Epigenetic Alterations in Cancer: The Therapeutic Potential of Epigenetic Drugs in Cancer Therapy

Abstract

To date, numerous studies have emerged that indicate the possible role of epigenetic modulation in the development and progression of several diseases, including cancer. Epigenetic alterations participate in various steps of carcinogenesis. They play important regulatory roles in processes like cell division, proliferation, angiogenesis, and metastasis. Thus, epigenetic modifications such as DNA methylation, histone modifications, and non-coding RNAs serve as attractive and promising targets for cancer prevention and anti-cancer therapy. Epigenetic drugs or epi-drugs possess the ability to reverse many such epigenetic alterations and thus can help manage the clinical manifestations of cancer. Epigenetic drugs broadly target epigenetic modifications, including DNA methylation and histone post-translational modifications, to manifest their effects. Several naturally occurring as well as chemically synthesized compounds have been recognized as epigenetic drugs. Some of them are clinically approved, while many are in their preclinical and clinical trials. In this review, we aim to present a broad overview of the epigenetic modifications implicated in carcinogenesis. The review also compiles various epigenetic drugs that are approved for clinical practice, as well as those that are in the preclinical and clinical stages of investigation for anti-cancer therapy.

1. Introduction

It has been well established that both genetic and epigenetic factors play a significant role in the development of disease. While genetic factors modify gene expression by making alterations in the DNA sequence, epigenetic factors contribute to phenotypic changes independent of genetic alterations. More recently, the term epigenetics has referred to the cellular events that control gene expression through structural modifications in chromatin. The correct regulation of such chromatin-based events is crucial for appropriate cell growth and development, and any de-regulation of epigenetic patterns leads to aberrant gene expression, resulting in cancer or other epigenetic diseases. Epigenetic processes are controlled by three distinct mechanisms, viz., DNA methylation, histone tail modifications and small noncoding RNAs or microRNAs [1]. These mechanisms function in a coordinated manner to fine-tune the chromatin structure and thus maintain precise control over gene expression and silencing. In particular, DNA methylation and histone tail modifications regulate the accessibility of transcription factors to the target genes via the modulation of heterochromatin to euchromatin transition and vice-versa [2,3].

Aberrant epigenetic modifications, along with genetic alterations, are key players in the development and progression of several diseases, including cancer [4]. Unlike irreversible genetic alterations or mutations, epigenetic modifications are more dynamic and thus can be reprogrammed to achieve a normal phenotype [5]. This unique and valuable feature makes scientists particularly interested in understanding the molecular features of epigenetic mechanisms. Better molecular insight into epigenetic factors would certainly be valuable in the development of drugs that fix the altered epigenetic regulation of gene expression. Moreover, targeting reversible epigenetic modifications is a more attractive and promising strategy compared to targeting stable genetic mutations, from a therapeutic standpoint [6].

Over the past decade, expanding research into chromatin biology has identified various new drug targets that largely belong to the chromatin regulatory factors. These include enzymes that covalently modify DNA and histones [6], chromatin remodeling complexes [7], and transcription activators or repressors [8]. Since these chromatin regulatory enzymes play crucial roles in epigenetic pathways of cancer and other human pathologies, targeting them with high-efficacy drugs is currently a research area with huge therapeutic relevance. In general, the term epigenetic drugs or epi-drugs is used to define a class of molecules that are used to reprogram the altered epigenome in a diseased state, leading to beneficial clinical manifestations. Currently, various epi-drugs are in clinical use, and many more are undergoing preclinical as well as clinical stages of drug development [9]. In this review, we discuss the current understanding of epigenetic alterations in carcinogenesis. A comprehensive list of the various epi-drugs that are currently in clinical practice for anti-cancer therapy is provided. The list also summarizes recent advances in epi-drugs at various stages of drug development in clinical settings.

2. Epigenetic Modifications

Epigenetics refers to the inherited genetic traits that arise due to alterations in gene expression rather than changes in the DNA sequence itself. An important regulatory mechanism for gene expression involves the temporal and spatial accessibility of DNA to various DNA-binding proteins, which in turn is regulated by the high-order chromatin structure, comprising DNA and histone proteins. The fundamental unit of chromatin is the nucleosome, which is made up of 147 base pairs of DNA wrapped around a histone octamer, comprising two units each of four canonical histones, i.e., H2A, H2B, H3, and H4. To facilitate a genome organization that fits into the nucleus, nucleosomes are arranged on DNA like “beads on a string”, which is further condensed to chromatin fibers and a high-order chromatin structure via inter-nucleosomal interactions (Figure 1) [10]. DNA accessibility is regulated by the degree of condensation of this high-order chromatin structure. Heterochromatin refers to the highly condensed chromatin state, which primarily comprises repetitive DNA sequences and silenced genes and is mostly inaccessible to DNA-binding machinery. In contrast, euchromatin represents de-condensed chromatin regions with actively transcribed genomic regions [11].

The fundamental epigenetic modifications that regulate chromatin accessibility include DNA methylation and post-translational modification of histone tails such as acetylation, methylation, citrullination, and phosphorylation [12]. Additionally, non-coding RNAs control gene expression at the level of protein translation by degrading mRNA or inhibiting its translation (Figure 1) [13]. The continuous interest in the growing field of epigenetics had led to the uncovering of the important role of epigenetic modifications in a multitude of diseases including cancer, neurodevelopmental disorders, neurological diseases, autoimmune diseases, endocrine disorders, and so on.

3. Epigenetic Modifications in Cancer

In cancers of various organs, genetic aberrations work in conjunction with epigenetic alterations—particularly of histone modifications—creating chaos in transcriptional control. Several biological processes are affected by epigenetic dysregulation, including cell proliferation, growth, differentiation, apoptosis, and DNA repair [4]. Unlike genetic perturbations, epigenetic mechanisms control transcription via changes in gene expression levels without compromising DNA sequence integrity [14]. Thus, it is wise to target dysregulated epigenetic functions rather than targeting stable and irreversible genetic mutations from a therapeutic standpoint. Altered histone modifications have been linked to the mis-regulated expression of multiple genes with crucial roles in tumorigenesis [15]. In fact, certain tumors exhibit specific altered histone modifications and DNA methylation patterns even at the individual cellular level. Such cell- and tumor-specific altered profiles of histone modifications act as markers for cancer prognosis as well as predicting the chances of relapse [16]. Moreover, DNA methylation is often observed to be dysregulated in various cancer types. Notably, different subtypes of breast cancer show different profiles of DNA methylation [17].

Because of the well-established link between epigenetic dysregulation and cancer development, various therapeutic strategies have been envisaged that target DNA methyltransferases (DNMT) and histone modifying enzymes, thereby reverting the pathogenic consequences of epigenetic alterations. Histone modifying enzymes are majorly categorized as histone methyltransferases (HMT), histone demethylases (HDM), histone acetyl transferases (HAT), and histone deacetylases (HDAC) [3]. The drugs that target DNA methyltransferase and various histone modifying enzymes are termed as epigenetic drugs or epi-drugs [18].

3.1. DNA Methylation

DNA methylation is a well-defined epigenetic marker that involves a family of enzymes known as DNA methyltransferases (DNMTs). DNMTs catalyze the transfer of methyl groups from S-adenosyl methionine (SAM) to the C-5 position of the pyrimidine ring of cytosine in CpG di-nucleotides in the genome [19]. Four different active isoforms of DNMTs have been identified in mammals, viz., DNMT1, TDRMT1 (or DNMT2), and DNMT3. DNMT1 and DNMT3 are localized in the nucleus, while TDRMT1 functions in the cytoplasm. DNMT1 is the most abundant form that maintains methylation patterns post-replication by predominantly methylating hemimethylated CpG di-nucleotides, whereas DNMT3 methylates both hemimethylated and unmethylated DNA with the same efficiency. Interestingly, TDRMT1 does not methylate DNA, it methylates aspartic acid transfer RNA (tRNA^Asp), despite sharing strong structural similarity with classical DNMTs. DNA methylation also occurs at sites other than CpG sites [20]. DNA methylation in non-CpG sites is enriched at gene bodies and depleted at enhancers and protein binding sites [21].

DNA methylation is an important mechanism for gene silencing, especially of tumor suppressing genes and genes involved in DNA repair processes. It is often observed to be dysregulated in various cancers [22,23,24]. Aberrant DNA methylation, including both site-specific hypermethylation (e.g., CpG islands in gene regulatory elements) and genome-wide hypomethylation (e.g., repetitive sequences and intergenic regions), are common characteristics of cancer cells (Figure 2). Differential DNA methylation patterns may exist in different cancer types of the same tissue. For example, different breast cancer subtypes show different DNA methylation patterns. Aberrant hypermethylation is more prominently observed in estrogen receptor alpha (Erα)-positive breast cancer as compared to Erα-negative subtypes [25]. Abnormal DNA methylation in the promoters of cancer-linked genes often lead to their altered expression, as well as to the inactivation of tumor suppressor genes. One example is gastric cancer, where abnormal methylation occurs in genes associated with transcriptional regulation (like Helicase-like transcription factor), DNA repair (like MGMT and hMLH1), cell growth and differentiation (such as HoxD10), and apoptosis (like BNIP3) [26]. Notably, the hypermethylation of the promoter region of MutL homolog 1 (MLH1), a DNA mismatch repair gene, is observed in sporadic cases of colorectal cancers [27]. Likewise, the hypermethylation of promoters of epithelium-specific genes and the hypomethylation of epithelial to mesenchymal transition (EMT) markers are observed in prostate cancer, resulting in EMT and stemness [28].

3.2. Histone Modifications

The nucleosome, the fundamental unit of chromatin, is composed of 147 base pairs of DNA wrapped around a histone octamer. Each histone octamer has two each of the canonical histone proteins, viz., H2A, H2B, H3, and H4. The N-terminal tails of histones that protrude out from the nucleosome are accessible on its surface for various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, and sumoylation. The enzymes that regulate various histone PTMs include histone methyl transferase (HMT), histone demethylase (HDM), histone acetyltransferase (HAT), histone deacetylase (HDAC), kinases, and E3-ubiquitin [16]. These histone modifications can affect the interaction of histones with DNA strands as well as the interaction of one nucleosome with another nucleosome. They also act as sites for the recruitment of transcription factors or other histone binding proteins such as PHD-, chromo-, and bromo-domain-containing proteins [3,29]. In this way, histone tail modifications can influence the chromatin state, and thus, gene expression. The chromatin organization or architecture depends upon histone configuration. Euchromatin, having a relaxed chromatin structure, facilitates the transcription process and usually contains high-expression genes. In contrast, heterochromatin has a denser configuration and is primarily associated with gene silencing. Notably, both euchromatin and heterochromatin are recognized by specific epigenetic markers. Euchromatin is rich in histone acetylation, whereas heterochromatin shows high methylation levels (Figure 3) [30].

Altered patterns of histone PTMs, both at specific gene loci and at the global genome level, have been implicated in various human cancers [31]. There is a proven role of altered histone modifications in the over-activation of oncogenes or suppression of tumor suppressors (Figure 3). Both transcriptionally active histone markers such as H3K4me3 and H3K36me3 as well as repressive markers including H3K9me2/3, H3K27me3, and H4K20me3 are involved in cancer progression [29]. MLL2, a H3K4 methyltransferase, is involved in breast cancer development; it interacts with estrogen receptor alpha, which regulates key genes involved in breast cancer proliferation [32]. One study also suggests that a H4K4 methyltransferase (MLL4) and a H3K27 demethylase (UTX) coordinate with each other to regulate the transcriptional programs for cell invasiveness in breast cancer MDA-MD-231 cells [33]. In the same line, it has been reported that the inactivation of KMT2C/MLL3, another H3K4 methyltransferase, promotes the development of colorectal cancer through transcriptional dysregulation in various pathways with established cancer relevance [34]. Deregulation of HMTs or HDMs is known to be associated with prostate cancer development and progression. SUV39H2 and SETDB1, which catalyze H3K9me1/2 and H3K9me3, respectively, are upregulated in prostate cancer [35,36]. Similarly, SMYD3, a H3K4 methyltransferase, is overexpressed in prostate, colorectal, hepatocellular, and breast cancer [37,38,39]. Reduced levels of H4K20me3 are found to be associated with HER-2-independent breast cancer. In fact, cancer cell invasiveness is overcome by the ectopic expression of the H3K20 methyltransferases SUV420H1 and SUV420H2 [40].

Functional dysregulation of histone acetyltransferases (HATs), just like HMTs and HDMs, is strongly correlated with carcinogenesis [41]. Mutations in HATs have been reported in various cancers, with involvement at various stages of cancer progression [42,43,44]. Mutations in CREBBP and EP300, two histone acetyltransferases (HATs), are linked to many lymphoid malignancies including acute lymphoblastic leukemia, B-cell lymphoma, and follicular lymphoma [43,45,46]. It has also been observed that the interaction of CBP/p300 with c-Myb activates its transforming potential and induces acute myeloid leukemia [47]. HAT1 is upregulated in pancreatic ductal adenocarcinoma (PDAC) and is associated with poor prognosis. Interestingly, the knockdown of HAT1 does in fact decrease cancer cell proliferation in PDAC both in vitro and in vivo [48]. A large body of growing evidence suggests that overexpression of HDAC occurs in cancers of various organs such as lung cancer, breast cancer, colon cancer, gastrointestinal cancer, neuroblastoma, and many more [49,50,51,52,53]. HDACs regulate various aspects of tumorigenesis ranging from cell growth/differentiation, autophagy, apoptosis, and angiogenesis to prognosis and chemo-resistance [54,55,56].

Other common post-translational modifications of histone proteins include ubiquitination and SUMOylation. Histone ubiquitination involves the addition of ubiquitin protein molecules onto target histone tails. Histones are frequently modified with one ubiquitin molecule (mono-ubiquitination), but can also acquire ubiquitin chains (polyubiquitination). The addition of ubiquitin to the histones is a reversible modification carried out by E3 ubiquitin ligases and ubiquitin-specific peptidases (deubiquitinating enzymes (DUBs)). It plays important roles in DNA replication, gene transcription, and DNA damage repair. It also affects the chromatin state as well as regulating the recruitment of repair proteins at DSB sites [57]. Moreover, histone ubiquitination regulates other histone PTMs including methylation and acetylation to preserve genome integrity. Dysregulation of histone ubiquitination is often observed in cancer and can promote cancer development by altering the expression of oncogenes and tumor suppressors. One example is the elevated expression of H2AK119ub1 in pancreatic ductal adenocarcinoma as compared to normal tissues. Interestingly, the tumors show reduced levels of H3K27Me3, indicating the importance of the intricate cross-talk between histone ubiquitination and methylation in carcinogenesis [58]. In gastric cancer, triple-negative breast cancer, and colorectal cancer, a loss of H2BK120ub has been observed [59,60,61]. Notably, the genes encoding E3 ubiquitin ligases are frequently mutated in cancer. E3 ligases regulate major growth-promoting pathways such as the MAPK or PI3K–AKT–mTOR pathways. Thus, dysregulated E3 ligases drive uncontrolled proliferation and genomic instability, which promote malignant transformation and tumor progression [62].

Another histone modification, i.e., SUMOylation, refers to the reversible addition of a small ubiquitin-like modifier (SUMO) moiety to a lysine residue on the histone protein. It is carried out by three principle enzymes over three main steps: activation via SUMO E1, conjugation via SUMO E2, and ligation via SUMO E3 [63]. SUMO-specific proteases (SENPs) can reverse this modification. There are seven SENPs identified in humans, of which, SENP5 is observed to be upregulated in oral cancer compared to normal epithelial cells [64]. Furthermore, Katayama et al. has shown that SUMO-1 is highly overexpressed in human oral squamous carcinoma and contributes to tumor cell proliferation [65]. In another study, Shiio et al. demonstrated that SUMOylation promotes transcriptional repression via recruitment of heterochromatin protein 1 and histone deacetylases [66]. SUMOylation regulates the functional activity of many of the transcription factors governing transitions between epithelial and mesenchymal states, which points towards the important role of this modification in tumor growth and metastasis [67,68]. Given the important role of histone modifying enzymes in cancer development and progression, they serve as key targets of epigenetic drugs.

3.3. Non-Coding RNAs

Recently, several studies have emerged that suggest that processes other than the deregulation of DNA methylation and histone modifications contribute to tumorigenesis and its progression. One such well-defined process is the altered expression of non-coding RNAs (ncRNAs). They are divided into two main classes based on the length of the nucleotides, viz., short ncRNAs (<200 nucleotides) and long ncRNAs (>200 nucleotides). Short ncRNAs include microRNA (miRNA), germline-restricted PIWI-interacting RNA (pi-RNA), and small-interfering RNA (siRNA). Of all these, miRNAs are the best studied and serve as key players in epigenetics, playing important roles in cell proliferation and differentiation [69].

3.3.1. MicroRNAs

MicroRNAs are ~22 nucleotides long and complementary to messenger RNA, which is obtained via the processing of short RNA hairpins. miRNA does not translate into proteins; instead, it downregulates or inhibits the translation of mRNA that bears complementary sequences [70]. About 60% of protein coding genes in the human genome are targeted by miRNAs [71]. miRNA targets the 3′-UTR region of the target mRNA and inhibits its translation via mRNA cleavage and degradation.

Various recent studies have suggested that miRNAs play a key role in various regulatory processes such as cell division, proliferation, and apoptosis. Abnormal expression of miRNAs is observed in several human diseases such as cardiovascular diseases, various inflammatory diseases, autoimmune diseases, rare genetic disorders, and cancers of various organs [72,73,74]. OncomiRs are miRNAs that are associated with carcinogenesis and metastasis. In normal cells, some oncomiR genes function as oncogenes in the sense that they lead to the development of cancer upon overexpression. One example is miR-155, which is upregulated in breast cancer [75]. Strikingly, some other oncomiR genes function as tumor suppressors, and their downregulation promotes cancerous growth (Figure 4). One example is the downregulation of miR-15a and miR-16-1, which negatively regulate the anti-apoptotic gene BCL2 in chronic lymphocytic leukemia patients [76]. Also, miRNAs show differential expression patterns in cancerous tissues as well as in the blood of cancer patients [77]. Some of the miRNAs that are overexpressed and contribute to tumor growth include miR-10b, miR-16, miR-21, miR-34a, miR-155, and miR-221/222 [75,78,79,80,81,82]. miRNAs can also indicate the presence and clinical stage of the tumor. Hence, they serve as key diagnostic biomarkers for cancer and offer new paths for the development of cancer therapies.

3.3.2. Long Non-Coding RNA

Another type of ncRNA, i.e., long non-coding RNA (lncRNA), is a very heterogeneous class and includes RNAs with both housekeeping and gene regulatory functions. Just like miRNAs, lncRNAs interact with the DNA via sequence complementarity and form non-canonical, triple-stranded structures such as RNA:DNA triplexes, R-loops, and G-quadruplexes (Figure 5) [84]. RNA:DNA triplexes bind directly to the major groove of the DNA double helix via Hoogsteen hydrogen bonding following base-pairing like Watson–Crick interactions. Thus, RNA:DNA triplex-forming lncRNAs act as sequence-specific labels that target their protein partners to specific genomic locations [85]. In fact, various studies have suggested that lncRNAs form stable ‘domains’ for protein binding and chromatin localization, which enables the sequence-specific localization of chromatin modifiers or transcription factors. This in turn alters the epigenetic signatures and thus regulates gene expression at distinct genomic sites [86]. Another non-canonical three-stranded nucleic acid structures are R-loops, which consist of a DNA–RNA hybrid and a displaced single-stranded DNA [84]. They are formed during transcription when the newly synthesized RNA transcript remains bound to its DNA template [87]. These DNA:RNA hybrids nucleate from G-rich clusters in the DNA template and extend across GC-rich spans of transcribed genes during elongation. R-loops are very stable structures and thus need to be resolved enzymatically to restore the native double helix DNA. A balance between the constant formation and removal of R-loops creates a homeostasis at nucleation sites throughout the genome. An alteration in R-loop homeostasis leads to their accumulation in cells. This promotes genomic instability significantly, which is often associated with neurodegenerative disorders and cancer (Figure 5) [88].

LncRNAs affect various cellular properties such as differentiation, proliferation, cell cycle, apoptosis, invasion, and migration, suggesting that they play an important regulatory role in cancer development [89]. For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is one of the best-characterized druggable lncRNAs found to be dysregulated in multiple human malignancies [90]. The interaction of MALAT1 with zeste homolog 2 (EZH2) downregulates E-cadherin expression and promotes cell metastasis in osteosarcoma [91]. Likewise, MALAT1 plays an important role in the development of malignant esophageal squamous cell carcinoma (ESCC) via the targeting of β-catenin and Lin28 via Ezh2 [92]. The upregulation of MALAT1 is also observed in various hematological malignancies, including multiple myeloma and lymphoma, and is often associated with disease progression and reduced overall survival of patients [90]. Lung cancer-related transcript 1 (LUCAT1) is another lncRNA involved in various malignant tumor types such as liver cancer, esophageal cancer, lung cancer, thyroid cancer, ovarian cancer, breast cancer, gastric cancer, and renal cell carcinoma. LUCAT1 is overexpressed in esophageal squamous cell carcinoma (ESCC), which downregulates the tumor suppressor genes GADD45G and SFRP2 by stabilizing DNMT1, thereby stimulating the progression of ESCC [93]. Recently, a lncRNA growth arrest-specific 5 (GAS5) has gained attention as it functions as a tumor suppressor with significantly low expression levels in several malignancies, including breast cancer, gastric cancer, cervical cancer, non-small cell lung cancer (NSCLC), ovarian cancer, renal cancer, colorectal cancer, and so on [94]. GAS5 regulates multiple cellular functions such as cell proliferation, migration, EMT, apoptosis, and migration through multiple mechanisms. These include transcriptional regulation of GAS5 through microRNA sponges, binding to target mRNAs, and riborepression of steroid (glucocorticoid) hormones, thereby regulating the expression of both coding and non-coding genes at the transcriptional and translational levels [95].

R-loops have been found to be associated with various malignancies. They strikingly affect cancer progression and therapeutic efficiency by regulating crucial genes such as BRCA1/2, TOP1, PARP, DHX9, and sex hormone receptors [96]. The progressing R-loop collides with the replication fork, leading to fork stalling or reversal, leading to DNA single or double- stranded breaks and threatening genomic stability [97]. BRCA1 binds to R-loops that are formed over a subset of termination regions (TRs) of actively transcribed genes. It then mediates the recruitment of the R-loop resolving protein Senataxin (SETX). Various insertion/deletion mutations of these genes around TRs are observed in BRCA1-mutated breast tumors. This suggests that BRCA1/SETX functions as a DNA repair mechanism that resolves R-loop-mediated DNA breaks at transcription termination/pause sites, thereby halting tumorigenesis [95,98].

RNA G-quadruplexes (rG4s) are another structural motif in lncRNAs that have gained immense attention recently due to their varying roles in cell behavior and disease pathogenesis. The four guanines in G-rich RNA connect via Hoogsteen hydrogen bonds to form a square planar G-quartet structure. Several G-quartets stack on top of one another to create non-canonical structures called G-quadruplexes (G4s) [99,100]. In the nucleus, rG4s play an important role in the splicing of mRNA and ncRNAs, as well as in transcriptional regulation. Sequential G-tracts within the R-loops can also promote the formation of rG4s and inhibit transcription [101]. rG4s regulate mRNA translation, stability, and RNA biogenesis in the cytoplasm. The formation of rG4 in the 3′-UTR prevents the miRNA binding, thereby regulating RNA interference and promoting RNA stability [102]. In the genome, G4 structures are enriched at transcription factor binding sites, promoters, and telomeres, and play regulatory roles in protein expression, genome stability, and telomerase activity. In around 90% of all cancer cells, telomerase activity is upregulated, which enables the cell to replicate without the shortening of telomeres [103]. The formation of G4 structures at telomeres prevents telomerase binding and blocks its activity [104]. This strategy can be applied in the therapeutic intervention of cancer, wherein G4 formation blocks the telomerase activity in tumor cells at the telomeric region, thereby preventing uncontrolled DNA replication. Since somatic cells lack telomerase, they do not get affected. G4s are also described to be formed in the promoter regions of several oncogenes such as c-Myc and K-Ras, thereby playing a potential role in regulating gene expression and contributing to cancer development. One therapeutic approach is to stabilize G4s using ligands or oligonucleotide aptamers. The stabilization of these structures hinders the replication and transcription processes, as polymerases face steric clashes while moving through these stabilized structures [105,106]. Several chemical agents are known to stabilize G4 structures in the telomeres and promoters of oncogenes, and thus, they can be used as promising therapeutics; the list includes pyridostatin, TMPyP4, MM41 (naphthalenediimide derivative), and GQC-05 (ellipticine analog). These molecules bind to G4 structures through π-π stacking interactions or ionic coordination and stabilize their unique topology [107].

G4 ligands that tend to stabilize G4 structures can be used as potential anti-cancer drugs; however, no G4 binder has yet been approved in clinical trials [108]. G4 ligands bind to G4 motifs and affect cancer cells in multiple ways: These include (1) inhibition of telomerase function; (2) regulation of oncogene expression via stabilization of G4 motifs at their promoters; (3) gene regulation via targeting of the mRNA 5′-untranslated region; or (4) regulation by blocking the helicase unwinding function [108,109,110,111].

There are three fundamental strategies to targeting lncRNA for therapeutic intervention: These include the use of (a) antisense oligonucleotides (ASOs) that bind to lncRNAs, thereby inducing their degradation by RNaseH1; (b) small interfering RNAs (siRNAs) (20–25 nucleotides long) that bind target lncRNAs based on sequence complementarity and promote its degradation via Argonaute endonucleases; and (c) small molecules that are targeted to a specific lncRNA and inhibit it binding to its molecular partner or interfere with correct structure formation [112].

4. Epigenetic Drugs

Unlike mutations in DNA, epigenetic modifications are reversible. They also play important regulatory roles in processes like cell division, proliferation, angiogenesis, and metastasis. Altogether, this makes these epigenetic modifications, i.e., DNA methylation, histone modifications, and miRNA, attractive and promising targets for cancer prevention and therapy (Figure 6). Several naturally occurring as well as chemically synthesized compounds have been recognized as epigenetic modifiers or epi-drugs. They target enzymes that catalyze various epigenetic modifications such as DNMT, HDAC, HDM, HAT, HMT, Ubiquitin ligases, and non-coding RNAs (Figure 6). Mutations in these enzymes result in the altered expression of important regulatory genes involved in carcinogenesis, including the re-expression of tumor-suppressor genes. Various emerging studies have shown that epigenetic alterations regulate the tumor microenvironment, which can influence the response to chemo/immunotherapy.

Table 1. List of epi-drugs targeting DNMTs in cancer therapy.

Modification	Epi-Drug	Target	Cancer	Clinical Phase	Other Drug(s)/Intervention(s)	Outcome(s)/Mechanism	Brand Name/Trial ID/Reference(s)
DNMT	Decitabine (5-aza-2′-deoxycytidine)	DNMT1	MDS, CML	FDA approved, 2020	In combination with cedazuridine	Prolonged overall survival	INQOVI, Astex Pharmaceuticals, Inc. [118] NCT02103478 [119] NCT03306264 [120]
	Decitabine	DNMT1	High risk MDS, CML	Phase 1/2 study	In combination with cedazuridine and venetoclax	Higher response rate in short period of time; displays tolerable toxicity and satisfactory activity	NCT04655755 [121]
	Decitabine	DNMT1	MDS	Phase 3	-	Higher overall response rate, prolonged survival; well-tolerated dosage, with a manageable toxicity profile	[122]
	5-fluoro-2′-deoxycytidine	DNMT1	Breast cancer and other solid tumors	Phase 2	-	Arrest cell cycle at G2/M phase; activates p53 signaling and DNA damage response pathway; upregulates tumor suppressor genes	[123]
	Guadecitabine (SGI-110)	DNMT1	AML, MDS, CMML	Phase 1/2	-	Well-tolerated with clinical and biological activity	NCT01261312 [124,125,126,127,128]
	Guadecitabine	DNMT1	AML, MDS, CMML	Phase 1/2	In combination with Atezolizumab	Acceptable tolerance and clinically active	NCT02935361 [124]
	Guadecitabine	DNMT1	Platinum refractory germ cell cancer	Phase 1	In combination with Cisplatin	Exhibits good tolerance and demonstrates clinical activity	NCT02429466 [129]
	Guadecitabine	DNMT1	AML	Phase 1b	In combination with Atezolizumab	Limited clinical activity and an overall unfavorable benefit–risk profile at tested doses	NCT02892318 [130]
	Guadecitabine	DNMT1	Solid tumors (NSCLS)	Phase 1	In combination with Pembrolizumab	Tolerable, with biological and anticancer activity	NCT02998567 [131]
	Guadecitabine	DNMT1	Colorectal cancer	Phase 1	In combination with Cy/GVAX (cyclophosphamide with GM-CSF secreting colon vaccine)	Tolerable, but no significant immunologic activity	NCT01966289 [132]
	Guadecitabine	DNMT1	Melanoma	Phase 1b	In combination with ipilimumab (anti-CTLA-4 antibody)	Safe and tolerable, with initial signs of clinical and immunologic activity	NCT0260843 [128,133]
	Guadecitabine	DNMT1	SCLC	Phase 2	In combination with carboplatin	Exhibits good efficacy along with possible adverse events	NCT03913455 [134]
	Guadecitabine	DNMT1	Urothelial carcinoma	Phase 2	In combination with atezolizumab	Possible prolonged patient survival	NCT03179943 [135]
	Guadecitabine	DNMT1	Ovarian, Primary Peritoneal, or Fallopian Tube Cancer	Phase 2	In combination with Pembrolizumab	Exhibits clinical activity and possibly activates antitumor immunity	NCT02901899 [136]
	Guadecitabine	DNMT1	Chondrosarcomas	Phase 2	In combination with Belinostat or ASTX727	Active trial; not recruiting	NCT04340843 [137]
	5-Azacitidine	DNMT1	MDS, AML, CMML	FDA approved, 2004; EMEA approved 2009	-	Satisfactory safety profile and provides clinical benefit	Vidaza®; Celgene Corp., Summit, NJ, USA [138]
	MG98	DNMT1	Renal carcinoma	Phase 2 (terminated)	-	Exhibits no anti-cancer activity	[139]
	MG98	DNMT1	Solid tumors	Phase 1	-	Shows early evidence of clinical activity with good tolerance	NCT00003890 [140]
	Hydralazine	DNMT1/3A/3B	Refractory solid tumors	Phase 2	In combination with Magnesium valproate	Shows clinical benefits with potential to overcome resistance to chemotherapy	NCT00404508 [141]
	Hydralazine	DNMT1/3A/3B	Hepatocellular carcinoma	Phase 2	In combination with valproic acid	Exhibits manageable toxicity and clinical efficacy	TPVGH97-07-07 [142]
	Hydralazine	DNMT1/3A/3B	Breast cancer	Phase 2 (terminated)	In combination with Magnesium valproate	Well-tolerated and appears to increase the efficacy of chemotherapy	NCT00395655 [143]
	Hydralazine	DNMT1/3A/3B	Cervical cancer	Phase 3	In combination with Magnesium valproate; placebo-controlled	Unknown	NCT00532818 (Unpublished)
	Hydralazine	DNMT1/3A/3B	Ovarian cancer	Phase 3	In combination with Magnesium valproate; placebo-controlled	Unknown	NCT00533299 (Unpublished)
	Epigallocatechin-3-gallate (EGCG)	DNMT	Colon cancer (HT-29 cells), esophageal cancer (KYSE 150 cells), and prostate cancer (PC3 cells)	Preclinical phase	-	Reactivation of some methylation-silenced genes	[144]
	Resveratrol-salicylate derivatives	DNMT3	Colon cancer (HT-29), liver cancer (HepG2) and breast cancer (SK-BR-3)	Preclinical phase	-	Unknown	[145]
	SGI-1027 (4-anilinoquinoline)	DNMT1/3A/3B	Hepatocellular carcinoma (Huh7)	Preclinical phase	-	Reactivates silenced tumor suppressor genes and induces apoptosis in cancer cells	[146]
	Laccaic acid A (LCA)	DNMT1	Breast cancer (MCF-7)	Preclinical phase	-	Reactivates the expression of silenced tumor suppressor genes	[147]
	RG-108	DNMT1	Colorectal cancer (HCT116) and acute lymphoblastic leukemia (NALM-6)	Preclinical phase	-	Reactivates key tumor suppressor genes	[148]
	Nanaomycin A	DNMT3B	Lung cancer (A549), leukemia (HL60), and colorectal cancer (HCT116)	Preclinical phase	-	Reactivates silenced tumor suppressor genes	[149]

,

Table 2. List of epi-drugs targeting histone modifying enzymes in cancer therapy.

Modification	Epi-drug	Target	Cancer	Clinical Phase	Other drug(s)/Intervention(s)	Outcome(s)/Mechanism	Brand Name/Trial ID/Reference(s)
HDAC	Vorinostat (suberoylanilide hydroxamic acid)	Pan HDAC	Cutaneous T-cell lymphoma (CTCL)	FDA approved 2006	-	Shows clinical benefit	Zolinza®; Merck & Co., Inc., Whitehouse Station, NJ [150]
	Belinostat	Pan HDAC	Peripheral T cell lymphoma (PTCL)	FDA approved 2006	-	Shows clinical benefit	Beleodaq; Spectrum Pharmaceuticals, Inc. [151]
	Romidepsin	HDAC I	T-cell lymphoma (both CTCL and PTCL)	FDA approved	-	Shows clinical benefit	Istodax, Celgene Corporation [152]
	Pracinostat (SB939)	HDAC I/II/IV	Leukemia, solid tumors	Phase 1	-	Unknown	NCT01184274 (Unpublished)
	Pracinostat	HDAC I/II/IV	Solid tumors, MDS, hematologic malignancies	Phase 1	Alone or in combination with azacitidine	Safe, with modest single-agent activity	NCT00741234 [153]
	Pracinostat	HDAC I/II/IV	Solid malignancies	Phase 1	-	Well-tolerated and show inhibitory effects	SCS-PN0022 [154]
	Pracinostat	HDAC I/II/IV	Solid tumors	Phase 1	-	Shows good tolerability	[155]
	Pracinostat	HDAC I/II/IV	MDS	Phase 2	In combination with azacitidine and decitabine	Exhibits improved efficacy and tolerance at reduced doses	NCT01993641 [156]
	Pracinostat	HDAC I/II/IV	MDS	Phase 2	In combination with azacitidine	Fails to improve outcomes with high rate of treatment discontinuation	NCT01873703 [157]
	Pracinostat	HDAC I/II/IV	Myelofibrosis	Phase 2	-	Exhibits modest tolerability and clinical activity	NCT01200498 [158]
	Pracinostat	HDAC I/II/IV	Translocation-associated sarcoma (TAS)	Phase 2	-	Stopped prematurely due to prolonged unavailability of pracinostat	NCT01112384 [159]
	Pracinostat	HDAC I/II/IV	AML	Phase 3	In combination with azacitidine	Terminated due to lack of clinical response	NCT03151408 [160]
	Panobinostat (LBH-589)	HDAC I/II/III/IV	Multiple myeloma	FDA and EMA approved	In combination with bortezomib and dexamethasone	Potential inhibitory activity	Farydac® (Novartis) [161,162]
	Resminostat	HDAC I/IIb/IV	Biliary tract or pancreatic cancer	Phase 1	In combination with chemotherapy (S1)	Well-tolerated	JapicCTI-152864 [163]
	Resminostat	HDAC I/IIb/IV	CTCL, MF	Phase 1	-	Unknown	NCT04955340 (Unpublished)
	Resminostat	HDAC I/IIb/IV	Hodgkin lymphoma	Phase 2	-	Exhibits acceptable tolerance and clinical activity	NCT01037478 [164]
	Resminostat	HDAC I/IIb/IV	Hepatocellular carcinoma	Phase 1/2	In combination with sorafenib	Display early signs of efficacy and well-tolerated	NCT00943449 [165]
	Resminostat	HDAC I/IIb/IV	NSCLC	Phase 1/2	In combination with docetaxel	Fails to improve progression-free survival and increases toxicity	JapicCTI-132123 [166]
	Resminostat	HDAC I/IIb/IV	Hepatocellular carcinoma cells	Preclinical phase	In combination with sorafenib	Blocked platelet-induced hepatocellular carcinoma cell invasion	[167]
	Quisinostat	HDAC I/IIb/IV	Solid Malignancies and Lymphoma	Phase 1	-	Intermittent schedules show better tolerance than continuous schedules	NCT00677105 [168]
	Quisinostat	HDAC I/IIb/IV	Multiple myeloma	Phase 1	In combination with dexamethasone and bortezonib	Shows clinical efficacy and safe tolerance	NCT01464112 [169]
	Quisinostat	HDAC I/IIb/IV	CTCL	Phase 2	-	Shows an acceptable safety profile	NCT01486277 [170]
	CDX101	HDAC I	Lymphoma or advanced solid organ cancers	Phase 2a	-	Shows acceptable tolerance with clinical efficacy	NCT01977638 [171]
	CDX101	HDAC I	Colorectal carcinoma	Phase 2	In combination with nivolumab	Well tolerated and efficacious	NCT03993626 [172]
	Abexinostat	Pan HDAC	NSCLC, melanoma, urothelial carcinoma, squamous cell carcinoma of head and neck	Phase 1	In combination with pembrolizumab	Unknown	NCT03590054 (Unpublished)
	Abexinostat	Pan HDAC	Multiple myeloma, Hodgkin, and non-Hodgkin lymphoma	Phase 1	-	Unknown	NCT01149668 (Unpublished)
	Abexinostat	Pan HDAC	Solid tumors	Phase 1	In combination with pazopanib	Shows good tolerability and anticancer effects	NCT01543763 [173]
	Abexinostat	Pan HDAC	Follicular lymphoma	Phase 2	-	Unknown	NCT03934567 NCT03600441 (Unpublished)
	Abexinostat	Pan HDAC	Non-Hodgkin lymphoma	Phase 1/2	-	Unknown	NCT04024696 (Unpublished)
	Abexinostat	Pan HDAC	Follicular lymphoma or mantle cell lymphoma.	Phase 1/2	-	Well-tolerated and exhibits significant clinical activity	NCT00724984 [174]
	Abexinostat	Pan HDAC	Sarcoma	Phase 1/2	In combination with doxorubicin and GCSF	Shows manageable toxicity and clinical response	NCT01027910 [175]
	Abexinostat	Pan HDAC	CML, Hodgkin and non-Hodgkin lymphoma	Phase 1/2	-	Exhibits manageable toxicity and partial clinical response	EudraCT 2009-013691-47 [176,177]
	Chidamide (Epidaza)	HDAC I	Peripheral T-cell lymphoma	Approved in China	-	Exhibits clinical benefit	[178]
	AR42	Pan HDAC	Neurofibromatosis type 2-associated tumors and advanced solid malignancies	Phase 1	-	Safe and well-tolerated	NCT01129193 [179]
	AR42	Pan HDAC	AML	Phase 1	In combination with decitabine	Exhibits multi-organ failure as severe adverse effect	NCT01798901 [180]
	AR42	Pan HDAC	Multiple myeloma	Phase 1	In combination with pomalidomide	Unknown	NCT02569320 (Unpublished)
	AR42	Pan HDAC	Neurofibromatosis type 2	Phase 2/3 (recruiting)	Placebo-controlled	Unknown	NCT05130866 (Unpublished)
	Entinostat	HDAC I	Castration-resistant prostate cancer	Phase 1	In combination with enzalutamid	Shows acceptable safety profile	NCT03829930 [181]
	Entinostat	HDAC I	Acute leukemias	Phase 1	-	Effective inhibition of HDAC in vivo	NCT00015925 [182]
	Entinostat	HDAC I	Solid tumors and lymphoid malignancies	Phase 1	-	Shows good tolerability at the tested doses	NCT00020579 [183]
	Entinostat	HDAC I	Breast cancer	Phase 2	In combination with exemestane; placebo-controlled	Shows acceptable safety	NCT03291886 [184]
	Entinostat	HDAC I	NSCLC	Phase 1/2	In combination with pembrolizumab	Exhibits clinical benefit	NCT02437136 [185]
	Entinostat	HDAC I	HR-positive breast cancer	Phase 3	In combination with exemestane; placebo-controlled	Unknown	NCT03538171 [186]
HDM	GSK2879552	LSD 1	AML	Phase 1 (terminated)	Alone or in combination with All-Trans Retinoic Acid (ATRA)	Exhibits toxicity and adverse effect	N CT02177812 (Unpublished)
	GSK2879552	LSD 1	SCLC	Phase 1 (terminated)	-	Shows many adverse effects with poor disease control	NCT02034123 [187]
	INCB059872	LSD1	Solid Tumors and Hematologic Malignancy	Phase 1/2 (terminated)	Alone or in combination with ATRA, azacitidine, and nivolumab	Unknown	NCT02712905 [188]
	Tranylcypromine	LSD1	AML	Phase 1/2	In combination with ATRA	Exhibits clinical response with acceptable toxicity	NCT02261779 [189]
HMT	Tazemetostat	EZH2	Epithelioid sarcoma (ES), follicular lymphoma	FDA approved 2020	-	Exhibits clinical benefit	TAZVERIK, Epizyme, Inc.
	MAK683	EED/PRC2	Diffuse large B-cell lymphoma (DLBCL) and epithelioid sarcoma (ES)	Phase 1/2	-	Well-tolerated with observed clinical activity	NCT02900651 [190]
	Pinometostat (EPZ-5676)	DOT1L	Hematologic malignancies, leukemia	Phase 1/2	-	Shows inhibition of tumor growth	NCT02141828, NCT01684150 [191]
	GSK3326595	PRMT5	MDS, CMML, AML	Phase 1/2	-	Exhibits limited clinical activity	NCT03614728 [192]
HAT	PU139 and PU141	Pan HAT	Neuroblastoma	Preclinical phase	-	PU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300, whereas PU141 is selective toward CBP and p300; Blocks growth of SK-N-SH neuroblastoma xenografts in mice	[193]
	Spiro Oxazolidinediones derivatives	EP300/CBP histone acetyltransferase	Lung squamous cell carcinoma cell line LK2-xenografted mouse model	Preclinical phase	-	Inhibits acetylation of H3K27 in the human lung cancer cell line LK2	[194]
	DCH36_06 (thiobarbituric acid derivative)	p300/CBP	Leukemia cell lines	Preclinical phase	-	Shows anti-tumor activity in leukemia xenograft	[195]
	C646 and its derivatives	p300	NSCLC (A549, H460 and H157 cells)	Preclinical phase	-	Radio sensitization of NSCLC cells by enhancing mitotic catastrophe through the abrogation of G2 checkpoint maintenance	[196]
	EML425	KAT3 (p300/CBP)	Leukemia (U937 cells)	Preclinical phase	-	Cell cycle arrest in G0/G1 phase	[197]
	A-485	p300/CBP	Hematological malignancies, androgen receptor-positive prostate cancer	Preclinical phase	-	Shows potent anti-tumor activity	[198]
Ubiquitin ligase	Tosyl-L-arginine methyl ester (TAME)	Anaphase-promoting complex (APC)	-	Preclinical stage	-	Induces mitotic arrest and cell death (xenopus cell extract)	[199]
	Nutlin, RG7112 (RO5045337)	MDM2	Advanced Solid Tumors and hematologic neoplasms	Phase 1	-	Inhibits p53-MDM2 interaction; induces cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts	NCT00559533, NCT00623870 [200]
	Idasanutlin, RG7388 (RO5503781)	MDM2	AML	Phase 3	In combination with cytarabine; placebo-controlled	Exhibits good clinical response with acceptable toxicity	NCT02545283 [201]
	AMG 232	MDM2	Breast carcinoma, malignant solid tumor, multiple myeloma	Phase 1	-	Exhibit adverse events, dose limiting toxicities, and clinically significant changes in vital signs	NCT01723020 [202]
	SAR405838	MDM2	Solid tumors	Phase 1	In combination with pimasertib	Unknown	NCT01985191 (Unpublished)

and

Table 3. List of epi-drugs targeting small non-coding RNAs (microRNAs) in cancer therapy.

Modification	Epi-Drug	Target	Cancer	Clinical Phase	Other Drug(s)/Intervention(s)	Outcome(s)/Mechanism	Brand Name/Trial ID/Reference(s)
miRNA	MRG-106 (Cobomarsen)	miR-155	CTCL, mycosis fungoides (MF) subtype	Phase 2 (terminated)	Vorinostat	Unknown	NCT03713320 (Unpublished)
	MRG-106	miR-155	Mycosis Fungoides MF, CLL, DLBCL or ATLL	Phase 1	-	Unknown	NCT02580552 (Unpublished)
	MRG-106	miR-155	CTCL, MF subtype	Phase 1 (Terminated)	-	Unknown	NCT03837457 (Unpublished)
	TargomiRs	miR-16	Malignant pleural mesothelioma (MPM) and NSCLC	Phase 1	-	Well-tolerated with early signs of clinical activity	NCT02369198 [203]
	MRX34	miR-34a	Liver cancer, SCLC, lymphoma, melanoma, MM, RCC, NSLCL	Phase 1 (Terminated)	-	Exhibits serious adverse effects	NCT01829971 [204]
	INT-1B3	miR-193a-3p	Advanced solid tumors	Phase 1	-	Potential clinical benefit	NCT04675996 [205]
	MRG-110	miR-92a-3p	Healthy adults	Phase 1	Placebo-controlled	De-repression miR-92a targets	NCT03494712 [206]
	MRG-201	miR-29b	Keloid	Phase 2	Placebo-controlled	Exhibits clinical response and manageable adverse effects	NCT03601052 (Unpublished)
	RGLS5579	miR-10b	Glioblastoma multiforme	Preclinical phase	-	Improved survival in animal model	[207,208]
	TTX-MC138	miR-10b	Metastatic breast cancer	Preclinical phase	-	Shows early signs of clinical activity	[209]

lists various epi-drugs as cancer therapeutics with their clinical status. Some of them are clinically approved, while many are in preclinical and clinical trials.

Table 4. List of epi-drugs targeting long non-coding RNAs in cancer therapy.

Drug Module	Target	Cancer	Reference
Antisense oligonucleotides (ASOs)	MALAT1	Lung cancer, breast cancer, multiple myeloma	[210,211]
	HOTAIR	Lung cancer	[212]
	TUG1	Glioma, glioblastoma, pancreatic cancer	[213,214]
	AC104041.1	Head and neck cancer	[215]
	NEAT1	Neuroblastoma, multiple myeloma, colorectal cancer	[216,217]
	GCAWKR, CHiLL1	Lung cancer	[218]
	LLNLR-299G3.1	Esophageal cancer	[219]
siRNAs	HOTAIR	Breast cancer	[220]
	MALAT1	Lung, cervical, esophageal, and colorectal cancer, osteosarcoma, glioblastoma and lymphoma	[112,221]
	CRNDE	Acute promyelocytic leukemia	[222]
	CCAT1	Gastric, bladder and colorectal cancer	[223,224]
	lncRNA-ATB	Liver cancer	[225]
Small molecules	XIST	Breast cancer	[226]
	MALAT1	Breast cancer	[227]

shows a list of lncRNAs targeted via these three strategies, viz., ASOs, siRNAs, or small molecules. Interestingly, it has been observed that epigenetic drugs that are either approved or undergoing clinical trials modulate the tumor immune microenvironment, thereby inducing a strong antitumor immune response [113,114,115,116,117].

Many therapies with epi-drugs have demonstrated encouraging results against hematological malignancies, but have also showed limited efficacy and poor clinical response against solid tumors [228]. This is partly attributed to the fact that solid tumors tend to originate from more differentiated or even terminally differentiated cells with a reduced capacity for epigenetic reprogramming [229]. Additionally, the first-generation epi-drugs were developed using a ‘one size fits all’ approach without considering biomarkers for patient selection. This probably hindered the development of first-generation and second-generation epi-drugs, which are almost exclusively DNMT or HDAC inhibitors. However, some success has now been achieved with third-generation epi-drugs, which have been developed following a precision-medicine approach [229].

Another major drawback with epi-drugs is their non-selectivity, which leads to severe toxic side effects [230]. Many of the first-generation DNMT and HDAC inhibitors are not biomarker-driven and target broadly all their isoforms. This lack of specificity causes severe toxicity in subjects/patients. Consequently, many clinical trials that have aimed to evaluate the efficacy of epi-drugs on solid tumors have either failed or terminated early. Early attempts have also been made to develop isoform-specific inhibitor molecules, but these were also largely disappointing, particularly under monotherapy settings [228]. This further necessitates the rigorous work needed to be done in the development of more selective inhibitors that specifically target single isoforms or proteins in a family. The therapeutic impact of the drug can be immensely enhanced by focusing on selective inhibition with fewer unwanted interactions.

Epi-drugs have also been tested in combination with other anticancer agents (chemotherapy, immunotherapy, radiotherapy, targeted therapy), but this has also not improved clinical outcomes significantly. Advancements in the field of precision medicine might unlock the potential of epi-drugs by mitigating their non-selective or off-target side effects [229]. The development of sophisticated drug delivery systems based on lipid-based nanoparticles or targeted delivery vehicles could improve the specificity and efficacy of epi-drug-based therapies by directing them to precise intracellular sites. Lipid nanoparticles elicit a range of benefits, including biocompatibility, biodegradability, and entrapment effectiveness. The use of liposomes, dendrimers, and small-molecule drug conjugates can also be explored as delivery systems to unleash their full potential for targeted drug delivery. The critical investigation of lower doses, sequential scheduling, and targeted delivery may certainly improve the therapeutic potential of epi-drugs.

5. Summary and Future Perspectives

Cancer is a complex disease that involves several factors. Various genetic alterations like gene fusion, deletion, duplication, and mutations in tumor suppressors and oncogenes are closely related to cancer development. Additionally, epigenetic mechanisms, as discussed in the review, also play an important role in carcinogenesis and metastasis. Genetic aberrations are irreversible and are difficult to manage or treat. Given that targeting dynamically reversible epigenetic alterations appears to be a more attractive and promising strategy to improve cancer therapy, some epigenetic drugs have been approved by the FDA and are currently in clinical use for the treatment of various hematological malignancies. Furthermore, many naturally occurring as well as chemically synthesized molecules are under investigation in preclinical and clinical studies. Although epi-drugs have shown better clinical outcomes in combination with other anticancer therapies including chemotherapy, immunotherapy, and radiotherapy, several issues must be addressed. Advancements in the field of precision medicine might unlock the potential of epi-drugs by mitigating their non-selective or off-target side effects. Improvements in drug engineering, drug targeting, delivery methods, and the designing of clinical trials to evaluate the safety and efficacy of epi-drugs would certainly be fruitful in the development of anticancer therapies for different cancer types and patients in need.