical trials, holding promise to enhance antitumor immune responses or to reverse acquired resistance to ICIs. Even though most clinical studies have simple designs with both agents used simultaneously and continuously, epi-drugs and immunotherapy combinations are generally well tolerated in human subjects, in contrast to regimens incorporating targeted therapies and immunotherapies [39]. However, long-term use of certain epigenetic agents may have detrimental effects on antitumor immune response; for instance, prolonged use of BETi was linked with T cell depletion [288]. Therefore, to use these epigenetic drugs in immuno-oncology, more scientific effort is required to understand how the dose and scheduling of these therapies modulate the immune response and adverse effects in the clinical setting of melanoma management.

**Author Contributions:** Conceptualization, E.-G.D. and M.N.; writing—original draft preparation, E.-G.D.; writing—review and editing, M.N., C.C. and M.C.; supervision, M.N., C.C. and M.C.; funding acquisition, M.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Core Program, implemented with the support of NASR, projects PN 19.29.01.01 grant PN-III-P1-1.2-PCCDI-2017-0341/2018. The authors also wish to acknowledge Horizon2020 funded projects COST 16120 (EPITRAN) and CA18127-International Nucleome Consortium (INC).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** The present study will be integrated into the PhD thesis of the first author and PhD student Georgiana Dobre.

**Conflicts of Interest:** The authors declare no conflict of interest.
