{'fraction_non_alphanumeric': 0.032240585207260905, 'fraction_numerical': 0.016346067009843764, 'mean_word_length': 5.785539215686274, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Advances in Epigenetic Cancer Therapeutics

Cancer has traditionally been hailed a genetic disease, dictated by successive genetic aberrations which alter gene expression. Yet, recent advances in molecular sequencing technologies, enabling the characterisation of cancer patient phenotypes on a large scale, have highlighted epigenetic changes as a hallmark of cancer. Epigenetic modifications, including DNA methylation and demethylation and histone modifications, have been found to play a key role in the pathogenesis of a wide variety of cancers through the regulation of chromatin state, gene expression and other nuclear events. Targeting epigenetic aberrations offers remarkable promise as a potential anti-cancer therapy given the reversible nature of epigenetic changes. Hence, epigenetic therapy has emerged as a rapidly advancing field of cancer research. A plethora of epigenetic therapies which inhibit enzymes of post-translational histone modifications, so-called ‘writers’, ‘erasers’ and ‘readers’, have been developed, with several epigenetic inhibitor agents approved for use in routine clinical practice. Epigenetic therapeutics inhibit the methylation or demethylation and acetylation or deacetylation of DNA and histone proteins. Their targets include writers (DNA methyltransferases [DNMT], histone acetyltransferases [HAT] and histone deacetylases [HDAC]) and erasers (histone demethylases [HDM] and histone methylases [HMT]). With new epigenetic mechanisms increasingly being elucidated, a vast array of targets and therapeutics have been brought to the fore. This review discusses recent advances in cancer epigenetics with a focus on molecular targets and mechanisms of action of epigenetic cancer therapeutics.


Introduction And Background
Cancer is a leading cause of avoidable premature death in the United Kingdom (UK) [1]. One in two people born after 1960 can expect to be diagnosed with cancer in their lifetime, with 367,167 new cancer cases and 164,901 cancer deaths occurring nationally each year in the UK [2]. Although risk factors vary between cancer types, cancer is primarily a genetic disease arising through successive genetic aberrations. Through projects such as the International Cancer Genome Consortium and The Cancer Genome Atlas Program, the consequences of cancer gene mutations are emerging. However, the role of the epigenome in reshaping gene expression profiles has also come to light ( Figure 1). This review aims to highlight recent advances in our understanding of cancer epigenetics and the key targets for and mechanisms of epigenetic cancer therapeutics.

Group
The writers and erasers responsible for regulating DNA methylation include DNMTs and ten-eleven translocation (TET) proteins. DNMTs (DNMT1/2/3) transfer methyl groups from the methyl donor Sadenosyl methionine (SAM) to the 5' position of cytosine, forming 5-methylcytosine (5mC). Although deposition of 5mC in the gene promoter is recognised as a cause of gene repression, gene activation may result from 5mC deposition in hypermethylated promoters and enhancer elements [9]. Generally, however, DNA methylation of 'CpG islands' (CGIs), which are highly concentrated clusters of cytosine-phosphateguanosine (CpG) dinucleotides, restricts binding of transcription factors at promoters, while promoter CGI hypomethylation allows transcription factor binding and gene activation.
Dysregulation of these processes occurs in cancer. A number of anti-cancer drugs have been developed that target DNMTs. DNMTIs, such as the cytosine analogues Aza (Vidaza) and 5-aza-2′-deoxycytidine (decitabine; Dacogen®), and the second-generation hypomethylating prodrug SGI-110 (guadecitabine) are classed as DNA hypomethylating agents. Their mechanism of action involves incorporation into DNA and irreversible binding to DNMT1, leading to DNA-DNMT1 adduct formation, DNMT1 degradation and, consequently, DNA demethylation ( Figure 2). Aza also incorporates into RNA, more efficiently than DNA, following phosphorylation by uridine-cytidine kinase into triphosphates, resulting in polyribosome disassembly, defective methylation and acceptor function of transfer RNA and translation inhibition [10,11]. DNMTIs reduce aberrant hypomethylation and reactivate of silenced genes, thus restoring the function of tumour suppressor genes and DNA repair genes. In addition to the reactivation of tumour suppressor genes, DNMTIs enhance tumour immunogenicity through the upregulation of major histocompatibility complex (MHC) class I, leading to the recruitment of macrophages, natural killer (NK) cells and CD8+ T cells that secrete a variety of chemotactic and cytotoxic cytokines [12]. In contrast to DNMTs, erasers, such as TET proteins, demethylate DNA. TET proteins include a family of enzymes (TET1/2/3) that utilise Fe(II) and 2-oxoglutarate (alpha-ketoglutarate) as cofactors to oxidise 5mC to 5-hydroxymethylcytosine (5hmC). 5hmC formation alters transcriptional activation of gene expression. TET proteins also convert 5hmC to 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) before an enzyme called thymine-DNA glycosylase excises 5fC and 5caC from DNA. TET protein function is supported by IDH enzymes, which provide the essential 2-oxoglutarate cofactors through conversion of isocitrate to 2oxoglutarate, and, together, these mechanisms complete DNA demethylation [13].

Therapeutics targeting modifications in histone tails
Writers responsible for covalent modifications on charged NH2 termini (tails) of histones include histone acetyltransferases (HATs; GCN5, MYST, p300/CBP families) and histone methyltransferases (HMTs; SET [(Su(var)3-9, enhancer of zeste, trithorax)] domain containing and non-SET-domain containing lysinespecific and arginine-specific families). In addition to acetylation and methylation, histone modifications include phosphorylation, ubiquitylation, sumoylation and biotinylation. The type and location of these modifications alters chromatin structure and gene expression. Gene-activating histone modifications include acetylation of lysine 27 in histone H3, methylation of lysines 4 and 36 in histone H3, and demethylation of lysine 9 of histone H3. Transcriptionally repressive histone modifications include methylation of lysine 9 and 27 in histone H3 and sumoylation of lysine 59 in histone H4.
Some histone modifications (H3K27 methylation) form docking sites for interactions with polycomb group proteins (PcGs). Once docked, polycomb repressive complex 1 (PRC1) compacts chromatin, causing the physical hinderance of RNA polymerase II, repressing gene transcription. PRC1 has E3 ligase activity, while polycomb repressive complex 2 (PRC2) has HMT activity. One of the best characterised PRC2 subunits is EZH2, which is involved in the methylation of lysine 27 on histone H3. PcG-target genes often contain both repressive (H3K27me3) and active (H3K4me3) modifications [22]. Thus, PcG-target genes exist in a poised ready-to-transcribe state with PcGs holding RNA polymerase II at the transcription start site [22].
HATs (e.g. p300/CBP) are classified into type A (nuclear) and type B (cytoplasmic), depending on whether they acetylate nucleosomal histones or newly translated non-nucleosomal histones, respectively. HATs transfer acetyl groups from acetyl-CoA donors to the amino group of lysine residues of histones. Acetylation of an ε-amino group neutralises the charge of lysine residues, reducing interactions between histones and DNA and making DNA less compact and more accessible to transcription factors. Histone acetylation is associated with gene activation, while deacetylation silences genes. Anti-cancer drugs that target HATs (HAT inhibitors) include the small molecule C646, which selectively inhibits p300/CBP, resulting in reduced acetylation of histone H3 [23]. C646 reduces cell survival and induces cell cycle arrest, mitotic catastrophe and apoptosis [23,24].

FIGURE 5: Mechanism of action of HDACIs
HDACIs inhibit the deacetylation of histones by HDACs, leading to an increase in activating histone marks (e.g. H3K9 and H3K16 acetylation) and adoption of an 'open' transcriptionally active chromatin state with downstream anti-cancer effects.
HDACIs, histone deacetylase inhibitors; HDAC, histone deacetylase HDM inhibitor anti-cancer drugs have been developed that target the HDMs lysine demethylase 1A (LSD1/KMD1A) and 5B (KDM5B) as well as JmjC domain-containing proteins. JmjC demethylases are protein hydroxylases involved in free radical-dependent histone modification reactions [43]. LSD1 inhibitors (ORY1001, GSK2879552, tranylcypromine) induce cell cycle arrest and apoptosis by regulating the hexokinase 2 expression and increase the expression of the transcriptional repressor GFI1 as well as the transcription factor PU.1, thus inducing differentiation [44,45]. However, a recent phase I open-label trial of the LSD1 inhibitor GSK2879552 was terminated early due to poor disease control and an unfavourable sideeffect profile [46]. JmjC domain-containing protein inhibitors (GSK-J1, GSK-J4) induce apoptosis and inhibit tumour growth by increasing global levels of repressive trimethylated H3K27 and downregulating cancerpromoting HOX genes [47,48].

Conclusions
Cancer epigenetics is a highly complex and rapidly evolving field, with many exciting developments that enhance our understanding of carcinogenesis and disease progression. By examining epigenetic networks, novel therapeutic approaches can be identified that encompass a wide variety of solid and haematological cancers. Therapeutics that normalise or disrupt epigenetic aberrations hold promise across several malignancies, with well-defined clinical efficacy established in distinct clinical settings. Considering the abundance of epigenetic targets and agents in development, a systematic approach for the identification and validation of potential drug targets is essential to optimise drug development and translate the promise of upcoming epigenetic agents to routine patient management. Future research should focus on achieving a deeper understanding of epigenetic mechanisms to yield better therapies as well as exploiting therapeutics that promote global epigenetic normalisation to counteract epigenetic aberrations. These approaches will enhance the utility of epigenetic drugs, maximising benefits in terms of returns of research investment and alleviating the burden of cancer on public health.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.