Epigenetic drugs as immunomodulators for combination therapies in solid tumors.

JPT-06647; No of Pages 12 Pharmacology & Therapeutics xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

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Associate editor: B. Teicher

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Luca Sigalotti a, Elisabetta Fratta a, Sandra Coral b, Michele Maio b,⁎ a b

Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico Aviano, National Cancer Institute, Aviano, Italy Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Istituto Toscano Tumori, Siena, Italy

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Continuously improving knowledge of the fine mechanisms regulating cross-talk between immune cells, and of their multi-faceted interactions with cancer cells, has prompted the development of several novel immunotherapeutic strategies for cancer treatment. Among these, modulation of the host's immune system by targeting immunological synapses has shown notable clinical efficacy in different tumor types. Despite this, objective clinical responses and, more importantly, long-term survival are achieved only by a fraction of patients; therefore, identification of the mechanism(s) responsible for the differential effectiveness of immune checkpoint blockade in specific patient populations is an area of intense investigation. Neoplastic cells can activate multiple mechanisms to escape from immune control; among these, epigenetic reprogramming is emerging as a key player. Selected tumor-associated antigens, Human Leukocyte Antigens, and accessory/co-stimulatory molecules required for efficient recognition of neoplastic cells by the immune system have been shown to be epigenetically silenced or down-regulated in cancer. Consistent with the inherent reversibility of epigenetic silencing, “epigenetic” drugs, such as inhibitors of DNA methyltransferases and of histone deacetylases, can restore the functional expression of these down-regulated molecules, thus improving the recognition of cancer cells by both the innate and adaptive immune responses. This review focuses on the immunomodulatory activity of epigenetic drugs and on their proposed clinical use in novel combined chemo-immunotherapeutic regimens for the treatment of solid tumors. © 2013 Published by Elsevier Inc.

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Keywords: DNA methylation Histone post-translational modifications Immunostimulatory antibodies Immunotherapy

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Epigenetic drugs as immunomodulators for combination therapies in solid tumors

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . 2. Immune response to cancer cells . . . . . . . . . . . . . . 3. Epigenetics . . . . . . . . . . . . . . . . . . . . . . . 4. Epigenetic drugs . . . . . . . . . . . . . . . . . . . . . 5. Epigenetic modulation of tumor associated antigens . . . . . 6. Epigenetic modulation of antigen processing and presentation 7. Additional immunotherapeutic advantages of epigenetic editing 8. Epigenetic immunotherapy . . . . . . . . . . . . . . . . 9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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50 Abbreviations: 5-AZA-CdR, 5-aza-2′-deoxycytidine; APC, antigen presenting cell; APM, Antigen Processing Machinery; CIITA, class II transactivator; CpG, cytosine–guanine dinucleotide; CTA, Cancer Testis Antigen; CTL, Cytotoxic T lymphocyte; DNMT, DNA methyltransferase; DNMTi, DNMT inhibitor; EGCG, (−)-epigallocatechin-3-gallate; ER, endoplasmic reticulum; ESCC, esophageal squamous cell carcinoma; FASL, FAS ligand; GC, gastric cancer; HAT, histone acetyltransferase; HATi, HAT inhibitor; HDAC, histone deacetylase; HDACi, HDAC inhibitor; HDM, histone demethylase; HLA, Human Leukocyte Antigen; HMT, histone methyltransferase; HMW-MAA, high molecular weight melanoma-associated antigen; IFN, interferon; MBD, methyl-CpG-binding proteins; NK, Natural Killer; SB, sodium butyrate; TAA, tumor-associated antigen; TAP, transporter associated with antigen processing; TCT, T cell receptor; Th, T helper; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor; TSA, trichostatin A; TSG, tumor suppressor genes; VPA, valproic acid. ⁎ Corresponding author at: Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Strada delle Scotte 14, 53100 Siena, Italy. Tel.: +39 0577 586336; fax: +39 0577 586303. E-mail address: [email protected] (M. Maio). 0163-7258/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.pharmthera.2013.12.015

Please cite this article as: Sigalotti, L., et al., Epigenetic drugs as immunomodulators for combination therapies in solid tumors, Pharmacol. Ther. (2013), http://dx.doi.org/10.1016/j.pharmthera.2013.12.015

L. Sigalotti et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

1. Introduction

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Neoplastic transformation is sustained by a series of genetic, epigenetic and regulatory alterations that frequently also result in de novo expression or over-expression of tumor-associated antigens (TAA), eventually recognized as non-self by the host's immune system (Buonaguro et al., 2011). This notion has prompted major research efforts aimed at understanding the subtle mechanisms regulating the activity of the immune system and its fine interaction with cancer cells; as a consequence, different immunotherapeutic approaches are finally proving effective in treating cancer. Among these “host immune-modulation” strategies, the anti-CTLA-4 antagonistic immunostimulatory monoclonal antibody (ipilimumab) and the autologous cellular vaccine Sipuleucel-T have recently been shown to improve significantly the survival of melanoma and prostate cancer patients, respectively, and have been approved by the FDA and the EMA (Sharma et al., 2011). These achievements clearly

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identify immunotherapy as a valid therapeutic strategy for cancer patients; however, its overall effectiveness is still far from optimal since only a minority of treated patients achieves long-term clinical benefit (Ascierto et al., 2011; Di Giacomo et al., 2013; Wolchok et al., 2013). Therefore, in spite of substantial progress, it is essential to identify the mechanism(s) underlying the failure of a large proportion of cancer patients to benefit from modulation of the host immune system. An important limiting aspect is the plethora of immune escape strategies used by neoplastic cells to survive their interaction with the host's immune system. Several strategies modulating the host immune system have proven effective in counteracting the ability of cancer cells to impair priming and/ or activation of effector T cells, as well as the induction of tolerance or anergy. However, several immune-escape mechanisms that suppress or down-regulate key molecules required for the efficient recognition and destruction of cancer cells by immune effectors (see below and Fig. 1) are intrinsic to transformed cells and, therefore, can be targeted only by

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proteasome intracellular protein Fig. 1. CTL recognition of cancer cells. CTLs recognize cancer cells by the engagement of antigen-specific TCRs on CTLs, with the tumor antigen presented on the surface of target cells as peptide associated with HLA class I molecules. Cytosolic tumor antigens are degraded to peptides via the ubiquitin/proteasome pathway, and are then transported to the ER lumen by the TAP1/2 heterodimer, where they are further trimmed to their final length by the ERAP1 and 2 aminopeptidases. In the ER, the HLA class I heavy chain associates with β2-microglobulin (b2), giving rise to a partly folded HLA class I molecule. This is then loaded with peptide, assisted by the chaperone molecules ERp57, tapasin and calreticulin. The trimeric HLA class I heavy chain/β2-microglobulin/peptide is finally delivered to the cell surface via the trans-Golgi apparatus. In addition to engagement of HLA class I/peptide by the TCR, CTL activation and cytotoxicity require additional stimulatory signals provided by interactions between co-stimulatory/accessory molecules (CD80, CD86, LFA3) on the antigen presenting cells and activating receptors on the CTL (e.g., CD2, CD28, LFA1).


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Immune elimination of cancer cells can be achieved both by the innate and the adaptive branch of the immune system. The innate immune response is immediately effective in the host, does not require antigen recognition, and relies mainly on Natural Killer (NK) cells. The latter use surface receptors [e.g., Killer Inhibitory receptors; NK group 2, member D (NKG2D)] to identify aberrant cells that expose “abnormal” signals, including absent expression of HLA class I molecules or exposure of stress-induced ligands [e.g., MHC class I-related chain A and B (MICA, MICB), UL16-binding proteins (ULBPs)]. Antibody dependent cell cytotoxicity is another way of recognizing cells to be cleared by NK cells, and relies on engagement of the NK receptor CD16 with antibodies covering the target cells. Killing of targets by NK cells is achieved through engagement of death receptors [e.g. FAS, TNF-related apoptosis-inducing ligand receptors (TRAIL-R)] on target cells by NKexpressed ligands [e.g., FAS ligand (FASL), TRAIL], and through the release of cytotoxic granules including perforin and granzymes, all of which ultimately trigger apoptosis in the target cell (Waldhauer & Steinle, 2008). Adaptive immune responses, on the other hand, rely on the specific recognition of “non-self” or “aberrant” antigens on target cells, involving a series of activations, intercellular interactions, and controlled maturation steps that renders them not immediately effective in the host. However, adaptive immune responses are extremely specific and result in an immunological memory that ensures rapid activation of an effective immune response on successive antigen encounter. Specific elimination of neoplastic cells by the adaptive immune response is mainly mediated by CD8+ cytotoxic T lymphocytes (CTLs) that kill target cells by mechanisms similar to those employed by NK cells. The first, mandatory requirement for target recognition and activation of antigen-specific CTLs is engagement of the T cell receptor (TCR) expressed by CTLs with the antigen presented on the surface of target cells as a peptide bound to HLA class I molecules. Generation of the peptide/HLA class I complex is a multistep process called antigen processing, which is carried out by different molecules collectively composing the Antigen Processing Machinery (APM, Fig. 1). The major route of antigen processing for presentation to CTLs starts with degradation of cytosolic proteins via the ubiquitin/proteasome pathway, generating peptides with HLA class I compatible C-termini, but extended N-termini. The repertoire of peptides generated by the proteasome may be modulated by IFN-γ, which triggers the exchange of constitutive proteasome subunits with the immunoproteasome subunits LMP2, LMP7 and MECL1. The peptides are then translocated into the Endoplasmic Reticulum (ER) lumen by a heterodimeric complex comprising transporter associated with antigen processing (TAP) 1 and 2, where they are further trimmed to their final length by the ER-associated aminopeptidases, ERAP1 and 2. In the ER, the HLA class I heavy chain is stabilized by binding with calnexin, until it associates with β2-microglobulin giving rise to a partly folded HLA class I molecule. The latter is next loaded with peptide, and then folded

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In 1942 the biologist Conrad Waddington defined epigenetics as “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (Waddington, 2012). A newer definition arose with the growth of molecular biology and, more recently, the term has been used to describe heritable and potentially reversible changes in gene expression that occur without altering DNA sequence (Sigalotti et al., 2010). Epigenetic changes play a critical role in the regulation of all DNA-based process and, as such, are early events in tumor development. The most widely studied epigenetic modifications in cancer are DNA methylation and histone modifications which act in a synergistic and cooperative manner to alter gene expression. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) and involves transfer of methyl groups to the C5 position of the pyrimidine ring of cytosine, predominantly within cytosine–guanine dinucleotides (CpG). There are three enzymatically active mammalian DNMTs: DNMT1 that is involved in maintaining DNA methylation patterns (maintenance DNMT), and DNMT3A and 3B that are implicated in generating new methylation patterns (de novo DNMT). The overall effect of DNA methylation is silencing in cis of the affected locus either by directly interfering with binding of specific transcription factors to their promoter recognition sites or by recruiting methyl-CpG-binding proteins (MBDs), which prevent gene expression by recruiting chromatin remodeling co-repressor complexes (Sigalotti et al., 2010). DNA methylation is linked to the maintenance of chromatin in a silent state; it is involved in establishing genomic imprinting, in maintaining X-chromosome inactivation, and in silencing tissue-specific genes in those cell types in which they should not be expressed. DNA methylation also protects genome integrity by silencing mobile genetic elements such as transposons. Finally, methylation of CpG dinucleotides within non-coding regions helps to maintain proper structure and integrity of the chromosomes (Rodriguez-Paredes & Esteller, 2011). Disruption of DNA methylation homeostasis is a hallmark of cancer and is closely associated with loss of both global and gene-specific DNA methylation, as well as with hypermethylation of tumor suppressor gene (TSG) promoters. Global hypomethylation is the first aberrant epigenetic alteration observed in cancer cells and represents one of the most common molecular alterations in human cancers (Ehrlich, 2009; Sigalotti et al., 2011). Some studies have demonstrated that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences (Ehrlich, 2002; Schulz et al., 2002). Reactivation of transposable LINE-1, SINE, Alu, and IAP retroviral elements through hypomethylation may lead to genomic instability, thus increasing the frequency of mutations, deletions, amplifications, inversions and translocations. On the other hand, demethylation of repetitive sequences located at centromeric and subtelomeric chromosomal regions is associated with enhanced

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through a series of coordinate transient interactions with the chaperone molecules ERp57, tapasin and calreticulin. The trimeric HLA class I heavy chain/β2-microglobulin/peptide is finally delivered to the cell surface through the trans-Golgi apparatus. Importantly, binding of the peptide is required to complete the folding of HLA class I molecules and is necessary for their export from the ER. As a consequence, a fully functional APM is mandatory for exposure of HLA class I molecules on the cell surface, and any defect in APM causes reduced or absent expression of HLA class I antigens on the cell membrane, preventing the recognition of cells by the adaptive cellular immune response (Bukur et al., 2012). Besides the first signal provided by the engagement of HLA class I/peptide complex with antigen-specific TCR, additional stimulatory signals are required for the CTL to be activated and deliver its cytotoxic activity (Fig. 1). These are mainly mediated by interactions between co-stimulatory or accessory molecules (CD80, CD86, LFA3) on the antigen presenting cells (APC) and activating receptors on the CTL (e.g., CD2, CD28, LFA1).

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cancer (rather than host) immunomodulatory approaches. Epigenetic alterations in cellular homeostasis during cancer onset and progression appear to play a prominent role, since they impair immune recognition and clearance of cancer cells, posing a major threat to the successful implementation of anticancer immunotherapies (Sigalotti et al., 2005). The need for functional epigenetic enzymes to maintain epigenetic alterations makes these changes reversible by pharmacologic inhibitors (Sigalotti et al., 2007). The proven efficacy of epigenetic drugs in restoring gene expression in multiple settings makes them attractive tools to counteract the challenges posed by epigenetic alterations to the immune recognition of tumor cells. In this review, we focus on current understanding of the epigenetic immunoediting of cancer cells and on the clinical potential of using tumor immunomodulation by epigenetic drugs in novel combinations to improve the efficacy of current and future immunotherapeutic strategies in solid tumors.

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As seen above, epigenetic alterations are a hallmark of cancer cells, contributing to the neoplastic phenotype by different mechanisms, of which silencing of key genes by DNA hypermethylation and histone hypoacetylation are the best-documented. Maintenance of such gene silencing requires continued activity of DNMTs and HDACs, and inhibition of these enzymes can be exploited to counteract the epigenetic alterations put in place by the tumor. Accordingly, different epigenetic drugs have been developed to inhibit DNMTs or HDACs, many of which are in clinical trials and some of which have been approved by the FDA for the treatment of cancer (Nebbioso et al., 2012). Different inhibitors of DNMTs (DNMTis) proved effective in reversing aberrant hypermethylation of a multitude of genes, restoring their expression and functional activity. Based on their molecular classification, DNMTis can be divided in two major classes: nucleoside and non-nucleoside analogs. Nucleoside analogs of cytidine were the first DNMTis to be described, are still the most widely used, and also appear to be the most effective DNMTis (Stresemann et al., 2006). They include 5-azacytidine (Vidaza), 5-aza-2′-deoxycytidine (5-AZA-CdR, Decitabine, Dacogen), 5-fluoro-2′deoxycytidine, 5,6-Dihydro-5-azacytidine, and zebularine; 5-azacytidine and 5-AZA-CdR have undergone intensive clinical development leading to their FDA approval for patients affected by MDS (Garcia et al., 2010). Second generation nucleoside analogs are also being actively developed to address both stability and tolerability issues. Of these, the 5-AZACdR-containing dinucleotide SGI-110 has demonstrated effective DNA demethylating activity and improved stability and tolerability in vivo (Coral et al., 2013). After cell uptake, nucleoside analogs are phosphorylated by cellular kinases to generate nucleotides, which are then incorporated into genomic DNA during S phase of the cell cycle. Once incorporated, these cytidine analogs behave as suicide substrates for the DNMTs, which, in the attempt to methylate them, become irreversibly inactivated by the formation of a covalent link between the enzyme active site and the modified pyrimidine ring. Since newly synthesized DNA strands cannot be methylated, the resulting cellular depletion of functional DNMTs leads to a passive demethylation of the genome following DNA replication (Sigalotti et al., 2007). Non-nucleoside DNMTis can be further classified as “small molecules”, “natural molecules”, or oligonucleotides, all of which deliver their hypomethylating activity without being incorporated into the DNA. The “small molecules” hydralazine and procainamide are FDAapproved drugs for cardiovascular diseases (Peng et al., 2010), for which the clinical relevance of DNMTi activity has only recently been recognized. Although the mechanism of action has not been completely elucidated, it appears that these DNMTis bind to CpG-rich sequences and act as partial competitive inhibitors, interfering with the binding of DNMTs to their substrates, reducing DNMT enzymatic activity and favoring their dissociation from the substrate DNA (Amatori et al., 2010). More specific targeting of DNMTs has recently been achieved using three-dimensional modeling of the catalytic site of DNMT1, enabling the design of the RG108 small molecule DNMTi which blocks the enzyme's activity without forming covalent bonds (Brueckner et al., 2005). The demonstrated DNA hypomethylating activity of RG108, both in vitro and in vivo, is encouraging further development of specific DNMTis which may circumvent the cytotoxic, nonspecific effects of nucleoside analogs that arise from covalent trapping of DNMTs in the genomic DNA (Juttermann et al., 1994). Among the natural molecules, the main polyphenol compound of green tea, (−)-epigallocatechin-3-gallate

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with both methylated DNA and a co-repressor complex containing HDACs to mediate chromatin condensation and transcriptional repression (Hashimoto et al., 2010). In progenitor cells, disruption of any of these distinct and mutually reinforcing epigenetic mechanisms may be determinant not only for cancer risk but also for tumor progression (Feinberg et al., 2006).

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transcription and subsequent accumulation of small minor satellite transcripts that may induce chromosomal abnormalities (Howard et al., 2008; Pogribny, 2010). In addition to global genomic DNA hypomethylation, normally silenced genes involved in growth-promoting pathways have been found to be hypomethylated in cancer cells, resulting in aberrant expression of the corresponding protein (Hanada et al., 1993; Watt et al., 2000; Oshimo et al., 2003; Scherf et al., 2013). DNA promoter hypomethylation can also induce de novo expression of different genes whose function is not yet completely understood, but may act as tumor rejection antigens [e.g., Cancer Testis Antigens (CTAs)] (Fratta et al., 2011). Aberrant DNA hypermethylation is a frequent event in cancer and acts as an alternate and complementary mechanism to gene mutation or deletion, leading to inactivation of specific TSGs. During the last few years, the list of genes inactivated by hypermethylation has grown considerably to include essentially all the pathways important for tumor biology such as cell cycle control, apoptosis, cell signaling, adhesion, motility, tumor cell invasion, metastasis, angiogenesis, and immune recognition (Sigalotti et al., 2007). Histone modifications include a multitude of covalent variations affecting the histone amino-terminal (N-terminal) tails protruding from the nucleosomes. These post-translational modifications comprise acetylation, methylation, phosphorylation, SUMOylation and ADP ribosylation, which alter both the electrostatic charge of the histone and its interactions with multiproteic complexes, thus modulating the activity of the associated DNA. Consistent with the modification patterns of histones, Strahl and Allis proposed a “histone code” hypothesis by which nonhistone proteins would be able to “write”, “read”, and “erase” histone modifications in order to regulate specific chromatin functions and, therefore, to determine the activation or repression of gene expression (Strahl & Allis, 2000). Among all the post-translational modifications on histone tails, histone acetylation and methylation of specific lysine residues on histones H3 and H4 are the most extensively studied. Histone acetylation depends on the balance of activity between histone acetyl transferases (HATs) and histone deacetylases (HDACs). In general, transcriptional activators recruit HATs, which alters nucleosomal conformation to produce an open chromatin structure, whereas transcriptional repressors associate with HDACs, which decreases the level of histone acetylation and promotes a more condensed and inactive chromatin state. HATs and HDACs target not only histone tails, but also chromatin and nonchromatin proteins regulating important pathways such as cell cycle progression, differentiation and apoptosis (Minucci & Pelicci, 2006). Histone methylation, catalyzed by histone methyl transferases (HMTs), occurs at both arginine and lysine residues on the tails of histone proteins H3 and H4. Similar to acetylation/deacetylation, histone methylation is reversible, and demethylation is catalyzed by histone demethylases (HDMs). Unlike histone acetylation, methylation of histone proteins can result in either activation or repression, depending on which residue is affected. Indeed, trimethylation of histone H3 lysine 9 (H3K9), histone H3 lysine 27 (H3K27), and histone H4 lysine 20 (H4K20) is associated with silent chromatin and transcriptionally inactive genes, while methylation of lysines 4, 36, and 79 on histone H3 (H3K4, H3K36 and H3K79) is closely linked with active transcription (Cheung & Lau, 2005; Vakoc et al., 2005). Although the epigenetic modifications described above involve different sets of enzymes, it has recently become evident that they work together to establish and maintain global and local chromatin states. The relationship between DNA methylation and histone modification might depend on interactions between HMTs and DNMTs, and might also be mediated partially through MBDs. In fact, in the context of H3K9 and H3K27 methylation, several HMTs recruit DNMTs to target promoters to silence genes stably (Lehnertz et al., 2003; Vire et al., 2006). In addition, HMTs and HDMs may regulate the stability of DNMT proteins, thus influencing DNA methylation levels (Esteve et al., 2009; Wang et al., 2009). DNA methylation and histone deacetylation are also linked through the MBD MeCP2, which associates

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immunotherapy should be highly immunogenic, homogeneously expressed within the tumor mass, frequently expressed in neoplastic tissues, regardless of tumor type, and have cancer-restricted expression to prevent autoimmune reactions to normal tissues. CTAs, which include the MAGE, NY-ESO-1, and SSX gene families and the GAGE/ PAGE/XAGE super-families, fulfill many of these requirements since they: i) encode both HLA class I and HLA class II restricted peptides, eliciting both cellular and humoral immune responses; ii) are expressed in neoplastic tissues of different histologic origin; and iii) are not expressed in normal tissues except testis and placenta (Fratta et al., 2011). The absence of HLA class I molecules on male germ cells, and the presence of the blood-testis barrier, which reduces access of immune cells to the testis, allow CTAs to be regarded as cancer-specific antigens from an immunological perspective (Fratta et al., 2011). Despite the above considerations, several issues limit the applicability and effectiveness of CTA-directed immunotherapies. Firstly, tumors from fewer than 50% of cancer patients analyzed typically express CTAs, with major variation between different tumor types (Fratta et al., 2011). As a result, anticancer immunotherapeutic strategies based on specific CTAs are restricted to only a fraction of patients. Secondly, CTA expression is often heterogeneous within a given tumor mass, with positive immunohistochemical staining below 50% of neoplastic cells appearing the rule rather than the exception (Fratta et al., 2011; Mengus et al., 2013). This intratumoral heterogeneity of CTA expression clearly impairs immunological targeting of cancer cells in the neoplastic tissue, and allows escape of CTA-negative cancer cells from CTA-directed immune eradication. Among the different classes of TAA described as epigenetically regulated (Nicolay, 2009), CTAs are undoubtedly the best characterized and those for which epigenetics definitively influences their expression (Fratta et al., 2011). Hypomethylation of CTA promoters has been strongly linked to CTA expression in neoplastic cells and was suggested to result from deregulation of cellular methylation homeostasis that itself is caused by the global genomic DNA hypomethylation frequently observed in cancer cells (Fratta et al., 2011). Hypermethylation of promoter regions has been clearly identified as the primary mechanism that shuts off the expression of CTAs in cancer cells, implying that hypomethylation of CTA promoters is sufficient to allow their expression in any cell (De Smet et al., 1996; Sigalotti et al., 2002; Loriot et al., 2006). Consistent with this theory, promoter hypomethylation was associated with CTA expression in melanoma stem cells, which are considered responsible for tumor initiation, progression, and metastasis of the disease (Sigalotti et al., 2008). Asymmetric cell division and divergent “differentiation” from cancer stem cells has also been suggested to contribute to or to generate the intratumoral phenotypic and functional heterogeneity of cancer cells typically observed in neoplastic lesions (Stingl & Caldas, 2007). In this regard, it is noteworthy that intratumoral heterogeneous promoter methylation patterns have been shown to determine the intratumoral heterogeneity of CTA expression (Sigalotti et al., 2004; Woloszynska-Read et al., 2008) and to maintain it by heritability of the CTA methylation status at single cell level (Fratta et al., 2010). The unique role of DNA methylation in regulating CTA expression makes these antigens ideal targets for pharmacologic reactivation by DNMTis. Supporting this notion, treatment with different DNMTis, in particular with 5-AZA-CdR, has been consistently proven to induce or to up-regulate CTA expression in neoplastic cells from solid tumors of different histologic origin, both at the mRNA and the protein level (Weber et al., 1994; Coral et al., 2002; Fratta et al., 2011). The multiCTA-positive phenotype deriving from concomitant induction of multiple CTAs in the treated cancer cell population, confirmed at single cell level, is particularly interesting from a therapeutic viewpoint as it enables concomitant immunological targeting of different CTAs and reduces the chance of tumor escape should clones lacking expression of an individual CTA arise. Importantly, expression of CTAs induced de novo by DNMTis is highly stable, being detectable several months after drug removal (Sigalotti et al., 2005), and seems to become a

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(EGCG), is the most well studied in terms of hypomethylating activity; its proposed mechanism of action involves blocking cytosine to be methylated from entering the DNMT catalytic site (Fang et al., 2003). EGCG activity has been shown to result in efficient re-expression of genes silenced by promoter methylation, though its kinetics is slower than those of 5AZA-CdR (Nandakumar et al., 2011). Finally, the antisense oligonucleotide MG98, specifically targeting DNMT1, and mir-29b, targeting DNMT3A and 3B, have been reported to decrease DNA methylation and induce expression of tumor suppressor genes (Amato, 2007; Garzon et al., 2009). Despite their activity, the clinical data obtained so far with MG98 are disappointing (Klisovic et al., 2008), raising questions about the efficiency of the in vivo delivery of these molecules. Compounds that target classical class I (HDAC1, 2, 3 and 8), II (HDAC4, 5, 6, 7, 8, 9 and 10), and IV (HDAC11) HDACs are commonly called HDAC inhibitors (HDACis). These inhibitors are currently being evaluated as anticancer agents in several clinical trials (for a review see (Nebbioso et al., 2012)). Based on their chemical structure, HDACis can be divided into several classes: i) short-chain and aromatic fatty acids [e.g. sodium butyrate (SB), 4-phenylbutyrate, and valproic acid (VPA)]; ii) hydroxamic acids [e.g., Trichostatin A, (TSA)] and hydroxamic acidbased hybrid polar compounds [e.g. suberoylanilide hydroxamic acid (SAHA, vorinostat), LBH589 (panobinostat)]; iii) cyclic tetrapeptides [e.g., FK288 (depsipeptide/romidepsin)]; iv) benzamides [e.g., MS-275 (SNDX-275), MGCD0103 (Mocetinostat)]; and v) miscellaneous compounds (e.g. depudecin). The mechanism underlying the anticancer effects of HDACis is still not entirely clear because they are active in the nucleus as well as in the cytoplasm, where they catalyze the deacetylation of both histones and many non-histone proteins involved in essentially every known cancer-related pathway. In 2006, vorinostat was the first HDACi approved by the FDA for the treatment of patients with cutaneous T-cell lymphoma (Mann et al., 2007). Recently, romidepsin, a natural product obtained from the bacterium Chromobacterium violaceum, was approved by the FDA for treating the refractory form of the same disease. Vorinostat and romidepsin are proposed to inhibit histone deacetylation by targeting the zinc ion located in the catalytic pockets of Class I and Class II HDACs (Lech-Maranda et al., 2007; Marks & Breslow, 2007; Bertino & Otterson, 2011). Two short-chain fatty acids, VPA and SB, and two benzamides, MS-275 and MGCD0103, are under active clinical development as single agents or in combination with other drugs (Nebbioso et al., 2012). Although HDACis have been extensively studied, little data are available for inhibitors of HATs (HATis). A natural p300-specific HATi, curcumin, has gained increasing attention as a potential anticancer drug and is currently being investigated in several clinical trials. Curcumin, which possesses intrinsic HATi activity specific for p300/ CBP, has been shown to inhibit histone acetylation both in vitro and in vivo (Nebbioso et al., 2012). In contrast to the large number of clinical trials investigating HDACi activity, clinical evaluation of HMT and HDM inhibitors is still limited and at an early stage, mainly due to the low specificity and relatively high toxicity observed (Nebbioso et al., 2012). In light of the key role altered histone methylation contributes to the neoplastic phenotype, substantial efforts have been made to target HMTs responsible for setting silencing histone marks. Inhibitors of selected HMTs are finally becoming available; among these, promising efficacy has been demonstrated by the fungal mycotoxin, chaetocin, and also by BIX-01294 that both specifically inhibit G9a HMT, leading to a reduction in the H3K9 methylation silencing mark. Significant antineoplastic activity has also been described for 3-Deazaneplanocin A (DZnep), that appears to selectively inhibit EZH2 (KMT6) HMT, reducing trimethylation of H3K27 and resulting in up-regulated expression of different TSGs (Nebbioso et al., 2012).

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The recognition and lysis of neoplastic cells by antigen-specific CTLs requires antigen presentation to T cells in the form of antigenic peptides associated with a specific HLA class I allospecificity on the tumor cell surface. However, cancer cells frequently exhibit specific alterations in the expression of HLA class I antigens including total loss or downregulation, as well as selective loss or down-regulated expression of distinct HLA class I allospecificities that significantly impair or even prevent their recognition by CTLs (Seliger, 2008). The most common molecular mechanisms underlying such alterations are regulatory or structural defects in various components involved in the biosynthesis and assembly of the peptide/β2-microglobulin/HLA class I heavy chain complex (Seliger, 2008). In addition to these wellcharacterized molecular defects, emerging data also suggest a relevant role for epigenetic events in the absent or down-regulated expression of HLA class I molecules in cancer cells. For example, epigenetic inactivation mediated by promoter hypermethylation seems to be an important mechanism for loss or down-regulated expression of HLA class I antigens in human esophageal squamous cell carcinoma (ESCC) and gastric carcinoma (GC). In fact, methylation of one or more HLA loci was found in 45% to 60% of ESCC or GC samples analyzed, and correlated with absence or down-regulation of the respective mRNA and/or protein (Nie et al., 2001; Ye et al., 2010; Qifeng et al., 2011). Emphasizing the causal link between HLA promoter methylation and silencing of HLA class I expression in ESCC and GC, treatment of HLA-deficient neoplastic cell lines with 5AZA-CdR resulted in demethylation of HLA promoters and in reexpression or up-regulation of the HLA-A or -B gene (Nie et al., 2001; Ye et al., 2010). Despite this important role of DNA hypermethylation in shutting off HLA class I gene expression in ESCC and GC, its direct relevance in other neoplasms is still debated. In melanoma, for instance, Serrano et al. reported that hypermethylation of HLA-A and -B loci in the MSR-3 mel cell line was accompanied by lack of expression of HLA class I antigens both at mRNA and protein level, which was reversed by combined treatment with 5-AZA-CdR and IFN-γ (Serrano et al., 2001). The clinical relevance of these findings, however, remains to be defined. In fact, shutting off of HLA genes in melanoma by DNA hypermethylation seems to be a rather uncommon event, as demonstrated by the inability of 5-AZA-CdR to restore HLA class I expression in a panel of eight human melanoma cell lines with either complete loss of HLA class I antigens or selective loss of HLA-A2 allospecificity (Fonsatti et al., 2003). Conversely, frequent involvement of DNA methylation has been reported in sustaining the reduced levels of HLA class I antigens observed in melanoma cells. Indeed, 5-AZA-CdR consistently proved effective in generating persistent up-regulation of HLA class I antigens and allospecificities in extended panels of human metastatic melanoma cells, although the molecular mechanism has not been investigated in detail (Coral et al., 1999). Likewise, accumulating evidence shows that epigenetic targeting by DNMTis and/or HDACis can up-regulate HLA class I antigens on different types of neoplastic cell, including neuroblastoma, cervical, ovarian and prostate carcinoma (Magner et al., 2000; Mora-Garcia Mde et al., 2006;

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targets for cancer immunotherapy. Among these, the high molecular weight melanoma-associated antigen (HMW-MAA) has been utilized as therapeutic target in clinical studies with HMW-MAA-mimicking anti-idiotypic antibodies (Mittelman et al., 1992). Hypermethylation of the HMW-MAA promoter was recently associated with lack of HMW-MAA expression in acral lentiginous melanomas. The causal link between promoter methylation and HMW-MAA gene expression was further confirmed by the ability of 5-AZA-CdR to demethylate the HMW-MAA promoter in treated melanoma cells, resulting in the reexpression of HMW-MAA mRNA and protein (Luo et al., 2006). Overall, the reported data suggest that epigenetic drugs are promising therapeutic agents that may be particularly useful in re-establishing homogeneous TAA expression in neoplastic tissues to be targeted by anticancer immunotherapies.

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constitutive feature of neoplastic cells when analyzed at the single cell level (De Smet et al., 1999; Fratta et al., 2010). Drug-induced transcription of CTA genes leads to synthesis of an immunologically functional protein, which is correctly processed by the tumor cell APM and properly presented to CTLs. Indeed, treatment with DNMTis ultimately leads to the immunological recognition and cytolysis of cancer cells that were otherwise resistant to CTA-specific CTLs (Weber et al., 1994; Coral et al., 2002; Adair & Hogan, 2009; Bao et al., 2011; Konkankit et al., 2011; Chou et al., 2012; Pollack et al., 2012). Of note, DNMTis have also been demonstrated to reverse functionally the intratumoral heterogeneous expression of CTAs, as shown by the ability of 5-AZA-CdR to homogenize CTA expression and recognition by MAGE-A-specific CTLs of melanoma cells, in an ex vivo model of intratumoral heterogeneity (Sigalotti et al., 2004). The recognized co-operation of DNA methylation and histone acetylation in controlling gene transcription prompted research into the effects of HDACis, given alone or in combination with DNMTis, on CTA expression and on the immunological recognition of cancer cells. The HDACi TSA was able per se to induce a weak de novo expression of MAGE-A genes in selected melanoma, prostate, breast, and colon cancer cell lines, which was much lower than that induced by 5-AZA-CdR alone (Wischnewski et al., 2006). It has also been reported that TSA enhanced the transcription of reporter genes driven by either methylated or unmethylated MAGE-A2 and -A12 promoters, indicating a role for histone acetylation in the control of MAGE-A gene transcription, and explaining the synergistic up-regulation of MAGE-A genes observed in selected cancer cell lines by 5-AZA-CdR/TSA combinations (Wischnewski et al., 2006; Sigalotti et al., 2007). Despite these observations, and a demonstrated synergistic effect of combined DNMTi and HDACi treatment on CTA expression in some cell lines, DNMTis remain the most effective epigenetic drugs in both inducing and up-regulating CTA expression in cancer cells (Sigalotti et al., 2007). The synergistic up-regulation of CTA expression observed by the addition of HDACis is usually only 2–3 fold higher than with DNMTi treatment alone and is not durable, and some cell lines display no synergism at all (Wischnewski et al., 2006; Sigalotti et al., 2007). In addition, it appears that even where synergistic up-regulation of target CTAs is observed following combined DNMTi/HDACi treatment, this does not necessarily result in increased recognition and lysis of neoplastic cells by CTA-specific CTLs compared with DNMTi treatment alone (Weiser et al., 2001). The contribution of histone methylation to CTA regulation has also been investigated. Different studies have addressed the effect of G9a and/or GLP HMTs knockdown; these enzymes contribute to transcriptional silencing by catalyzing H3K9 dimethylation (H3k9me2). The results were in part contradictory, showing that HMT knockdown is sufficient to induce Mage-A genes in mouse embryonic stem cells, but not on human colon cancer cells (Tachibana et al., 2005; Link et al., 2009). However, combined HMT inhibition and DNMTi treatment was able to synergistically increase CTA expression in human colon cancer cells (Link et al., 2009). Similarly, minor effects on CTA expression were observed in lung cancer cells following RNA interference-mediated depletion of HDMs KDM1 and KDM5B, which mediate removal of the activatory signals H3K4me, me2, me3, or of HMT KMT6/EZH2, which sets the H3K27me3 silencing mark; when combined with 5-AZA-CdR treatment, however, synergism was observed resulting in increased recognition by CTA-specific CTLs. The synergism was also confirmed when using DZNep drug, which removes the H3K27 mark by inhibiting EZH2/KMT6 (Rao et al., 2011). Furthermore, inhibition of EZH2/KMT6 by DZNep or RNA interference increased the expression of GAGE genes when combined with both HDACis and DNMTis, as compared to either the single agents alone or the double combinations (Sun et al., 2009). These data, together with those on HDACis, clearly demonstrate crosstalk and a degree of cooperative activity for the various layers of epigenetic control, strengthening the notion that promoter DNA methylation usually functions as a gatekeeper for allowing CTA expression. Besides CTAs, epigenetic regulation is now recognized as an important factor influencing the expression of other TAA that are utilized as

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7. Additional immunotherapeutic advantages of epigenetic editing 685 In addition to the above reported effects on adaptive immune recognition of cancer cells, recent data indicate that epigenetic drugs may be exploited to increase or allow eradication of tumor cells by cells of the innate immunity. In particular, HDACis have been involved in targeting tumor cells by NK cells through engagement of the activating receptor NKG2D expressed on NK cells and the stress-induced MICA, MICB or ULBP1-3 ligands expressed on neoplastic cells (Armeanu et al., 2005). Histone deacetylation appears to prevail over promoter methylation in reducing expression of NKG2 ligands (e.g., ULBP1-3) by neoplastic cells, with specific HDACs being crucial for the suppression (LopezSoto et al., 2009). Accordingly, different HDACis, including VPA, depsipeptide, MS-275, TSA, romidepsin, vorinostat and PDX101, were able to up-regulate the expression of MICA, MICB and/or ULBP1-3 in neoplastic cells from different solid malignancies, including glioblastoma, medulloblastoma, sarcoma, cervical, hepatocellular, and prostate carcinoma. This induction or up-regulation of MICA and MICB and/or ULBP1-3 on neoplastic cells allowed or increased their lysis by NK cells (Armeanu et al., 2005; Skov et al., 2005; Schmudde et al., 2008; LopezSoto et al., 2009; Zhang et al., 2009; Yamanegi et al., 2010; Berghuis et al., 2012; Horing et al., 2013). The mechanism by which HDACis upregulate NKG2D ligands on neoplastic cells is still not completely elucidated, but appears to include both direct hyperacetylation of histones associated with promoters of NKG2 ligands (Yamanegi et al., 2010), and indirect effects such as HDACi-mediated activation of the ATM/ ATR-dependent DNA damage response (Berghuis et al., 2012), as well as activation of GSK3 kinase (Skov et al., 2005). Despite histone acetylation apparently being the major regulatory mechanism for NKG2D ligand expression, treatment with DNMTis, alone or in combination with different HDACis, also proved effective in demethylating the MICB promoter, up-regulating MICA and MICB expression, and downregulating their decoy soluble forms in neoplastic cell cultures from different solid tumors, finally leading to neoplastic cell sensitization to NKmediated cytotoxicity (Tang et al., 2008; Chavez-Blanco et al., 2011; Yamanegi et al., 2012). As seen for HDACis, the DNA damage response also appears to contribute to DNMTi-mediated upregulation of NKG2D ligands (Tang et al., 2008). Notably, irrespective of the underlying mechanism, no up-regulation of NKG2D ligands or of NK-mediated lysis was observed on normal cells, suggesting a tumor-specific immunomodulatory activity of HDACis (Armeanu et al., 2005; Skov et al., 2005). In addition to influencing the activation of effector cells, epigenetic drugs appear to contribute significantly to improving the effectiveness of cytotoxic pathways mediating both innate and adaptive immune responses. Engagement of death receptors, including FAS and TRAIL receptors, on target cells by their ligands expressed on CTLs and NK cells

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triggered by the drug, including induction of HLA class II promoter hyperacetylation (Chou et al., 2005; Gialitakis et al., 2006). The picture of immunomodulatory activities exerted by epigenetic drugs is enriched by their ability to induce or up-regulate expression of accessory or co-stimulatory molecules on cancer cells (Fig. 1). In this respect, DNMTis and HDACis appear to have different tropisms. Indeed, ICAM-1 appears to be mainly targeted by DNMTis; 5-AZA-CdR and its newly derived pro drug SGI-110 have consistently been shown to upregulate ICAM-1 on neoplastic cells of different origin (Coral et al., 1999; Arnold et al., 2001; Coral et al., 2002; Coral et al., 2013). On the other hand, HDACis, including TSA and SB, preferentially up-regulate CD40, CD80, and CD86, as seen in human melanoma and neuroblastoma cell lines (Magner et al., 2000; Kalbasi et al., 2010). This different behavior of HDACis and DNMTis could well provide a rationale for their combined use to achieve optimal tumor immunomodulation. Overall, the impact of epigenetic drugs on antigen processing and presentation to immune cells is composite and exhaustive, and cancer cell recognition by immune cells is likely to embrace all the above reported effects (Fonsatti et al., 2007).

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Kitamura et al., 2007; Adair & Hogan, 2009). As expected from the key role played by HLA class I antigens in the immune recognition of cancer cells, the above phenotypic modifications triggered by epigenetic drugs are characterized by a strong functional immunomodulatory potential, which is sufficient per se to restore or to up-regulate the recognition of neoplastic cells by TAA-specific CTLs (Serrano et al., 2001; Mora-Garcia Mde et al., 2006; Fonsatti et al., 2007). In addition to defects directly targeting HLA class I molecules, their reduced surface expression may also be related to defective expression of APM components (Fig. 1) (Bukur et al., 2012). While structural defects of APM components have rarely been reported, epigenetic disturbance appears to be widely responsible for reduced expression of these molecules, via direct and indirect mechanisms (Bukur et al., 2012). A direct role of DNA hypermethylation or histone hypoacetylation has been reported for down-regulation of ERp57, LMP7, TAP1, TAP2 and tapasin in selected cases of ESCC, renal cell carcinoma, melanoma, cervical carcinoma, and oral squamous cell carcinoma (Setiadi et al., 2007; Seliger, 2008; Jiang et al., 2010; Bukur et al., 2012; Hasim et al., 2012; Zheng et al., 2013). Interestingly, DNMTis and HDACis were consistently effective in inducing or upregulating the expression of APM components in a broad spectrum of tumor types, regardless of the constitutive epigenetic status of the corresponding genes, suggesting a contribution by indirect epigenetic triggering mediated by molecules still to be identified (Tomasi et al., 2006; Khan et al., 2008; Seliger, 2008; Bukur et al., 2012). Although most studies have focused on the role of HLA class Ipresented peptides and CD8+ CTLs in achieving tumor rejection, other research has identified the important role of CD4+ T helper (Th) lymphocytes as key regulators of immune responses, which are able to drive long-lasting anticancer immunity. Activation of CD4+ Th lymphocytes requires recognition of the antigenic peptides associated with HLA class II molecules expressed on the cell surface of professional APCs. Activation results in Th1 responses, which in turn activate anticancer CD8+ CTLs, and Th2 responses, which may promote humoral immunity to cancer cells. The effects of Th activation are completed by their ability to recruit cells of the innate immune system, which contribute to the immune response to neoplastic cells, and by the ability of selected CD4+ T cells to exert direct cytotoxic activities on cancer cells (Knutson & Disis, 2005; Marshall & Swain, 2011). Cancer cells themselves can function as nonprofessional APC if they express HLA class II antigens on their membranes, and expression of HLA class II antigens may also render them susceptible to the activity of CD4+ T cells that exert their antitumoral activity by different mechanisms (Thomas & Hersey, 1998). As seen with HLA class I antigens, neoplastic cells rely on epigenetic alterations to escape Th cell activation or recognition. This is usually accomplished by epigenetic inactivation of the class II transactivator (CIITA), whose expression is required for both constitutive and cytokine-induced expression of HLA class II antigen (Sigalotti et al., 2005; Wright & Ting, 2006). Different mechanisms have been proposed for the lack of CIITA expression in cancer cells, and were primarily defined in hematologic malignancies, where CIITA promoter IV (PIV) methylation and associated histone hypoacetylation cooperated in preventing IFN-γ-induced HLA class II expression (Morimoto et al., 2004; Cycon et al., 2013). Data in neuroblastoma, uveal melanoma, rhabdomyosarcoma, breast, colorectal, and head and neck carcinoma support a similar regulation for solid tumors, with varying contributions of CIITA-PIV DNA hypermethylation, histone hypoacetylation and H3K27me3 histone methylation depending on tumor type (Croce et al., 2003; Kanaseki et al., 2003; Satoh et al., 2004; Radosevich et al., 2007; Meissner et al., 2008; Londhe et al., 2012). Depending on the regulatory mechanism involved, DNMTis and HDACis proved effective in re-establishing CIITA expression and the sensitivity of neoplastic cells to IFN-γ-triggered expression of HLA class II genes (Kanaseki et al., 2003; Satoh et al., 2004; Chou et al., 2005; Radosevich et al., 2007; Londhe et al., 2012). Additional studies reported that TSA is able to stimulate transcription of HLA class II genes by a mechanism that does not involve CIITA, but instead relies on chromatin modifications

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As seen above, robust data support the role of epigenetic drugs in facilitating immunological targeting of cancer cells by their ability to modulate different molecules and pathways involved in the interaction between tumor cells and the immune system. Furthermore, the distinctive ability of epigenetic marks, and especially of DNA methylation patterns, to be physiologically inherited during cell division (Fratta et al., 2010) renders epigenetic modifiers particularly attractive from a clinical perspective. In fact, durable modification of the tumor phenotype triggered by epigenetic drugs is expected to allow continuous immunological targeting and elimination of neoplastic cells even in the absence of concomitant epigenetic treatment. In the light of these considerations, researchers have proposed the clinical use of epigenetic drugs to overcome some major limitations of currently available immunotherapeutic regimens, and second generation epigenetic drugs with improved efficacy and clinical tolerability are now being developed (Coral et al., 2007; Coral et al., 2013). Preclinical data generated using mice models

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strongly support the feasibility and effectiveness of the proposed approaches. In particular, systemic administration of 5-AZA-CdR proved effective in modifying the immune phenotype of human metastatic melanoma xenografts, by inducing or up-regulating cellular CTA expression and expression of HLA class I antigens and HLA A1 and A2 allospecificities (Coral et al., 2006, 2007). These in vivo modifications were remarkably durable; NY-ESO-1 expression and HLA class I antigen up-regulation were still detectable on melanoma xenografts 30 days after the end of 5-AZA-CdR administration (Coral et al., 2006). Emphasizing the notion that epigenetic modification of tumor cells strongly up-regulates their immunogenicity, injection of BALB/c mice with 5AZA-CdR-treated human melanoma cells generated high-titer antiNY-ESO-1 antibodies (Coral et al., 2006), while systemic treatment with TSA delayed glioblastoma xenograft growth by enhancing tumor recognition by NK cells following HDACi-mediated up-regulation of NK-activating ligands on tumor cells (Horing et al., 2013). Importantly, recent whole genome expression profiling in syngeneic mouse models confirmed a comprehensive modulation of immune-related genes in tumors by 5-AZA-CdR, while normal tissues were substantially unaffected. These observations further support the tumor immunomodulatory potential of epigenetic drugs, and their safe use alongside immunotherapies, since autoimmune reactions to normal tissues appear unlikely (Coral et al., 2012). In agreement with the above background, in vivo preclinical data support the likely synergistic activity of epigenetic tumor immunomodulation and recipient immunomodulation using different strategies. Initial data reported synergistic antitumor activity of low doses of 5-AZA-CdR and IL12 in B16F10 mouse melanoma, which was dependent on generation of tumor reactive CD4+ and CD8+ T cells (Kozar et al., 2003). Similarly, 5-AZA-CdR combined with immunotherapy comprising CpG oligodeoxynuclotides or IL2-producing cellular vaccine, showed increased therapeutic efficacy compared with each agent alone in a mouse model of HPV16-associated tumors. In the combined regimens, but not with 5-AZA-CdR alone, reduction in the tumor mass mostly relied on CD8+ T and NK cells, suggesting the need for cooperation between 5-AZA-CdR and immunotherapy in achieving effective immune-mediated tumor eradication (Simova et al., 2011). Adoptive cell therapy with effector T cells targeting different TAAs was demonstrated to benefit from both DNMTis and HDACis. Indeed, induction of murine CTA P1A in 4 T1 mammary tumor grafts by systemic 5-AZA-CdR enabled tumor targeting by adoptively transferred anti-P1A CTLs, resulting in potent and significant synergistic reduction in the number of lung metastases by 4 T1 cells (Guo et al., 2006). HDACis, on the other hand, appear to improve the in vivo therapeutic efficacy of adoptive cell therapy by concomitantly acting on: i) upregulation of target TAA on neoplastic cells, ii) depletion of endogenous competing lymphocytes in recipient mice, giving a proliferative advantage to the transferred cells; and iii) improving the cytotoxic activity of adoptively transferred T cells (Vo et al., 2009). In view of the promising clinical data obtained with mAbs targeting immunologic checkpoints, a particularly relevant topic is the potential synergism between tumor immunomodulation by epigenetic drugs and host immunomodulation via immunostimulatory mAbs. With respect to this, Johnstone and colleagues recently did an important study which combined HDACi treatment (vorinostat or panobinostat) with administration of an immunomodulatory cocktail composed of agonistic anti-CD40 and anti-CD137 mAbs with the aim of concomitantly promoting APC function and survival and activation of T cells (Christiansen et al., 2011). Using a syngeneic mouse model, the authors showed only minimal efficacy with each agent alone, while strong antitumor activity against established mammary, colorectal and renal carcinoma tumors was observed for the combination. The effect was clearly immunemediated, being abrogated in immunodeficient mice, and relied mainly on the generation of tumor-specific CD8+ T cells with a minor contribution from NK cells. In addition to epigenetic modulation of tumor phenotype, the authors proposed that immunogenic cell death (Kroemer et al.,

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is a key mechanism for eradication of neoplastic cells via induction of apoptosis. In this scenario, HDACis, with or without DNMTis, appear to contribute to enhanced killing by NK cells or CTLs by up-regulation of TRAIL R2 and FAS in neoplastic cell cultures of different histologic derivation (Lundqvist et al., 2006; Yamanegi et al., 2012; Yang et al., 2012). In support of the above mechanism, epigenetic treatment sensitized neoplastic cells to apoptotic death by recombinant FASL and/or TRAIL, as well as by agonistic antibodies directed at death receptors that are currently in clinical development (Konkankit et al., 2011; Koshkina et al., 2011; Rao-Bindal et al., 2012; Yang et al., 2012; Jazirehi & Arle, 2013). Concomitant induction of death receptors and ligands on neoplastic cells by HDACi treatment was also able to contribute to the anticancer effects of these drugs, eliciting an “autocrine” apoptotic loop (Kim et al., 2006; Gillenwater et al., 2007). The up-regulation or induction of death receptors upon epigenetic treatment also appears to play a major part in mediating cancer cell apoptosis triggered by IFNs. IFNs are key players in both the innate and the adaptive immune response, and are frequently used in cancer patients because of their immunomodulatory properties and direct anticancer action, which relies on their growth-suppressing and pro-apoptotic activities. The latter are mainly mediated by the induction of death receptors and ligands on target cells. Borden and colleagues have demonstrated that epigenetic events can target IFN response genes, blocking their transcription and resulting in resistance of cancer cells to IFN-triggered apoptosis (Reu et al., 2006a,b; Bae et al., 2008). Of these events, epigenetic silencing of TRAIL-R1 was shown to be a major block to efficient killing of melanoma cells by IFNs, and its re-expression induced by 5-AZA-CdR appeared to be necessary for restoring the sensitivity of neoplastic cells to apoptosis triggered by IFN-α2b and IFN-β (Bae et al., 2008). Demethylating treatment also restored and/or up-regulated the expression of other pro-apoptotic genes, including XAF-1, RASSF1A, and TRAILR2, which all appeared to contribute to the overall sensitization of melanoma and renal cell carcinoma cells to IFNs (Reu et al., 2006a,b). Indeed, the general ability of epigenetic drugs to shift the balance of proapoptotic and anti-apoptotic molecules towards a pro-apoptotic cellular environment seems to be an important contribution to improved immunological elimination of cancer cells. Despite the robust data described above, other data suggest that HDACi tumor immunomodulation may also impair NK recognition and lysis of tumor cells. Fiegler and colleagues have recently demonstrated that HDACis (vorinostat, TSA, VPA, apicidin) as well as small interfering RNA-mediated knockdown of HDAC 2 or 3 down-regulate expression of the B7-H6 activating ligand for the NK receptor NKp30 on tumor cells, resulting in reduced degranulation of NK cells (Fiegler et al., 2013). Accordingly, it is conceivable that the ability of NK cells to recognize and lyse HDACi-treated tumor cells will depend on the final balance of activator ligands expressed on treated tumor cells.

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Fig. 2. Tumor immunomodulatory properties of epigenetic drugs. DNMTis and/or HDACis can concomitantly modulate tumor cell expression of different molecules that are engaged by their respective “receptors” on immune effectors cells. These modifications include the induction/up-regulation of HLA class I and class II antigens, tumor antigens presented as immunogenic peptides, the co-stimulatory/accessory molecules CD40, CD54, CD58, CD80, and CD86, and the activating NKG2D ligands MICA, MICB, and ULBPs. Engagement of these molecules by their “receptors” on effector cells prompts effective recognition and killing of neoplastic cells.

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Epigenetic alterations are frequently used by neoplastic cells to evade host immune surveillance. Epigenetic drugs, by acting concomitantly on different molecules and pathways involved in host-tumor interactions, are powerful agents to induce or increase immunologic targeting of cancer cells in vivo (Fig. 2). Their long lasting effects on the phenotype of cancer cells, including induction, up-regulation and intratumoral homogenization of CTA expression, as well as modulation of HLA class I and class II molecules, accessory or co-stimulatory molecules, NK cell activating ligands, and of cytokine signaling, point to epigenetic drugs as appealing clinical potentiators of both the innate and adaptive immune response to cancer. Although these activities of DNMTis and HDACis have themselves proved immunologically effective, their combination with immunotherapeutic approaches appears

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LFA-1 (CD11a/CD18)

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2013) of cancer cells triggered by HDACis might contribute to mounting of the antitumor immune response (Christiansen et al., 2011). As well as tumor cell-directed activity, HCACis have also been reported to modulate the host immune system, reducing regulatory T cells in vivo and thus improving the efficacy of immunotherapies with IL-2 or with a survivinbased vaccination in murine models of renal and prostate carcinoma (Shen et al., 2012). Following their comprehensive characterization in preclinical mice models, the immunomodulatory activities of epigenetic drugs are now also being identified in the clinic. Initial support for the immunological efficacy of DNMTis, alone or combined with HDACis, came from studies in hematologic malignancies, particularly AML and MDS; 5-azacytidine and 5-AZA-CdR have both been approved by the FDA for the treatment of MDS. We have demonstrated that a single course of 5-AZA-CdR was able to induce de novo expression of the CTAs MAGE-A1, NY-ESO-1 and SSX in AML and MDS patients, and that the acquired CTA phenotype was maintained 30 days after the end of 5-AZA-CdR-treatment in most patients (Sigalotti et al., 2003). These observations led us to propose that, in addition to its well-known cytotoxic or cytostatic, proapoptotic and differentiating activities, 5-AZA-CdR could, at least in part, sustain long-term disease control through the activation of immunological mechanisms (Sigalotti et al., 2003). In support of this theory, anti-MAGE CD8+ T cell responses were induced in ten out of 20 AML patients following treatment with 5-AZA-CdR combined with VPA, and this immune response appeared to correlate with clinical response (Goodyear et al., 2010). These data were first confirmed in solid tumors in research by Schrump et al. Using a continuous 72 h infusion of 5-AZACdR, they obtained de novo induction of MAGE-A3 and NY-ESO-1 in tumor biopsies from thoracic cancer patients. De novo expression of the NY-ESO-1 protein was accompanied by the appearance of antiNY-ESO-1 circulating antibodies in three of five patients, confirming the in vivo immunogenicity of the de novo induced CTAs and their potential role in immunological tumor rejection (Schrump et al., 2006). In addition to its direct role in modulating cancer immune phenotype, 5-AZA-CdR has recently been evaluated as a potential modulator of the immune activating properties of high-dose IL-2 in melanoma and renal cell carcinoma patients. 5-AZA-CdR pre-treatment resulted in demethylation of genomic DNA and alteration of immunomodulatory genes in PBMC from treated patients that was still present at the time of IL-2 administration, and was therefore expected to modulate response to the cytokine. Gene expression profiling identified complex modulation of gene expression patterns. While up-regulation of chemokines and genes involved in IL-1, IL-17, IL-22 and IFN signaling might favor the activity of administered IL-2, down-regulation of IL2Rα, CD3-ε, CD2 and genes involved in IL-12 signaling can be expected to impair IL-2 activity (Gollob et al., 2006). On the other hand, the CTLA4 down-regulation observed in PBMC from 5-AZA-CdR treated patients may have a generally positive impact on different immunotherapeutic regimens by the reduction of CTLA-4-mediated immune suppression (Gollob et al., 2006).

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to be an ideal strategy that is obtaining preliminary support in mouse models. Acting on the immune evasion mechanism intrinsic to cancer cells via epigenetic tumor immunomodulation on the one hand, and acting on escape strategies relying on the host's immune system deficiency via host immunomodulation on the other hand, could have a dramatic synergistic effect on long-term immunologic control of cancer. Autologous cellular vaccines and mAbs targeting immunological checkpoints that have already demonstrated significant clinical efficacy are clearly the most desirable partners for combination with DNMTis and/ or HDACis. Although additional preclinical data are needed to assess the efficacy and toxicity of these combinations in vivo, the high specificity of epigenetic drugs for neoplastic cells combined with the different toxicity profiles of DNMTis and HDACis from those of immunotherapies augur well for their likely safe and effective clinical use to treat cancer.

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Conflict of interest statement

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The authors declare that there are no conflicts of interest.

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This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC IG 11746 to MM and MFAG 9195 to LS), Regione Toscana “Regional Health Research Program 2009”, and the Ministero della Salute. The funding sources had no involvement in the conduct of the research or in the preparation of the article.

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Potential of epigenetic therapies in the management of solid tumors.

IKK inhibition therapies in solid tumors.

Epigenetic targeting drugs potentiate chemotherapeutic effects in solid tumor therapy.

Targeted drugs in combination with radiotherapy for the treatment of solid tumors: current state and future developments.

Modeling the effects of space structure and combination therapies on phenotypic heterogeneity and drug resistance in solid tumors.

Harnessing the potential of epigenetic therapy to target solid tumors.

Epigenetic polypharmacology: from combination therapy to multitargeted drugs.

Polypharmacology: the rise of multitarget drugs over combination therapies.

Myeloid Cells as Targets for Therapy in Solid Tumors.

Genomic aberrations in the FGFR pathway: opportunities for targeted therapies in solid tumors.

Self-assembled nanoparticles as multifunctional drugs for anti-microbial therapies.

Prevention of Nausea and Vomiting in Patients Undergoing Oral Anticancer Therapies for Solid Tumors.

Epigenetic Drugs for Multiple Sclerosis.

Epigenetic-based therapies for Friedreich ataxia.

Mitochondrial D310 mutation as clonal marker for solid tumors.

Mast cells as targets for immunotherapy of solid tumors.

Epigenetic Therapy for Solid Tumors: Highlighting the Impact of Tumor Hypoxia.

Epigenetic pathway inhibitors represent potential drugs for treating pancreatic and bronchial neuroendocrine tumors.

Novel receptor tyrosine kinase targeted combination therapies for imatinib-resistant gastrointestinal stromal tumors (GIST).

Peptides and peptidomimetics as immunomodulators.

Combination therapies in oncology.

Epigenetic therapies as a promising strategy for overcoming chemoresistance in epithelial ovarian cancer.

At the tipping point for epigenetic therapies in cancer.

Neuregulin expression in solid tumors: prognostic value and predictive role to anti-HER3 therapies.