Targeting Cancer with Epi-Drugs

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Targeting Cancer with Epi-Drugs: A Precision Medicine Perspective Lisa Gherardini1, Ankush Sharma2, Enrico Capobianco2,3,* and Caterina Cinti1 1

Experimental Oncology Unit, Institute of Clinical Physiology, National Council of Research, Siena, Italy; 2Laboratory of Integrative Systems Medicine (LISM), IFC-CNR, Pisa, Italy; 3Center for Computational Science (CCS), University of Miami, Miami, FL, USA Abstract: Recent pan-cancer studies have shown the importance of coupling DNA methylation patterns with transcriptome profiles to reveal tumor subgroups with clinically relevant distinct characteristics. While the coupling patterns remain in most cases matter for further study and/or interpretation, it is emerging that all associations between epigenetic changes and specific cancer histotypes can facilitate the development of novel epidrugs. In particular, together with chemotherapy and chemoprevention of cancer, these epidrugs will target specific enzymes involved in the complex regulation of gene expression. This perspective surveys recent cancer epigenetic findings on target drugs and therapeutic strategies, and focuses on the epigenetic modifications that can reverse a stable differentiated state of adult cell towards neoplastic phenotypes. The relevance of such developments may thus pave the way for patient’s customized personalized therapies.

Keywords: Epigenetic Memory, Cancer Epigenomics, Cancer Therapy, Epi-drugs, Precision Medicine, Translational Medicine. Received: February 17, 2015

Revised: May 06, 2016

REPROGRAMMED EPIGENETIC MEMORY DURING CANCER DEVELOPMENT Epigenetics promises to enable radical changes in therapy once its complex influence exerted on gene expression is better revealed. In particular, chromatin regulation and DNA methylation, as a mechanism inhibiting gene expression and often appearing dysregulated in cancer, are central to epigenetic drug development. Below, we report salient aspects worth in-depth analysis in the future. EPIGENETIC MODIFICATIONS Epigenetics deals with the non-Mendelian inheritance of DNA modifications that co influencing the adult differentiated phenotype, playing a crucial role in both development and tissue determination. It is known that epigenetic marks are acquired throughout life, and have properties of both relative reversibility and stability [1]. Considering the epigenetic impact means to assign centrality to functional relevant modifications of gene expression. These are modification not involving a change in DNA sequence but rather in DNA methylation, histone modifications, ATP-dependent chromatin remodeling and non-coding RNA (ncRNA). Also, among the epigenetic regulatory mechanisms, covalent posttranslational modifications occurring on lysine amino acid residues of histone protein tails exert an impact on chromatin *Address correspondence to this author at the Center for Computational Science (CCS), University of Miami, Miami, FL, USA; Tel: ?????????????; E-mail: [email protected] 1389-2010/16 $58.00+.00

Accepted: May 23, 2016

structure and function. Chromatin can be influenced by various modifications, but the most common ones found in cancer are methylation-induced deregulation of DNA, acetylation and deacetylation of DNA-binding proteins [2]. Specifically, DNA methylation is achieved by the mechanism of covalently binding a methyl group to 5position of cytosine base, a reaction catalyzed by DNA methyl-transferase protein family (DNMTs). Following this modification, the methylated site acts as a regulatory mark regulating the gene expression on the basis of the genomic location and density. In mammalian cells, the majority of 5methylcitosine is located within CG rich sequences, which are named CpG islands. These are mainly present at the promoter and at the intragenic regions of genes, but also at both the intergenic regions and in relation with repetitive sequences. Under normal conditions, occurrence of methylation determines genomic imprinting, X-chromosome inactivation, suppression of repetitive elements, as well as lineage specific gene silencing. Notably, DNA methylome changes have been reported in various human cancers, involving hyper-methylation of tumor-suppressor genes and hypo-methylation of intragenic and intergenic repetitive sequences. In particular, the hypermethylation of tumor-suppressor genes induces either physical inhibition of the binding of transcription factors or the recruitment of proteins that have transcription-repressive properties, thereby silencing genes [3]. Also, the hypomethylation of the highly repetitive sequences, such as long interspersed nucleotide elements-1 (LINE-1) and short interspersed nucleotide elements (SINE), is a mechanism induc© 2016 Bentham Science Publishers

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ing the transcriptional activation of such elements, contributing to genomic instability and facilitating tumor progression [4]. Note that a low level of histone acetylation increases the affinity binding with DNA backbone, causing DNA condensation, and therefore preventing transcription. Instead, a high level of acetylated histones is characteristic of de-condensed chromatin and activation of transcription. The enzymes Histone Acetyl Transferase (HAT) and Histone Deacetylase (HDAC) cooperate on reversible histone lysine modification and on the control of the level of histones acetylation, hence gene expression [5]. At either global or specific gene scale, histone covalent modifications change accompanied by mutations in gene encoding regulators, such as DNMT3, IDH1/2 or H3.3. These are epigenetic hallmarks frequently found in multiple cancer types [6]. NcRNA IN REGULATING EPIGENETIC EVENTS Substantial evidence indicates that transcriptional gene silencing through epigenetic changes is mediated by ncRNAs [7]. Various ncRNAs have been shown to play a regulatory role, with evidences from tissue- or cell type-specific expression levels, and from their involvement in defining space, time and developmental stages. Recently, epigenetics and ncRNAs have stimulated analyses on very complex transcriptomes, aimed for instance at identifying and assigning functionality to the rich RNA repertoires of normal and disease states. Such advances have extended our knowledge on both the contribution of ncRNA-driven chromatin remodeling toward the establishment of chromatin structure and the maintenance of epigenetic memory. Among the findings, ncRNAs appear as genomeorganizing architectural factors of transcribed chromosomal domains [8, 9]. In particular, a functional role has emerged with an origin from enhancers (eRNAs) [10]. In general, the class of long ncRNA (lncRNAs) seems not to overlap with genes and indeed to activate transcription (ncRNAactivating). It has been found that the knockdown of these structures is attenuating the expression of nearby genes. In turn, eRNA may play a role in enhancer-promoter interactions and nearby gene transcription. Notably, they may regulate transcription by promoting chromatin accessibility and RNA Polymerase II recruitment, which are processes required for the stabilization of enhancer-promoter interactions. Moreover, there is growing evidence that chromatin modifiers and remodelers bound to nuclear RNA play a key role in determining histone post-translational modification positions along the chromatin [11–13]. Of interest to our scopes, ncRNAs play a crucial role in maintaining genomic stability, an essential condition for cell survival and for preventing tumorigenesis. Through an extensive crosstalk between ncRNAs and the canonical DNA damage response (DDR) signaling pathway, DDR-induced expression of ncRNAs can provide a regulatory mechanism to enable accurate control of the spatio-temporal expression of DNA damage responsive genes. At a mechanistic level, DNA damage alters the expression of a variety of ncRNAs at multiple regulation levels, including transcriptional and posttranscriptional regulation, and RNA degradation.

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In parallel, ncRNAs can directly regulate cellular processes involved in DDR by altering the expression of their targeting genes. This holds especially for micro (miRNAs) and lncRNAs. miRNAs are required for almost every aspect of cellular responses to DNA damage, with reference to: •

Sensing DNA damage

•

Transducing damage signals

•

Repairing damaged DNA

•

Activating cell cycle checkpoints

•

Inducing apoptosis.

lncRNAs control transcription of DDR relevant genes by four different regulatory models, including signal, decoy, guide, and scaffold. Due to the crucial role played by all such ncRNAs in the response to DNA damage, it is expected that novel and very promising therapeutic targets may counteracting drug resistance of cancer. DISRUPTION OF EPIGENETIC MEMORY DURING CARCINOGENESIS An epigenetic feature (restriction) of gene expression was proposed to be the mechanism of how cells establish identity and their differentiated states [14]. The most relevant property of the epigenetic controlled gene expression profile is inheritance. Therefore, cells retain through generations the original template by a sort of epigenetic memory [15]. Interestingly, the latter is exerting multilevel influence, at single cell lineage, at tissue, and at whole individual level. During cell cycle DNA sequence, but also DNA methylation patterns, histone modifications and nucleosome positions are faithfully duplicated and transmitted to daughter cells. Consequently, restriction of gene expression during development and tissue differentiation is maintained in a lineage-specific manner, thus ensuring epigenetic stabilization of the adult phenotype [16]. Moreover, epigenetic gene expression profile can be maintained also through meiosis, therefore determining the transmission of the epigenetic state from multiple generations of individuals. It is widely accepted that epigenetic modifications take place during early embryonic and primordial cell differentiation, and that there are critical stages during which epigenetic marks are cleared and then re-established. Mitosis has been identified as a critical stage during which a shift in gene expression between cellular generations may be achieved by changing the complement of chromatin binding proteins present in the cells [17]. Disruption of DNA methylation profile through replicative cell cycles has a global impact on heterochromatin stability and hyper-variability of gene expression, thus opening to cancer transformation. When this epigenetic state reverts to a gene expression pattern typical of an earlier lesser-differentiated stage, the conditions for cancer onset are created. In fact, cancer is also thought to be the result of the loss or reversal of a stable differentiated state of adult cells mediated in part by epigenetic changes, in turn modifying the expression profile of specific genes involved in the control of cell viability, proliferation and invasiveness [18]. The controlled induction of embryonic gene expression in differentiated cells by nuclear reprogramming drives a

Targeting Cancer with Epi-Drugs: A Precision Medicine Perspective

shift from somatic transcription patterns to embryonic patterns. When these changes in gene expression do not occur, the somatic identity is lost and the cell moves toward a dynamic and reversible transition to pluripotent phenotype. Similarities in gene expression between pluripotent and cancer cells have been found, in particular with regard to embryonic master regulatory transcription factors (especially MYC) turned on [19, 20]. Notably, there are evidences linking the degree of de-differentiation with both aggressive cancer progression and embryonic gene expression patterns [21]. In short, carcinogenesis is a process by which normal cells acquire a new malignant identity and give rise to a clonal aberrant population. This event is only possible if the initiating cell has the necessary plasticity to undergo such changes, and if the oncogenic events that initiate cancer have the essential reprogramming capacity to be able to lead a change in cellular identity [22]. The molecular mechanisms underlying tumor cell reprogramming are the pathological counterparts of the normal processes regulating developmental plasticity. Reprogramming of the cellular identity is mediated by signals from the environment and/or by internal changes at the transcriptional and epigenetic levels. Virtually all human cancer types show epigenetic abnormalities that contribute, in concert with genetic alterations, to the disease onset and progression. Moreover, even the risk of disease consequent to exposure to environmental stress in older generations may predispose subsequent generations to disease so as to involve the transgenerational inheritance of epigenetic information. Despite the fact that targeting DNA methylation or histone modifications alone are not sufficient for therapeutics purposes, cancer epigenome studies have already started to diversify the control and therapy approached to cancer, by providing potential molecular markers aimed to assess cancer risk and early cancer detection. As a matter of fact, reversibility of the epigenetic alterations renders such detections viable therapeutic targets. INTEGRATED EPIGENOME ANALYSIS TO IMPROVE CANCER PATIENT STRATIFICATION It is now evident that alterations in the genome and in the epigenome cooperate to promote oncogenic transformations. The disruption of epigenomic control is pervasive in malignant conditions and can be classified as an enabling characteristic of cancer cells, akin to genome instability and mutation [23]. Recent innovations in chromatin immunoprecipitation paired with microarrays and high-throughput sequencing have enabled unprecedented insights into protein–DNA interactions and chromatin architecture in a wide range of biological models, and particularly in cancer-related ones. The emerging role played by epigenetics can be observed by looking at Next Generation Sequencing. Contrary to gene mutations, cancer-inducing events can be reversed by drugs targeting epigenetic mechanisms. Notably, targeting the reversal of epigenetic alterations suggests a rationale for cancer therapies aimed to achieve epigenetic reprogramming, thus normalizing gene expression and impeding tumorigenesis. It can be hypothesized that epigenetic changes in genes

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and ncRNAs form additional variation layers compared to mutations, revealing different molecular mechanisms responsible for cancer development. The identification of such expression signatures can further characterize patient groups according to diagnostic, prognostic and therapeutic factors. The idea that each tumor type has a characteristic DNA methylation pattern, has been recently confirmed by genome-wide sequencing projects involving multiple cancers, especially with reference to DNA methylation subgroups. However, uncertainty remains over a two particular aspects, among others: (i) Whether these DNA methylation patterns are unique for a specific tumor type or instead comparable across different types of cancers; (ii) How epigenetic modifications could facilitate cancer development. Advances in genome-wide DNA methylation profiling are expected to complete our understanding of the molecular mechanisms responsible for the epigenetic defects linked to aberrant DNA methylation. Recently, an atlas of DNA methylation was generated across a variety of samples from 82 cell lines and tissues, providing insight on gene regulation aspects and disease, and leading to the identification of methylation signatures in part cancer-associated and in part cell-type specific [24]. Current computational efforts are directed towards building integrative and comparative analyses [25, 26]. Highthroughput techniques, including array and sequencing-based technologies, provide genome-scale whole methylomes, confirming the role of aberrant methylation as a cancer hallmark to identify novel types of biomarkers. Especially pan-cancer methylomes are expected to shed light over new cancer subtyping analyses, even if they present several computational challenges. These may be summarized as follows: a)

The integration of evidences from different platforms

b) The expansion of datasets needed to avoid restriction of the outputs to overlapping CpG sites c)

The treatment of heterogeneity through the control of likely abundance of false positives

d) The design of algorithmic pipelines for the detection of biomarkers clinically relevant for patient stratification [27]. The Epigenomic Roadmap Consortium (http://www. roadmapepigenomics.org/) represents a public resource of human epigenomic data (> 120 epigenomes) designed to decipher origins and functions of new important health, susceptibility and disease landmarks and the regulation underlying mechanisms. The key thing is that causation instead of correlation is the real goal to be scrutinized when studying coupled profiling between epigenetic and transcriptomic data. This initiative, together with another major initiative named Blueprint (http://www.blueprint-epigenome.eu/, EUfunded), is going to be the backbone of the International Human Epigenome Consortium (http://ihec-epigenomes.org/) targeting 1000 epigenomes in the next decade. Chromatin regulation and thus control of gene expression have been central to epigenetic drug developments, but other epigenetic mechanisms have therapeutic potential that “Big Omics Data” are destined to reveal. This new development


will expand cancer phenotyping, through increased marker detection, deeper subtyping and more stratified methylation, and promises to exert a huge clinical impact on diagnosis and prognosis. Also, promoting therapy advances with new candidate druggable targets is a goal, together with generating hypotheses for innovative clinical trials design. Computational tools designed to investigate causal relationships between epigenetic changes might be related with driver events for cancer development and progression. Challenging aspects remain critical for an overall assessment, and they refer to platform sensitivity, selection of sample sources, differentiation in tumor heterogeneity, integration with clinical data. However, inference can also find benefit from the emerging role of usually lowly expressed lncRNAs which are detected in wide-spectrum transcriptome profiling, and further help to our understanding can be derived by more refined topological characterization of epigenetic regulatory networks in terms of complex modular configurations or simple connectivity motifs. NOVEL EPIGENETIC THERAPIES Overview The galaxy of epigenetics is rapidly expanding (see Table 1 for a sketched representation) promising to put forth new candidates for clinical trials. Observing epidrugs’ development by the growth of scientific publications and interest by social media, we might legitimately expect to witness soon clinical success. An appealing concept is provided by the words “reverse and restore”, in the direction of personalized medicine The rationale is offered by reversal effects from epi-modifications on cancerous cells designed to reprogram disease epi-profiles, thus restoring the natural cells faith. Several strategies have been investigated leading to epienzyme activity modulation and in turn to chromatin state regulation in cancer. This was seen with RNAi-mediated DNMT3b knockdown used as adjuvant for chemotherapeutic treatment in breast cancer and in glioma [28, 29]. Similarly, regulation of HDAC though the use of epi-miRNA have been reported to benefit cancer development [30] particularly in hepatocarcinoma, HDAC regulation by several different miRNAs cause cell cycle arrest and proliferation inhibition [31]. Nonetheless, the consolidated way of epigenetic regulation of gene expression is now through the use of different classes of chemical inhibitors that have gradually evolved into specific and effective solutions. DNMTs enzymatically add a methyl group to cytosine in CpG dinucleotides in DNA. DNMT inhibitors induce hypomethylation at low concentrations: AZA and DAC, two DNA methyltransferases (DNMT) inhibitors (Azacitidine and Decitabine, respectively) that have been approved for the treatment of acute myeloid leukemia (AML) [32, 33] enter the cell via nucleoside transporters and after phosphorylation by the respective enzymes, are incorporated into RNA (AZA), or into DNA (DAC). Even minor substitution of 5azacytosine for cytosine in DNA seems sufficient for DAC to inactivate DNMTs. Fig. 1a shows a 3D re-construction of DNA (blue) and DMNT (purple) interaction. Yoo et al. [34] computationally reconstruct the interaction of the enzyme with the decitabine at active site (inset in Fig. 1, adapted

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[34]). The 3D pocket (Fig. 1a green) irreversibly accommodates azacytosine bases and inactivates methylation (Chimera [35] molecular modelling reconstruction processed following [34]). Inactivated DNMT is destined to proteasomal degradation. At higher dosage, DAC is incorporated into the DNA of dividing cells. By forming covalent complexes with the DNMTs, DAC exerts direct cytotoxicity, which is mediated by induction of apoptosis through DNA double strand break [36]. Although already approved and clinically applied, AZA and decitabine are focus of clinical trials (ClinicalTrials.gov Identifier: Oct; 25(13): 1605-12. NCT01409070). HDACs are enzymes acting on a plethora of cytosolic proteins with many cellular functions. These enzymes regulate the acetylation states of histone proteins and other nonhistone protein targets. Most relevantly histone deacetylase can regulate gene expression at the chromatin level contributing to chromatin remodelling. Mammals have 18 known HDACs divided among these four classes each with different localization in the cell, role and structure. Classes I and II (IIa and IIb) and IV depend on zinc for the catalytic action. Differently, class III couples protein deacetylation with NAD onto which to transfer the acetyl group, thus making them important for energy metabolism positively regulating transcription factors such as NF-κB, p53 and forkhead box (FOX) proteins. The above classes all share nuclear localization except for HDAC 6 (class IIb) that is found in the cytoplasm. Class III HDACs, the ‘sirtuins’ (SIRTs1-7) can be found in the nucleus (SIRT 1,2 6,7), however SIRT1 and SIRT2 can shuttle between the nucleus and cytoplasm. SIRT 3,4,5 are located in the mitochondria [36]. Overexpression of SIRT1/2 has been shown to predict poor prognosis in a wide variety of solid tumors, and malignant hematological diseases. SIRT 1 is implicated in the development of drug resistance, blocking of cell aging and apoptosis and promoting angiogenesis [37]. A recent protein-protein interaction study demonstrated that this protein family shares an intense interacting network with at least hundred different kinases implicated in metabolism, aging and circadian rhythm regulation. Since HDACs often act broadly on the transcription of many genes, their inhibition results downstream changes in gene-expression conferring their therapeutic properties [38, 39] HDAC inhibitors (HDACIs) are substances which inhibit the function of HDACs (see Table 1). The mechanism of action strictly correlates with the chemical nature of those molecule as shown in [40]. The interaction between the hydroxamic acid progenitor vorinostat and the enzyme ternary structure (blue in Fig. 1b) was experimentally described docking into an active pocket of the catalytic domain (green in Fig. 1b). Zinc interaction and hydrogen bonds (yellow in Fig. 1b) stabilize the structure at low energy level. Inhibition of deacetylation leads to an accumulation of both hyperacetylated histones and transcription factors. Together with Vorinostat, approved in 2006, other HDACIs such as Romidepsin (2009), Belinostat (2014), have been approved for the treatment of lymphomas. Recently, Farydak (panobinostat, 2015) has been approved for the treatment of multiple myeloma because claimed to be effective at nanomolar concentration. As an aside note, the


Table 1.


????????????????????????????????????????????????????????????????????????. Chemical Classification of epigenetic drugs Non-Nucleoside A nalogue Inhibitors

DNMT inhibitors

Nucleoside Analogue Inhibitors

Antisense Oligonucleotides

Chemical name

Other names

Chemical name

Other names

Chemical name Other names


5-azacytidine

AZA (Vidaza)

Procainamide

Procan-SR (Procanbid)

RG108

RG108

MG98

MethylGene

5-aza-2'-deoxycytidine

Decitabine (DAC; Dacogen)

Procaine hydrochloride

Novocan (SP01A)

5,6-dihydro-5-azacytidine

DHAC

Epigallocatechin- EGCG 3-gallate

5-fluoro-2'-deoxycytidine

FdCyd

1-hydrazinyl-phthalazin3

Hydralazine (Apresoline)

Arabinosyl-5-azacytidine

Fazarabine

NSC 309132

Zebularine Aliphatic Acids (Short-Chain Fatty Acid)

Hydroxamates

HDAC ihnibitors

Small Molecules

Cyclic Tetrapeptides and Analogues

Chemical name

Other names

Chemical name

Other names


Suberoylanilide hydroxamic acid

Vorinostat (SAHA; Zolinza)

Sodium butyrate

Phenylbutyrate depsipeptide Buphenyl

suberoyl-3aminopyridinamine hydroxamic acid

PXD101

valproic acid

Romidepsin Istodax

Benzamides Chemical name Other names MS-275

Entinostat Benzamidine

VPA

PX105684

Divalproex

Belinostat

Depakote

Oxamflatin

Metacept 3

AN-9

Pivaloyloxymethyl butyrate Pivanex

LAQ824

Dacinostat

OSU-HDAC42

AR-42

LBH-589

Panobinostat

ITF2357

Givinostat Gavinostat

Fig. (1). (1a) 3D reconstruction of DMNT1 structure. The interaction with DNA helix (blue) is shown. Accordingly to [34], the active pocket residues are shown in green. The computational reconstruction of Decitabine interaction with the site is shown in the inset. (1b) 3D reconstruction of HDAC chain (blue). It holds the active site (green) for the docking of Vorinostat according to [40]. Hydrogen bonds (yellow) stabilize the low energy conformation of the complex.


new entry farydak is accompanied by a variety of biological side effects. Apart from being regulators of gene expression, HDACs also act on a wide range of proteins that regulate many cellular functions, including, autophagy, apoptosis induction and cell cycle arrest, DNA damage and repair, angiogenesis and immune responses. PERSPECTIVE In general, the clinical success obtained with the first generation of epigenetics-based drugs used in various cancers might mask existing risks, such as the one of inducing a widespread genome-scale effect potentially leading to the emergence of off-targets and toxicity effects. Moreover, chemical and pharmacological instability of these molecules can contrast systemic delivery. Despite these limitations, epiprogenitors are paving the way to the development of an increasing number of epi-enzyme inhibitors for use in monotherapy and combinatory strategies. It is well-known that a chemical modification of the original molecule leads to a more effective molecule, serving both pharmacokinetic and toxicity scopes. Clinicians and researchers often refer to those agents as second-generation HDAC inhibitors. Consequently, next-generation epigenetic therapies are currently under clinical development [41]. An example is provided by 2'3'5'triacetyl-5-azacytidine, formulated to be delivered in oral form so to yield a more effective DNMT inhibiting pro-drug, by improving bioavailability, solubility, stability, and also by reducing general toxicity. Then, another example is SGI-110 (2-deoxy-5azacytidylyl-(3-5)-2-deox-yguanosine sodium salt), a dinucleotide of 5-AZA-cytidine and deoxyguanosine linked with a natural phosphodiester linkage (Astex Pharmaceuticals Dublin, CA). The molecular modification had protective effects, by activating moiety from environmental systemic cytidine deaminase, and by preventing activity degradation [42]. Moreover, this molecular arrangement has endowed the drug with immunomodulatory properties that contribute to its anticancer activity[43]. For HDACIs, classification comprises different structural groups: the hydroxamic acids, cyclic peptides, benzimides and short-chain fatty acids. Among those, hydroxamates are able to target and affect all classes of HDACs. HDACs and SAHA (N'-hydroxy-N-phenyloctanediamide, Suberoylanilide Hydroxamic Acid) is one of the prominent member of the family, deriving from trichostatin A (TSA) with improved solubility and halved life with respect to TSA. HDACIs preferentially targeting a single HDAC have been recently developed inducing HDAC isoform specificity by chemically modifying progenitor molecules of first generation HDAC inhibitors. For example, it was recently reported the specific effect of a highly potent HDACI, named ST7612AA1 as prodrug of ST7464AA1, which shows potency toward all class I HDACs and class IIb HDACs [44]. This oral antitumor agent, synthetized as a thioacetate-ω (γlactam amide) derivative, presents high cytotoxic activity on NCI-H460 (NSCLC) and HCT116 (colon carcinoma). Also, newly designed drug such as N-hydroxy-3-acrylamide (MPT0G157) had improved molecular stability. In HCT116 cells, MPT0G157 was found to induce anti-angiogenesis

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effect by hyperacetylation of heat shock protein 90 (Hsp90) and induction of hypoxia-inducible factor-1α (HIF-1α) degradation that resulted in by down-regulation of vascular endothelial growth factor (VEGF) expression, confirming the profile of HDACi holding pleiotropic activity [45]. Finding a suitable delivery method would lead to minimizing side effects and achieving a higher therapeutic index [46, 47]. Environmental susceptibility and decay are common challenges for many epi-drugs as well as for new anticancer agent such as antibodies or miRNAs [48]. In this view, recently gastro-resistant nanoparticles containing 5Aza-2'-deoxycytidine and 5-FluUridine have been tested for gastrointestinal cancer cells [49]. Similarly Decitabine was delivered in nanoparticles to fight breast cancer [50]. In a recent preclinical animal study, it was demonstrated the potential of an erythrocyte-based drug delivery system to administer extremely effective low dose of Decitabine as a potential new therapy in human prostate cancer (details in [51]). Then, erythrocyte loaded with Decitabine in combination with trichostatin A is also being used to reduce WeriRB1 cell proliferation in vitro and in vivo in human retinoblastoma cancer model, and preliminary results confirm cellular-carrier mediated combinatory therapy feasibility (work in progress). In the case of human erythrocyte, the use of homologous carrier would suggest a direct impact in translational personalized medicine. In fact, not only erythrocytes are able to deliver customized nucleotide bio-drugs, as for synthetized epigenetic tools, but the therapy could be personalized through the carrier sourced by patient auto-transfusion. Furthermore, target delivery localization would stimulate the use of guidable carrier. For example, erythrocytes can be modified with magnetic nanoparticle and engineered on the surface with fusogenic hemagglutinin protein so to successfully increase localization of therapeutic release, thus maximizing intracellular DNMT inhibition [52]. A common path visible in the described drug delivery examples is the use of a nanocellular delivery system allowing combinatory protocols. Beyond the most parsimonious and time effective strategy, a combinatory strategy also leads to a reduced efficacy dosage for each of the drug components, and consequently to limited and/or undesired side effects [53]. COMBINATION IS THE WATCHWORD In the past five years, 20 clinical trials were instructed (https://clinicaltrials.gov/) to evaluate the potential of epigenetic drug, mostly those already approved by FDA in combinatory protocols. The rationale behind might be the desire to activate a two-pronged strategy. Thus, oral Azacitidine to be given in combination with Fulvestrand in post menopause women with metastatic mammalian cancer positive to hormone receptors that have been already treated with Aromatase inhibitors (NCT02374099). Also, Decitabine and the new generation HDAC inhibitor, LBH589, to be used with the aim of inducing reactivation of estrogen receptor (ER) in triple negative breast cancer where its silencing occurs. A trial has been designed so that Tamoxifen will be administered after ER re-expression to limit cancer growth (NCT01194908). Similarly, combinatory strategies were



Table 2. ???????????????????????????????????????????. New epigenetic drugs

demethylation

Class

Name

DNMT First Decitabine inhibitors generation (Dacogen FDA) Azacytidine (Vidaza FDA) Second 2'3'5'triacetyl-5generation azacytidine SGI-110 (2-deoxy-5azacytidylyl-(3-5)-2deox-yguanosine sodium salt)

acetylation

HDAC First Vorinostat (Zolinza) inhibitors generation Romidepsin (Isodax)

FDA Use/Authorisations approval

Adm. Route

Clinical trial with results

Open clinical trials

References

2006

myelodysplastic syndrome/Acute myeloid leukemia

I.V.

30

76

Clinicaltrial.gov.

123

www.fda.gov

2004

Chronic/acute myeloid leukemia

S.C.

54

(AML) NSCLC

Oral

0

0

Ziemba et al., 2011

Metastatic colorectal cancer S.C.

1

50

ClinicalTrial.gov. 2016 Issa et al., 2015

2006

Cutaneous T cell Lynphoma Oral

59

45

ClinicalTrial.gov.

2009

Peripheral T cell Lynphoma

I.V.

10

35

Zinzani et al., 2016

Belinostat (Beleodaq) 2014

Peripheral T cell Lynphoma

I.V.

11

6

Panobinostat (Farydak)

2015

Multiple myeloma

Oral

13

19

Human solid and haematologic malignancies

Oral

0

0

Second ST7612AA1 generation (thioacetate-ω-γlactam amide) Givinostat

Chronic Myeloprolifer active Neoplasms

designed for Decitabine and Vemurafenib in several stages of melanomas (NCT01876641), while Azacitidine was tested in different stages of Non-Small Cell Lung Cancer together with either Paclitaxel (NCT02250326) or Entinostat (NCT01207726). Combinatory development of epi-drugs and chemotherapeutic or bio-drugs might also represent a cost-effective advancement in cancer treatment, thus revamping old molecules for new therapeutic strategies. Clinical trials already shed light on cancer epigenetics in relation with local epidemiology (NCT01904968), environmental connections (NCT01374074), as well as comorbidities (NCT01411943). The rule of “nurture and nature” in cancer etiology is therefore confirmed. A fundamental aspect is to get information on clinical additional and secondary findings that might lead to identify epigenetic relevant aspects, due to the ground-breaking role of clinical trial in the development of personalized medicine in cancer. The high-throughput investigation of epigenome from trial participants allows comparisons between normal and tumor tissues, and represents a rich source of patient-specific biomarkers and therapeutic solutions, inspiring strategies for detecting epigenetic signatures. HOW TO ENABLE PERSONALIZED EPIGENETIC TREATMENT? Knowing the target of a drug is beneficial to both improving its efficacy and reducing unwanted toxicity. Understanding the mechanisms of anticancer activity of epidrugs, such as HDAC and DNMT inhibitors, is essential for drug

Vesci et al., 2015 ClinicalTrial.gov.

1

2

Pinazza et al., 2016

design in targeted therapies. Knowledge of the involved patterns of activity is needed to design optimized clinical protocols as well as to develop new classes of drugs. In this view, the screening of already approved library of epigenetic active molecules could represent a cost-effective approach to discovery suitable candidates for alternative clinical applications. Disease stratification studies aimed at identifying patterns of DNA methylation associated with an increased risk of cancer development, could pinpoint specific driver mutations. Such investigations are mostly taking place during clinical trials, at a stage when patient cohort genetic profiles might indicate shared epi-mutations to be targeted with specific drugs [54, 55]. Progresses in NGS and Omics approaches have allowed systematic analysis and identification of novel epigenetic marks (top-down mapping of epigenetic changes). The most salient achievement is however to define epi-modification in space to define landmark distribution across the genome. This is more easily achievable using bottom-up epigenetic analysis to map the spatial reconstruction of localized changes on the epigenome [56]. By this approach, epigenetic marks can be introduced by directing a genetic modifier to a specific DNA binding domain, thus targeting an effector domain to the desired locus. EPIGENOME EDITING RETROSPECTIVELY Editing the epigenome means to place phenotypes under artificial control. This means employing artificial tools for regulating the expression of endogenous genes. Precision genome editing tools have also become invaluable for cancer


biology to determine the genetic basis underlying pathological traits [57]. Epigenetic engineering has been the topic of recent reviews listing pros and cons of the use of sitespecific nucleases in association with DNA specific binding domains [10, 58]. This development should induce the double strand break in specific site, thus modifying the epigenetic state of the target locus. The first successful examples of predictable interaction tools were the zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) technologies [59, 60]. In general, ZFN proteins recognize a specific sequence on DNA (DNA binding sites, DBS) and are able to reach a unique sequence on the compact chromatin structure by matching triples of bases with their amino acid sequences. In the past few years ZFN became an interesting tool for molecular genetics due to extensive use to re-activate epigenetically silent genes, such as tumor suppressor genes. Similarly TALEN-mediated genome editing relies on the ability of recognising nucleotides on target DNA, which direct the activity of a comprised nuclease FokI. Recently, TALEN was used against HPV-infected cells to cleave viral oncogene E6/E7 DNA sequence and suppress cancer proliferation in vitro and in vivo. TALENs would also interfere with host repairing mechanisms of DNA breaks via the non-homologous end-joining to cause mutation and therefore inhibiting E6/E7 oncogene functions [59]. In order to target epigenetic modifications at specific loci, the catalytic domain of epi-enzymes have been linked to these sequence-specific DNA-binding domains to ensure deposition of the corresponding epigenetic mark on the chromatin [56, 61]. However both TALEN and Zinc fingers–DNA interactions are not fully specific and might bind to their cognate DNA triplets with different affinity, justifying the fact that off-target side effects have been reported. More recently the ability of the bacterial system named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated with a endonuclease protein Cas9 was exploited as a more sophisticated RNA-guided nuclease technology for targeted (epi)genomes, even in eukaryotic cells [62]. This system belongs to the defense strategy of bacteria for facing viruses and plasmids infections. The complex also includes a guiding RNA, with length of 20 bases normally used to address DNA recognition. However, longer sequences might increase the chances of hitting specific targets. In this case, high specificity is required to avoid off-target interactions. The ribonucleoproteic Cas-CRISPRRNA complex (crRNA) is able to recognize nucleotidic target sequence, opposite to the protospacer-adjacent motif (PAM) and, through the activity of two nicking recognition site, to generate double strand brakes. At this stage, eukaryotic cells would activate their repairing mechanisms, in particular non-homologous end joining (NHEJ) and homologous, respectively, thus inserting programmed mutation. By fusing any enzyme of choice to Cas9, one will be able to alter histone modification changing chromatin states. Moreover this system would also influence DNA methylation. The CRISPR-Cas9 system is sufficiently powerful for biologists to allow access to a sophisticated repertoire of regulation of cellular functions. In a few years, the CRISPR-Cas9 technology has become the tool of choice

Gherardini et al.

for different genetic purposes. Availability of reliable animal models able to recapitulate cellular regulations by mimicking physio-pathological strategies is one of the major strategies for tackling genetic diseases. Using this technique either direct insertion of mutation or chromosomal rearrangements are possible to generate reliable in vitro and in vivo models for preclinical investigation on specific features such as drug resistance or sensitivity [63, 64]. For example, in a therapeutic perspective, CRISPR-Cas9 has been used as gene knockout or to increase expression of specific endogenous genes [65]. When CRISPR-Cas9 is used to target epi-enzymes such as DNMT in human embryonic cells, this will interfere with their expression, limiting their functions and redesigning cell destiny [66]. This structureguided approach could be optimized and extended to a bioengineering design of Cas9-based transcription activation system for achieving robust multiple RNA-mediated gene up-regulation. To this purpose, numerous specific RNA sequences can be coupled within the same CRISPR Cas9 system, assembled in a synthetic transcription activation complex with multiple distinct effector domains. This would allow a simultaneous reactivation of expression of those RNA targeted genes, thus offering new insight on cancer epigenetic therapeutic targets [67]. By the use of multiple reference sequences for the construction of CRISPR-Cas9 it would be possible to identify novel regulatory gene cluster enrichment related to specific cancer features. For example this strategy was used to identify those mutations capable of mediating resistance against the BRAF inhibitor PLX-4720 in melanoma cells [68]. Although CRISPR-Cas9 has become the golden standard technology in bio-molecular engineering, it is not yet fully exploited within cancer research [58, 69]. It is in fact foreseeable that this type of genome screening could have an even greater potential impact on identification of epigenetic marks at the cancer genome scale when associated with system biology approaches and bioinformatics pipelines. CONCLUSIONS Advances in genome-wide DNA methylation profiling are expected in the future 5-10 years to complete our understanding of the molecular mechanisms responsible for the epigenetic machinery defects in cell transformation and carcinogenesis. Computational efforts are currently directed towards building integrative comparative analyses of multiplatform outsourced datasets, paving the way for the design of algorithmic pipelines aimed to detect clinically relevant biomarkers and to provide more accurate patient stratification. Editing the epigenome and its built-in players is the new frontier of drug discovery, the one shaping the roadmap of personalized medicine in cancer. Epigenetic memory participates in determining the adult differentiated phenotype, playing a crucial role in maintaining stability of cellular identity. Its dysregulation facilitates cancer transformation. The comparative analyses of DNA methylation patterns and transcriptome, including non-coding RNA, across pan- cancers are clarifying the extreme complexity of disease biology and patient groups sharing distinct epigenetic patterns.


Notably, while the literature describes diversity of molecules that can be classified as epigenetic drugs and whose activity interferes with posttranslational changes, only 5 molecules have been approved by FDA and are currently used in clinics. Pre-clinical studies are in progress on novel epi-drugs targeting specific epigenetic regulators with the aim to solve the off-target side effects and to develop personalized epigenetics as a new profitable layer in patient’s tumor profiling.


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CONFLICT OF INTEREST

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The author(s) confirm that this article content has no conflict of interest.

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ACKNOWLEDGEMENTS Declared none. [25]

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PMID: 27229488