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Volume 6, Issue 6 p. 657-682
Open Access

Trials with ‘epigenetic’ drugs: An update

Angela Nebbioso

Angela Nebbioso

Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. de Crecchio 7, Napoli 80138, Italy

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Vincenzo Carafa

Vincenzo Carafa

Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. de Crecchio 7, Napoli 80138, Italy

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Rosaria Benedetti

Rosaria Benedetti

Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. de Crecchio 7, Napoli 80138, Italy

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Lucia Altucci

Corresponding Author

Lucia Altucci

Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. de Crecchio 7, Napoli 80138, Italy

Istituto di Genetica e Biofisica, Adriano Buzzati Traverso, IGB, Via P. Castellino, 80131 Napoli, Italy

Corresponding author. Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. de Crecchio 7, Napoli 80138, Italy.Search for more papers by this author
First published: 06 October 2012
Citations: 192


Epigenetic inactivation of pivotal genes involved in correct cell growth is a hallmark of human pathologies, in particular cancer. These epigenetic mechanisms, including crosstalk between DNA methylation, histone modifications and non-coding RNAs, affect gene expression and are associated with disease progression. In contrast to genetic mutations, epigenetic changes are potentially reversible. Re-expression of genes epigenetically inactivated can result in the suppression of disease state or sensitization to specific therapies. Small molecules that reverse epigenetic inactivation, so-called epi-drugs, are now undergoing clinical trials. Accordingly, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for cancer treatment have approved some of these drugs. Here, we focus on the biological features of epigenetic molecules, analyzing the mechanism(s) of action and their current use in clinical practice.

1 Introduction

It is well established that both genetic and epigenetic changes contribute to the development of human diseases, such as cancer (Hahn and Weinberg, 2002; Jones and Baylin, 2002).

The term “epigenetics” refers to all heritable alterations in gene expression and chromatin structure due to chemical modifications that do not involve changes in the primary gene nucleotide sequence (Nightingale et al., 2006). The correct regulation of these alterations ensures appropriate cell growth (Bernstein et al., 2007; Jenuwein and Allis, 2001; Strahl and Allis, 2000). In contrast, deregulation of epigenetic patterns leads to induction and propagation of disease state (Feinberg et al., 2006; Hahn and Weinberg, 2002; Jones and Baylin, 2002).

Epigenetic inheritance can be classified into three distinct types: DNA methylation, histone modifications, and non-coding RNAs. All of these are crucial mechanisms that ensure the stable propagation of gene activity from one generation of cells to the next (Feinberg et al., 2006; Jenuwein and Allis, 2001). Disruption of any of these three distinct and mutually reinforcing epigenetic mechanisms leads to inappropriate gene expression, resulting in cancer development and other “epigenetic diseases” (Egger et al., 2004; Feinberg et al., 2006; Feinberg and Tycko, 2004; Jones and Baylin, 2002).

Although precise underlying mechanisms are not yet clear, in recent years, scientific interest in epigenetics has increased insofar as it represents an important tool to advance our understanding of pathogenesis, in particular tumorigenesis, and to help in the development (Egger et al., 2004; Strahl and Allis, 2000) of strategies for cancer treatment and prevention.

2 Chromatin structure and regulation

In eukaryotic cells, nuclear DNA associates with histones to form a compact complex called chromatin. Histones constitute a family of basic proteins, which are generally highly conserved across eukaryotic species. The core histones, termed H2A, H2B, H3, and H4, associate to form an octamer structure comprising two molecules each of H2A, H2B, H3 and H4 histones. DNA winds around this protein core, with the basic amino acids of the histones interacting with negatively charged phosphate groups of the DNA. Approximately 146 bp of DNA wrap around a histone core to make up a nucleosome particle, the repeating structural motif of chromatin. Histone H1 assembles such nucleosomes into a higher ordered structure. The N-terminal tails of nucleosomal histones are subject to different modifications including acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation. Of these, acetylation has been the most extensively investigated (Shukla et al., 2008). Recent studies in the field of chromatin research provided experimental evidence that led to a concept known as the “histone code” (Jenuwein and Allis, 2001; Strahl and Allis, 2000). It postulates that different histone modifications are combinatorial and consistent, generating a code that is read by cellular machineries to dictate functional outcomes. Different histone modifications were shown to be essential for normal cellular processes because crucially involved in transcriptional regulation. In view of this fact, attempts have been made to regulate the transcription of disease-related genes (e.g., oncogenes and tumor suppressor genes (TSGs)) by using epigenetic regulators to treat cancers and other diseases.

3 Histone modificators in clinical trials

Epimutations play a role in the etiology of human cancers. In contrast to DNA mutations, which are passively inherited through DNA replication, epimutations must be actively maintained because they are reversible. The reversibility of epimutations by small-molecule inhibitors provides the foundation for their use in novel cancer therapy strategies. Among the compounds that inhibit epigenetic processes, the most extensively studied are DNA methyltransferase inhibitors and HDAC inhibitors (HDACi). Here, we focus on epigenetic modulators used in clinical trials (either completed or terminated) to treat human diseases (

3.1 HDAC inhibitors

Histone deacetylases are known to play a key role in the transcriptional machinery for regulating gene expression, to induce histone hyperacetylation and to affect gene expression. Therefore, they represent the target of therapeutic or prophylactic agents, HDACis, for diseases caused by abnormal gene expression such as inflammatory disorders, diabetes, diabetic complications, homozygous thalassemia, fibrosis, cirrhosis, acute promyelocytic leukemia (APL), autoimmune diseases and tumors as well as organ transplant rejections and protozoal infections.

Acetylation and deacetylation of histones are carried out by “writers” (histone acetyl transferases (HATs)) and “erasers” (histone deacetylases (HDAC)) enzymes. The state of acetylation of histones is an important determinant of gene transcription. Deacetylation is generally associated with reduced transcription of genes whereas increased acetylation of histones induced by the action of HDACi results in greater transcription of genes. Thus, HDACi affect multiple processes in the cell, which are likely to depend upon its dynamic state with respect to its capabilities of replication and differentiation.

The term “HDAC inhibitors” is commonly used for compounds that target the classical class I (HDAC1, 2, 3 and 8), II (HDAC4, 5, 6, 7, 8, 9 and 10), and IV (HDAC11) HDACs and are currently being evaluated in clinical trials. A number of structurally diverse HDACi have been identified, many of which are or derive from natural products. These can be classified, according to their chemical structure, into the following categories:

Increasing evidence supports distinct biological roles for each of the mammalian HDACs and it is probable that inhibition of specific members of the HDAC family will have specific functional consequences such as on gene expression, cell cycle regulation, proliferation, differentiation and apoptosis. A number of HDAC isoform-selective or -specific inhibitors are currently in development (Butler and Kozikowski, 2008; Estiu et al., 2008; Haggarty et al., 2003; Jones et al., 2008; Khan et al., 2008; Kozikowski et al., 2007; Moradei et al., 2008; Rasheed et al., 2007; Somoza et al., 2004). One question in the field of HDACi development, which remains unanswered, is whether selective inhibition of HDACs would be advantageous over broader-acting HDACi as anti-cancer agents.

HDACi have a plethora of biologic effects resulting from alterations in patterns of acetylation of histones and many non-histone proteins, which include proteins involved in regulation of gene expression, pathways of extrinsic and intrinsic apoptosis, cell cycle progression, redox pathways, mitotic division, DNA repair, cell migration, and angiogenesis (Blackwell et al., 2008; Bolden et al., 2006; Dokmanovic et al., 2007; Glozak and Seto, 2007; Jones and Baylin, 2007; Minucci and Pelicci, 2006; Shankar and Srivastava, 2008; Xu et al., 2007). HDACi also have immunomodulatory activity that may contribute to mediating their anti-cancer effects. Furthermore, in contrast to most cancer-therapy agents, HDACi can induce the death of transformed cells in both proliferative and non-proliferative phases of cell cycle (Burgess et al., 2004). The mechanism(s) of action of HDACi are complex and not completely clear.

Mechanism(s) of resistance to HDACi are not well elucidated. Resistance to HDACi may reflect drug efflux, epigenetic alterations, stress response mechanisms, and anti-apoptotic and pro-survival mechanisms (Rosato et al., 2006).

According to results of pre-clinical studies and early clinical trials, HDACi in combination therapy with targeted anti-cancer drugs, cytotoxic agents, anti-angiogenesis drugs or radiation is potentially very useful (Bolden et al., 2006; Dokmanovic et al., 2007; Glozak and Seto, 2007; Jones and Baylin, 2007; Minucci and Pelicci, 2006; Nolan et al., 2008; Xu et al., 2007). In pre-clinical studies, HDACi have been shown to be synergistic with diverse chemical and biological therapeutic agents. HDACi will likely be most effective therapeutically when used in combination with other anti-cancer agents (Carew et al., 2008; Nolan et al., 2008).

Based on published reports, there are at least 20 structurally different HDACi currently in clinical trials as monotherapy and combination therapy for hematologic and solid cancers.

3.1.1 Short-chain fatty acids

Their mechanism of action of short-chain fatty acids is based on the carboxylic group, occupying the acetate escaping tunnel, that can have a zinc-binding function or compete with the acetate group released in the deacetylation reaction (Mai and Altucci, 2009).

Two short-chain fatty acids, valproic acid (VPA) and sodium phenylbutyrate, are in clinical trials (Table 1).

Table 1. Short-chain fatty acids in clinical trials.
Drug Conditions Recruitment NCT number Phases
Valproic acid & fludarabine Chronic lymphocytic leukemia Terminated NCT00524667 Phase 2
Azacitidine, valproic acid Advanced cancers Completed NCT00496444 Phase 1
Temozolomide + valproic acid Brain metastases Terminated NCT00437957 Phase 1
5-Azacytidine + valproic acid + ATRA Myelodysplastic syndromes Completed NCT00439673 Phase 2
5 azacytidine + valproic acid + retinoic acid AML, MDS Completed NCT00339196 Phase 2
Enfuvirtide + valproic acid HIV infections Terminated NCT00312546 Phase 1
Valproic acid HTLV-I-associated myelopathy Terminated NCT00519181 N/A
Karenitecin + valproic acid Malignant melanoma Terminated NCT00358319 Phase 1, 2
Valproic acid ALPS|hypersplenism|lymphadenopathy Completed NCT00605657 Phase 1, 2
Raltegravir + valproic acid HIV infections Terminated NCT00614458 Phase 2
Hydralazine + magnesium valproate Cervical cancer Completed NCT00404326 Phase 2
Hydralazine + magnesium valproate Locally advanced breast cancer Terminated NCT00395655 Phase 2
Hydralazine + magnesium valproate Refractory solid tumors Completed NCT00404508 Phase 2
Sodium phenylbutyrate Huntington's disease Completed NCT00212316 Phase 2
Sodium phenylbutyrate Amyotrophic lateral sclerosis Completed NCT00107770 Phase 1, 2
Pivanex Leukemia, lymphocytic, lymphoma, small lymphocytic Terminated NCT00083473 Phase 2
Pivanex Malignant melanoma Terminated NCT00087477 Phase 1, 2
Pivanex/docetaxel Carcinoma, non-small-cell lung Completed NCT00073385 Phase 2

VPA, first used as an anticonvulsant and mood-stabilizing agent, is a pan-HDACi. Phase 1–2 clinical trials tested VPA alone or in combination treatment for lymphocytic leukemia (Bouzar et al., 2009; Lagneaux et al., 2007; Stamatopoulos et al., 2009), AML and myelodysplastic syndromes (MDS) in combination with 5-azacytidine (Kuendgen et al., 2011), melanoma (Daud et al., 2009), HIV infection (Archin et al., 2010), autoimmune lymphoproliferative syndrome (ALPS) (Dowdell et al., 2009), human T-lymphotropic virus type-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Olindo et al., 2011).

Some phase 2 clinical trials involved the combination therapy of hydralazine, a DNA demethylating agent, and magnesium valproate for treatment of cervical cancer (Candelaria et al., 2010), breast cancer (Arce et al., 2006) and refractory solid tumors (Candelaria et al., 2007).

There are also two clinical trials with sodium phenylbutyrate: one in phase 2 for treatment of Huntington's disease (Gardian et al., 2005; Hogarth et al., 2007) and one in phase 1–2 for amyotrophic lateral sclerosis (ALS) (Cudkowicz et al., 2009; Ryu et al., 2005).

Pivaloyloxymethyl butyrate (Pivanex, AN-9) is in phase 1–2 clinical trials for chronic lymphocytic leukemia and lymphoma (Rabizadeh et al., 2007), malignant melanoma (Tarasenko et al., 2008), and non-small cell lung carcinoma (NSCLC) (Reid et al., 2004).

3.1.2 Hydroxamic acids

Hydroxamic acids include the majority of HDACi currently in clinical trials for the treatment of several human diseases such as cancer. Peculiarities of hydroxamate-based molecules are the polar hydroximic group, a 4–6 carbon hydrophobic methylene spacer (CU, polar connection unit), a second polar site, and a terminal hydrophobic group. In the majority of HDACi, the CU can interact with amino acids in the tunnel, and a linker of 4 or 6 units of carbon causing the following zinc-binding group to bind the zinc ion inhibiting the enzyme (Mai et al., 2005).

The HDACi Vorinostat is at the most advanced stage in the clinical development (Tables 2 and 3) (Siegel et al., 2009). Vorinostat, a second-generation polar-planar compound, binds to the catalytic domain of histone deacetylases (HDACs). This allows the hydroxamic moiety to chelate zinc ion located in the catalytic pockets of HDACs, thereby inhibiting deacetylation and leading to an accumulation of both hyperacetylated histones and transcription factors. Vorinostat was the first HDACi approved by the Federal Drug Administration in 2006 for clinical use in treating patients with advanced cutaneous T-cell lymphoma (Duvic and Vu, 2007; Marks and Breslow, 2007). Tables 2 and 3 show about 60 Vorinostat clinical trials, either alone or in combination, completed or terminated against multiple myeloma (MM) (Badros et al., 2009; Mazumder et al., 2010; Richardson et al., 2008), head and neck cancer (Borbone et al., 2010; Gillenwater et al., 2007), pelvic cancer (Bratland et al., 2011; Ree et al., 2010), lymphoma (Dummer et al., 2012; Kirschbaum et al., 2011; Stathis et al., 2011), leukemia and myelodysplastic syndromes (MDS) (Garcia-Manero et al., 2008b; Prebet and Vey, 2011), breast cancer (Munster et al., 2011; Shi et al., 2010), small cell lung cancer (SCLC) (Gray et al., 2012), brain and central nervous system tumors (Friday et al., 2012), prostate and urothelial cancers (Schneider et al., 2012), colorectal cancer (Fakih et al., 2012), kidney cancer (Sato et al., 2010), pancreatic cancer (Tinari et al., 2012), and ovarian cancer (Modesitt et al., 2008).

Table 2. Vorinostat in clinical trials.
Drug Conditions Status NCT number Phases
Vorinostat Glioblastoma, gliosarcoma, brain tumor Completed NCT00641706 Phase 2
Vorinostat Ovarian cancer, primary peritoneal cavity cancer Completed NCT00132067 Phase 2
Vorinostat Lymphoma Completed NCT00771472 Phase 1
Vorinostat Leukemia, myelodysplastic syndromes Completed NCT00305773 Phase 2
Vorinostat Advanced cancer relapsed and refractory Completed NCT00632931 Phase 1
Vorinostat Carcinoma, non-small-cell lung Terminated with result NCT00251589 Phase 1, 2
Vorinostat Glioblastoma, gliosarcoma, brain tumor Completed NCT01647100 Phase 2
Vorinostat Mesothelioma|lung cancer Completed NCT00128102 Phase 3
Vorinostat Breast cancer Completed NCT00132002 Phase 2
Vorinostat Lung cancer Completed NCT00138203 Phase 2
Vorinostat Brain and central nervous system tumors Completed NCT00238303 Phase 2
Vorinostat Myelodysplastic syndromes Completed NCT00776503 Phase 1, 2
Vorinostat Cancer Completed NCT00045006 Phase 1
Vorinostat Head and neck cancer Completed NCT00134043 Phase 2
Vorinostat Pelvic cancer|radiotherapy Completed NCT00455351 Phase 1
Vorinostat Hematologic and solid cancer Terminated NCT01116154 Phase 1
Vorinostat Tumors Completed NCT00127127 Phase 1
Vorinostat Brain, CNS, intestine tumors, lymphoma Completed NCT00499811 Phase 1
Vorinostat MDS, blood and bone marrow disease Terminated NCT00486720 Phase 2
Vorinostat Advanced cancer Completed NCT00907738 Phase 2
Vorinostat Lymphoma Completed NCT00127140 Phase 1
Vorinostat Colorectal cancer Completed NCT00336141 Phase 1
Vorinostat Hematologia and solid cancer Completed NCT00005634 Phase 1
Vorinostat Kidney cancer Completed NCT00354250 Phase 2
Table 3. Vorinostat in combineted clinical trials.
Drug Conditions Status NCT number Phases
Isotretinoi + vorinostat Brain, neuroblastoma, CNS, hematologic tumors Completed NCT00217412 Phase 1
Idarubicin + vorinostat Leukemia, myelodysplastic syndromes Completed NCT00331513 Phase 1
Vorinostat + gemcitabine + platinum NSCL cancer Completed NCT00423449 Phase 1
Vorinostat + pemetrexed + cisplatin Advanced cancer Completed NCT00106626 Phase 1
MK0683 + vorinostat Breast cancer, colorectal cancer, NSCL Terminated NCT00126451 Phase 2
Vorinostat + docetaxel NSCL, prostate, bladder, urothelial cancers Terminated NCT00565227 Phase 1
Vorinostat + tamoxifen Breast cancer Terminated NCT01194427 Phase 2
MK0683 + vorinostat B-cell lymphoma Completed NCT00097929 Phase 2
Carboplatin + paclitaxel + vorinostat Unspecified adult solid tumor, protocol specific Completed NCT00287937 Phase 1
Vorinostat, vinorelbine Malignant solid tumor Terminated NCT00801151 Phase 1
Azacitidine + vorinostat Leukemia, myelodysplastic syndromes Suspended NCT00392353 Phase 1, 2
Gemtuzumab ozogamicin + vorinostat Hematologic cancer Terminated NCT00673153 Phase 2
MK0683, vorinostat Cutaneous T-cell lymphoma, mycosis fungoides Completed NCT00091559 Phase 2
Vorinostat + decitabine Leukemia, acute myelodysplastic syndromes Completed NCT00479232 Phase 1
Fluorouracil, leucovorin calcium, oxaliplatin, vorinostat Colorectal cancer Completed NCT00138177 Phase 1
Carboplatin + paclitaxel + vorinostat Lung cancer Completed NCT00481078 Phase 2
Vorinostat + paclitaxel + carboplatin Stage IIIB or IV non-small cell lung cancer Terminated NCT00473889 Phase 2, 3
Chemoradiation + vorinostat Pancreatic cancer Terminated NCT00831493 Phase 1, 2
Vorinostat – bexarotene Lymphoma Terminated with with result NCT00127101 Phase 1
Vorinostat + tamoxifen Breast cancer Completed with result NCT00365599 Phase 2
Gemcitabine + vorinostat Unspecified adult solid tumor, protocol specific Completed NCT00243100 Phase 1
Vorinostat + alvocidib Unspecified adult solid tumor, protocol specific Completed NCT01645514 Phase 1
Vorinostat + alvocidib Leukemia, myelodysplastic syndromes Completed NCT00278330 Phase 1
Topotecan + vorinostat Small cell lung cancer Terminated NCT00697476 Phase 1, 2
Vorinostat + alvocidib Unspecified adult solid tumor, protocol specific Completed NCT00324480 Phase 1
Fluorouracil + leucovorin calcium + vorinostat Hematologic and solid cancer Completed NCT00942266 Phase 2
MK0683, vorinostat Neoplasms Terminated NCT00424775 Phase 1
MK0683, vorinostat Cancer, advanced Completed NCT00750178 Phase 1
Vorinostat + bortezomib + dexamethasone Relapsed or refractory multiple myeloma Completed NCT00773838 Phase 2
Vorinostat + bortezomib Lymphoma, sarcoma, Wilms tumor, neuroblastoma Completed NCT01132911 Phase 1
Vorinostat + bortezomib Unspecified adult solid tumor, protocol specific Completed NCT00227513 Phase 1
Vorinostat + bortezomib Multiple myeloma Completed NCT00111813 Phase 1
Bortezomib + dexamethasone + vorinostat Multiple myeloma and plasma cell neoplasm Completed NCT00310024 Phase 1
Vorinostat + bortezomib Multiple myeloma Completed NCT00858234 Phase 1
Vorinostat + bortezomib Leukemia, myelodysplastic syndromes Terminated NCT00818649 Phase 2
Vorinostat + bortezomib Lymphoma Terminated NCT00810576 Phase 2
Dasatinib + vorinostat Leukemia Completed NCT00816283 Phase 1

Panobinostat (LBH589) is currently in phase 1 and 2 clinical trials as monotherapy and combination therapy for hematologic tumors such as non-Hodgkin's lymphoma (Ellis et al., 2008; Shao et al., 2010), acute lymphoblastic or acute myeloid leukemia (Fiskus et al., 2009; George et al., 2005; Rosato et al., 2012; Scuto et al., 2008), MM (Maiso et al., 2006), for advanced solid tumors (Fukutomi et al., 2012), breast cancer (Tate et al., 2012; Zhou et al., 2007) and lung cancer (Crisanti et al., 2009) (Table 4).

Table 4. Hydroxamic acids in clinical trials.
Drug Conditions Recruitment NCT number Phases
Panobinostat (LBH589) Breast cancer Terminated NCT00993642 Phase 0
Panobinostat (LBH589) Cutaneous T-cell lymphoma Completed NCT00490776 Phase 2, 3
Panobinostat (LBH589) Non-Hodgkin's lymphoma Terminated NCT01090973 Phase 2
Panobinostat (LBH589) Leukemia Completed NCT00723203 Phase 2
Panobinostat (LBH589) Cancer, advanced solid tumor Completed NCT00739414 Phase 1
Panobinostat (LBH589) Lymphoma, leukemia, multiple myeloma Completed NCT00621244 Phase 1, 2
LBH589 + trastuzumab + paclitaxel HER-2 positive breast cancer, metastatic breast cancer Completed NCT00788931 Phase 1
Panobinostat + trastuzumab Breast cancer Completed NCT00567879 Phase 1
Panobinostat (LBH589) Cancer Completed NCT00570284 Phase 1
Panobinostat (LBH589) Cutaneous T-cell lymphoma, leukemia–lymphoma Terminated NCT00699296 Phase 2
Panobinostat (LBH589) Tumors, cutaneous T-cell lymphoma Completed NCT00412997 Phase 1
Panobinostat (LBH589) Recurrent malignant gliomas Terminated NCT00848523 Phase 2
Panobinostat (LBH589) Non-Hodgkin lymphoma, neoplasms Completed NCT00503451 Phase 1
Panobinostat (LBH589) Small cell lung carcinoma Completed NCT01222936 Phase 2
CHR-3996 Solid tumor Completed NCT00697879 Phase 1
CHR-2845 Hematological disease, lymphoid malignancies Completed NCT00820508 Phase 1
ITF2357 (givinostat) Hodgkin's lymphoma Terminated NCT00496431 Phase 1, 2
ITF2357 (givinostat) Myeloproliferative diseases Completed NCT00606307 Phase 2
ITF2357 (givinostat) Chronic lymphocytic leukemia Terminated NCT00792831 Phase 2
ITF2357 (givinostat) Hodgkin's lymphoma Completed NCT00792467 Phase 1, 2
ITF2357 (givinostat) Polycythemia vera Completed NCT00928707 Phase 2
Belinostat or PXD101 De novo myelodysplastic syndromes Completed NCT01647009 Phase 2
Belinostat Myelodysplastic syndromes Completed NCT00357162 Phase 2
PXD101|dexamethasone Multiple myeloma Completed NCT00131261 Phase 2
Belinostat, carboplatin, paclitaxel and bevacizumab Non-small-cell lung carcinoma Terminated NCT01090830 Phase 1, 2
Belinostat + erlotinib Non small cell lung cancer Terminated NCT01188707 Phase 1, 2
JNJ-26481585 Hematologic cancer Terminated NCT00676728 Phase 1
SB939 Solid tumor Completed NCT00504296 Phase 1
SB939 + azacitidine Solid tumors and hematologic malignancies Completed NCT00741234 Phase 1

CHR-3996 is a second-generation hydroxamic acid-based HDACi. Specifically, it is a selective class I HDAC inhibitor with potential antineoplastic activity (Moffat et al., 2010), currently in clinical trials for the treatment of advanced tumors (Banerji et al., 2012) (Table 4).

CHR-2845 is a novel hydroxamic acid derivative HDACi, which is a selective substrate for the intracellular carboxylesterase hCE-1, whose expression is restricted to cells of the monocyte–macrophage lineage. One phase 1 clinical trial has been completed for this drug in treatment of hematological diseases and lymphoid malignancies (Table 4).

SB939 is a new hydroxamic acid-based HDACi with improved physico-chemical, pharmaceutical and pharmacokinetic properties. In vitro, SB939 inhibits class I, II, and IV HDACs, with no effects on other zinc-binding enzymes, and shows significant antiproliferative activity against a wide variety of tumor cell lines (Novotny-Diermayr et al., 2010). It is in phase 1 clinical trials for treatment of solid tumors (Razak et al., 2011), hematologic malignancies and MDS (Novotny-Diermayr et al., 2012) (Table 4).

ITF2357 (Givinostat) is a novel hydroxamic acid-based HDACi that inhibits both class I and II HDACs. It has been used in five phase 1–2 clinical trials for the treatment of hematological diseases (Galli et al., 2010; Rambaldi et al., 2010) (Table 4).

PXD101 (Belinostat) is another inhibitor of class I and II HDACs that has been included in phase 2 clinical trials for MDS (Cashen et al., 2012), MM (Feng et al., 2007; Gimsing et al., 2008) and phase 1–2 for NSLC (Force et al., 2011; Luchenko et al., 2011) (Table 4).

JHJ-26481585 is a pyrimidyl-hydroxamic acid analog showing a pan-HDACi activity and is in phase 1 for leukemia and MDS (Arts et al., 2009; Stuhmer et al., 2010; Tong et al., 2010) (Table 4).

3.1.3 Benzamides

This class is composed of HDACi containing a characteristic 2′-aminoanilide moiety able to contact specific aminoacids in the tube-like active site of the HDAC core, with or without coordination/chelation of zinc ion (Pontiki and Hadjipavlou-Litina, 2012).

At least two benzamides, MS-275 (SNDX-275, Entinostat) and MGCD0103 (Mocetinostat) are in clinical development. These agents are in trials as single agents and in combination with other drugs.

Clinical trials with MS-275, a class I selective inhibitor, include patients with a wide variety of hematologic and solid neoplasms (Knipstein and Gore, 2011) such as leukemia (Gojo et al., 2007; Lucas et al., 2004), melanoma (Gore et al., 2008), MDS (Fandy et al., 2009) and colorectal cancer (Bracker et al., 2009) (Table 5).

Table 5. Benzamides in clinical trials.
Drug Condition Status NCT number Phases
Entinostat Melanoma Completed NCT00185302 Phase 2
Entinostat Cancer Completed NCT00020579 Phase 1
Entinostat Leukemia, multiple myeloma and plasma cell neoplasm, MDS Completed NCT00015925 Phase 1
Entinostat + isotretinoin Lymphoma, small intestine cancer Completed NCT00098891 Phase 1
Azacitidine + entinostat Leukemia, myelodysplastic syndromes Completed NCT00101179 Phase 1
Azacitidine + entinostat Colorectal cancer Completed NCT01105377 Phase 2
MGCD0103 + docetaxel Solid cancer Terminated NCT00511576 Phase 1
Azacitidine + MGCD0103 Acute myeloid leukemia (AML)|myelodysplastic syndrome (MDS) Terminated NCT00666497 Phase 2
MGCD0103 Hodgkin's lymphoma Terminated NCT00358982 Phase 2
MGCD0103 Myelodysplastic syndrome, acute myelogenous leukemia Completed NCT00324220 Phase 1, 2
MGCD0103 Leukemia, myelodysplastic syndromes Completed NCT00324129 Phase 1
MGCD0103 Tumors, non-Hodgkin's lymphoma Completed NCT00323934 Phase 1
MGCD0103 Lymphocytic leukemia, chronic Completed NCT00431873 Phase 2
MGCD0103 Lymphoma Completed NCT00359086 Phase 2
MGCD0103 Leukemia, myelodysplastic syndromes Completed NCT00324194 Phase 1
MGCD0103 Myelogenous leukemia, acute, myelodysplastic syndromes Terminated NCT00374296 Phase 2
MGCD0103 + gemcitabine Tumors Completed NCT00372437 Phase 1, 2
MGCD0103 + azacitidine Hodgkin lymphoma Terminated NCT00543582 Phase 2

MGCD0103 is an isotype-selective HDACi that potently targets human HDAC1 but also exerts inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro (Fournel et al., 2008). Some phase 1–2 clinical trials are for treatment of hematological diseases such as leukemia (Blum et al., 2009; El-Khoury et al., 2010; Garcia-Manero et al., 2008a), lymphoma (Buglio et al., 2010; Younes et al., 2011) and also for solid cancers (Siu et al., 2008) (Table 5).

3.1.4 Cyclic peptides

Romidepsin (Depsipeptide or FK228) acts as an HDACi with the disulfide bond undergoing reduction within the cell to release a zinc-binding thiol. Thiol interacts with a zinc atom in the binding pocket of zinc-dependent HDAC to block its activity. Romidepsin is a natural product obtained from the bacteria Chromobacterium violaceum. On November 5, 2009, it was approved by the FDA for the treatment of cutaneous T-cell lymphoma (CTCL). Terminated clinical trials show the activity of this compound in treating lymphoma (Coiffier et al., 2012), MM (Khan et al., 2004; Kikuchi et al., 2010; Niesvizky et al., 2011), CTCL (Piekarz et al., 2011; Prince and Dickinson, 2012), MDS (Klimek et al., 2008), and solid tumors such as pancreatic (Sato et al., 2004), colorectal (Whitehead et al., 2009), lung (Otterson et al., 2010; Schrump et al., 2008), prostate (Lai et al., 2008), renal (Stadler et al., 2006), bladder (Karam et al., 2007), brain (Iwamoto et al., 2011), thyroid (Kitazono et al., 2001) and ovarian cancers (Son et al., 2010) (Table 6).

Table 6. Romidepsin in clinical trials.
Drug Conditions Recruitment NCT number Phases
Romidepsin + bortezomib Multiple myeloma Terminated NCT00765102 Phase 2
Romidepsin + ketoconazole Hematologic cancer Completed NCT01324310 Phase 1
Romidepsin (FR901228) Mycosis fungoides, CTCL, neoplasms Terminated NCT01445340 Phase 1
Romidepsin Peritoneal, epithelial, ovarian cancer Completed NCT01645670 Phase 2
Romidepsin + rifampin Hematologic cancer Completed NCT01324323 Phase 1
Romidepsin (FR901228) Lymphoma Completed NCT00383565 Phase 2
Romidepsin + gemcitabine Pancreatic cancer Completed NCT00379639 Phase 1, 2
Romidepsin (depsipeptide, FK228) Cutaneous T-cell lymphoma Completed NCT00106431 Phase 2
Depsipeptide, FR901228, FK228 Neoplasms Completed NCT00048334 Phase 1
Romidepsin + rituximab + fludarabine Lymphoma Completed NCT00079443 Phase 2
Romidepsin Brain and nervous system tumors Completed NCT00085540 Phase 2
Romidepsin Lung cancer Completed NCT00086827 Phase 2
Romidepsin Colorectal cancer Completed NCT00077337 Phase 2
Romidepsin Bladder, renal pelvis and urethral cancer Terminated NCT00087295 Phase 2
Romidepsin Leukemia, lymphoma Completed NCT00024180 Phase 1
Romidepsin Lymphoma Completed NCT00019318 Phase 1
Romidepsin Leukemia, lymphoma, MDS syndrome Completed NCT00042822 Phase 2
Romidepsin + decitabine Leukemia, lymphoma, MDS syndrome Completed NCT00114257 Phase 1
Romidepsin Breast cancer Completed NCT00098397 Phase 2
Romidepsin Esophageal cancer, gastric cancer Suspended NCT00098527 Phase 2
Romidepsin Ovarian, primary peritoneal cavity cancer Completed NCT00091195 Phase 2
Romidepsin Lymphoma Completed NCT00077194 Phase 2
Romidepsin Head and neck cancer Completed NCT00098813 Phase 2
Decitabine + depsipeptide Small cell lung carcinoma Completed NCT00037817 Phase 1
Romidepsin Nervous system tumors, leukemia Completed NCT00053963 Phase 1
Romidepsin (depsipeptide, FK228) Prostate cancer, metastases Completed NCT00106418 Phase 2
Romidepsin (FR901228) Small cell lung carcinoma Completed NCT00020202 Phase 2
Romidepsin (depsipeptide, FK228) Renal cell carcinoma, metastases Completed NCT00106613 Phase 2

3.2 Sirtuins

Class III HDACs, also known as sirtuins, are the silent information regulator 2 (Sir2) family of proteins. Distinct from other HDACs, sirtuins are NAD+-dependent protein deacetylases, not modulated by HDACi. The mammalian sirtuin family comprises seven proteins (SirT1-7), which differ widely in their cellular localization, activity and functions, and are subdivided into 4 classes (Carafa et al., 2012). The deacetylase activity of sirtuins is controlled by the cellular [NAD+]/[NADH] ratio; NAD+ works as an activator, whereas nicotinamide and NADH inhibit their activity. Two reactions are catalyzed by sirtuins: deacetylation and ADP-ribosylation. In both, the cleavage of NAD+ is the initial chemical step. SirT1, 2, 3, 5 and 7 catalyze a deacetylation reaction, in which they deacetylate lysine residues of target proteins using NAD+ as cofactor and releasing nicotinamide with the production of 2′-O-acetyl-ADP ribose. In contrast, SirT4 and 6, catalyze ADP-ribosylation reaction, during which ADP-ribosyl moiety is transferred to the protein substrate (Yamamoto et al., 2007).

Expressed from bacteria to humans (Vaquero, 2009), sirtuins seem to preferentially target non-histone proteins, though little is known about target specificity and selectivity. Sirtuins are connected to chromatin regulation as they are responsible for controlling two post-translational histone modifications crucial for chromatin structure: H4K16ac and H3K9ac. SirT1, 2, 3 and 6 are involved in chromatin regulation (McGuinness et al., 2011). SirT1, the best-studied family member, is responsible for heterochromatin formation by a deacetylating process.

In the last decade interest for sirtuins has grown, mainly due to their critical role in several biological processes, such as regulation of gene expression, control of metabolic processes, apoptosis and cell survival, DNA repair, development, neuroprotection and inflammation. Sirtuins control many vital functions and are involved in many disorders such as metabolic diseases, neurodegenerative diseases and cancer (Stunkel and Campbell, 2011). SirT1 displays contradictory roles, and has been suggested either as tumor suppressor or tumor promoter (Bosch-Presegue and Vaquero, 2011; Deng, 2009). The initial evidence of SirT1 as a tumor promoter derived from its repressive effect on the tumor suppressor p53. However, a potential tumor suppressor role has also been proposed for other human sirtuins (McGuinness et al., 2011). This hypothesis is mainly sustained by reduction of SirT2 in a large number of human brain tumor cell lines, and its involvement in cell cycle progression. SirT3 is the only mitochondrial sirtuin implicated in tumorigenesis. Its reduction in several cancers leads to an increase in reactive oxygen species (ROS) production, which results in enhanced tumor growth. SirT5 overexpression has been implicated in a study of pancreatic cancer (Kim et al., 2010). Recently, the role of SirT6 and SirT7 in tumorigenesis has been demonstrated. SirT6 controls NF-kB pathway and plays a role in DNA double-strand repair, indicating that this sirtuin has a key function in tumorigenesis. However, very little is known about the specific correlation with cancer. mRNA levels of SirT7 have been inversely correlated with the ability to undergo tumorigenesis in mouse cell lines, and levels of SirT7 have been found elevated in some forms of breast cancer (Ashraf et al., 2006).

Sirtuins are involved in aging diseases such as metabolic disease, neurodegeneration and aging itself. It is well known that overexpression of Sir2 (or its orthologs) can extend organism lifespan in a wide range of lower eukaryotes (Bosch-Presegue and Vaquero, 2011; Vaquero, 2009). Sir2 functions are often correlated to calorie restriction (CR). The link between the role of sirtuins, CR and longevity was first described in Saccharomyces cerevisiae. In yeast, CR leads to increased replicative lifespan. Lifespan extension was not observed in yeast lacking the Sir2 gene. Currently, the role of sirtuins in the regulation of mammalian lifespan is not clear. However, starting from the premise that sirtuins are an evolutionary conserved protein family, it is fair to assume that they play a role in the modulation of aging-related processes in higher organisms (Brooks and Gu, 2009; Westphal et al., 2007). In humans, the aging process is associated to telomere erosion. SirT1 and 6 are involved in the maintenance of telomeres and telomeric function, and are implicated in the aging process. Recent studies have demonstrated that reduction or removal of SirT6 results in telomere dysfunction and end-to-end chromosomal fusions. In terms of symptoms, the absence of SirT6 is similar to a disease characterized by premature aging, Werner's syndrome. Although very little is known about other sirtuins, no evidence suggests their involvement with telomere function, formation and stability (McGuinness et al., 2011).

Given that SirT1 has been reported to increase tumorigenesis, and despite its role as tumor suppressor or promoter, it is important to identify small chemical compounds that inhibit or activate SirT1. To date, a number of specific inhibitors of SirT1 have been proposed for cancer therapy (Alcain and Villalba, 2009). Moreover, both activators and inhibitors of sirtuins might act beneficially against different types of neurodegenerations. Thus, in addition to nicotinamide, the physiological inhibitor, some specific inhibitors have been characterized, including splitomicin and its analogs, tenovins, AGK2, sirtinol, suramin, the indole derivative EX-257, salermide and UVI5008.

SIRT activators are being studied in the hope of providing benefit to patients with neurodegenerative, inflammatory, metabolic and autoimmune diseases, and some tumor types.

Phenol derivates, including quercetin, piceatannol, and resveratrol, have been shown to posses SirT1-activating properties (Alcain and Villalba, 2009). The most potent SirT activator, and the first to be characterized, is resveratrol, a polyphenol found in grapes, and grape products. Subsequently, much more potent and efficacious SirT1 activators were reported as potential therapeutics for treatment of metabolic diseases (SRT1720, SRT2183, and SRT1460) (Saunders and Verdin, 2007). However, their activity is still debated. The question is whether they are SIRT activators or assay artifacts. Given that the activation of SirT1 by activators requires the use of fluorescently labeled substrate in a fluorescence assay, it was demonstrated that they do not activate SirT1 when using native peptide or protein substrate conjugated with a non-physiological fluorophore (Borra et al., 2005). To establish their effective modulation of SirT1, other technical approaches are necessary. Despite these issues, resveratrol is now in clinical trials (Table 7). Resveratrol can exhibit benefits against cardiovascular diseases (CVDs) or in its prevention, although its cardioprotective role as part of the human diet is not yet clear (Ruana et al., 2012). CR is a low calorie diet (about 30% fewer calories than the American Dietetic Association (ADA) recommends). CR has also been linked to health benefits (enhanced cardiovascular and metabolic health) and an extended lifespan. Many studies have compared the health benefits of both resveratrol and CR to determine whether resveratrol mimics some of the health benefits shown with CR. The consumption of this polyphenol could modulate cerebral blood flow and this in turn could influence cognitive performance by increasing access to blood-borne metabolic fuel. Research shows that resveratrol is able to induce vasodilation (and therefore blood flow) by interacting with nitric oxide (NO). Because tumors develop resistance to chemotherapeutic agents, the aim of cancer research is to discover effective chemosensitizers. One promising possibility is to use dietary agents, such as resveratrol, that sensitize tumors to chemotherapeutics (Aggarwal et al., 2004). Through its ability to modulate multiple cell-signaling molecules such as cell survival proteins, members of NF-kB and STAT3 pathways, resveratrol is able to prevent cancer (Gupta et al., 2011). Tumors shown to be sensitized by resveratrol include lung and breast cancer, AML, promyelocitic leukemia, MM, prostate, pancreatic and epidermoid cancers (Fulda and Debatin, 2004). Patients with colon cancer received treatment with resveratrol, and correlative laboratory studies examined its effects directly on colon cancer and normal colonic mucosa. Resveratrol may stop the growth of tumor cells by blocking some of the enzymes required for cell growth, and by inducing cell cycle arrest, apoptosis, inhibition of cell proliferation, stimulation of antiangiogenic responses and increased antioxidant and anti-inflammatory activity (Talero et al., 2012). The activity of resveratrol shows great potential in the prevention and therapy of a wide variety of human diseases.

Table 7. Resveratrol in clinical trials.
Drug Conditions Status NCT number Phases
Resveratrol and midazolam Healthy Completed NCT01173640
Resveratrol Cognitive and cerebral blood flow Completed NCT01010009
Resveratrol Cardiovascular diseases Completed NCT01449110 Phase 2
Resveratrol Colon cancer Completed NCT00256334 Phase 1
Resveratrol Chronic subclinic inflammation Completed NCT01492114 Phase 3
Resveratrol Type 2 diabetes mellitus Completed NCT01038089
Resveratrol Healthy Completed NCT01331382
Resveratrol Sports concussion Completed NCT01321151 Phase 1
Resveratrol Neoplasms, colorectal cancer Completed NCT00920803 Phase 1
Resveratrol Sedentary lifestyle Completed NCT01615445 Phase 2
Resveratrol Melanoma (skin) Completed NCT00721877 Phase 1
Resveratrol Metabolic syndrome, obesity Completed NCT01150955
Resveratrol Multiple myeloma Terminated NCT00920556 Phase 2
Resveratrol Healthy Completed NCT01640197 Phase 1
Resveratrol Colorectal cancer Completed NCT00433576 Phase 1
Resveratrol Unspecified adult solid tumor Completed NCT00098969 Phase 1
Resveratrol Obesity, metabolic syndrome, diabetes Completed NCT00823381
Resveratrol Obesity Completed NCT00998504
Resveratrol Cardiovascular disease Completed NCT01199549
Resveratrol Cellulite (orange peel skin) Terminated NCT01321268
Resveratrol Type 2 diabetes Completed NCT00937222

3.3 HAT inhibitors

Of the several known covalent histone modifications, the reversible acetylation of key lysine residues in histones holds a pivotal position in transcriptional regulation. Histone acetylation is a distinctive feature of transcriptionally active genes, whereas deacetylation indicates the repressed state of a gene. The balance between the acetylation and deacetylation states of histones regulates transcription. Dysfunction of enzymes involved in these events is often associated with the manifestation of several diseases, including cancer, cardiac hypertrophy and asthma (Kramer et al., 2001; McKinsey and Olson, 2004; Yang, 2004). These enzymes are therefore potential new targets for therapy. Acetyl transferases (HATs) modulate gene expression by catalyzing targeted acetylation of the ϵ-amino group of lysine residues on histone and non-histone proteins. HATs can be classified into several families on the basis of number of highly conserved structural motifs. These include the GNAT family (Gcn5-related N-acetyltransferase, e.g., Gcn5, PCAF), the MYST group (MOZ, YBF2/SAS3 and TIP60) and the p300/CBP family (Kramer et al., 2001; Sterner and Berger, 2000; Yang, 2004).

Although a wide number of transcriptional co-activator proteins are now recognized to possess HAT activity, very few HAT inhibitors (HATi) have been identified to date. Availability of recombinant HATs made it possible to synthesize and test more target-specific inhibitors: Lys-CoA for p300 and H3-CoA-20 for PCAF (Lau et al., 2000). Though Lys-CoA has been extensively employed for in vitro transcription studies, it is unable to permeate cells (Cebrat et al., 2003). In the early 2000s, two important HATis were isolated: anacardic acid from cashew nut shell liquid and garcinol from Garcinia indica, which are both non-specific inhibitors of p300/CBP and PCAF but are capable of easily permeating cells in culture (Balasubramanyam et al., 2004a, 2003). Different chemical modifications of these inhibitors were attempted to identify enzyme-specific inhibitors, but it serendipitously led to the synthesis of the only known p300-specific activator, N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) (Balasubramanyam et al., 2003) (Souto et al., 2010, 2008). None of the above is in clinical trials.

Curcumin (diferuloylmethane) is the major curcuminoid of turmeric, Curcuma longa, a characteristically orange-yellow colored spice often found in curry powder. In recent years, considerable interest has been focused on this substance due to its use in treating a wide variety of disorders without any side effects. It was used in ancient times to treat various illnesses such as rheumatism, body ache, skin diseases, intestinal worms, diarrhea, intermittent fevers, hepatic disorders, biliousness, urinary discharges, dyspepsia, inflammation, constipation, leukoderma, amenorrhea, and colic. Curcumin has the potential to treat a wide variety of inflammatory diseases including cancer, diabetes, cardiovascular diseases, arthritis, Alzheimer's disease and psoriasis through modulation of numerous molecular targets. Curcumin was identified as the first p300/CBP-specific cell permeable HATi (Balasubramanyam et al., 2004b). It does not affect the HAT activity of PCAF or histone deacetylase and methyltransferase activities. However, p300 HAT activity-dependent chromatin transcription is efficiently repressed by curcumin but not transcription from DNA template. Curcumin could also inhibit histone acetylation in vivo. It is the only HATi in clinical trials (Table 8) and exhibits great promise as a therapeutic agent. Its applications include atopic asthma (Kobayashi et al., 1997), chronic obstructive pulmonary disease (Rennolds et al., 2012), multiple myeloma (Ghoneum and Gollapudi, 2011), irritable bowel syndrome (Binion et al., 2008; Rapin and Wiernsperger, 2010), ulcerative colitis (Baliga et al., 2012), Crohn's disease (Mouzaoui et al., 2012), breast cancer (Nagaraju et al., 2012), Alzheimer's disease (Darvesh et al., 2012; Huang et al., 2012), pancreatic cancer (Dandawate et al., 2012; Veeraraghavan et al., 2011), colorectal cancer (Guo et al., 2012; Lin et al., 2011), diabetes (Abdel Aziz et al., 2012), and psoriasis (Kurd et al., 2008).

Table 8. Curcumin in clinical trials.
Drugs Conditions Recruitment NCT number Phases
Curcumin Atopic asthma Completed NCT01179256 N/A
Curcumin + bioperine Multiple myeloma Completed NCT00113841 N/A
Curcumin Irritable bowel syndrome Completed NCT00779493 Phase 4
Curcumin Healthy Completed NCT00181662 N/A
Curcumin Healthy Completed NCT00895167 Phase 1
Curcumin Breast cancer Completed NCT01042938 Phase 2
Curcumin Alzheimer's disease Completed NCT00164749 Phase 1 and 2
Curcumin Inflammatory bowel disease|ulcerative colitis|Crohn's disease Completed NCT00889161 Phase 1
Curcumin + bioperine Chronic obstructive pulmonary disease Completed NCT01514266 N/A
Curcumol Chemotherapy induced mucositis Completed NCT00475683 Phase 3
Curcumin + bioperine Mild cognitive impairment Completed NCT00595582 N/A
Curcumin C3 complex Alzheimer's disease Completed NCT00099710 Phase 2
Curcumin + fluoxetine Major depressive disorder Completed NCT01022632 N/A
Curcumin + gemcitabine Pancreatic cancer Completed NCT00192842 Phase 2
Curcumin Familial adenomatous polyposis Terminated NCT00248053 Phase 2
Curcumin Colorectal cancer Completed NCT00027495 Phase 1
Curcumin Aberrant crypt foci Terminated NCT00176618 N/A
Curcumin Healthy, no evidence of disease Completed NCT00768118 N/A
Curcumin + quercitin + sulindac Colorectal cancer Terminated NCT00003365 N/A
Observational study Diabetes Completed NCT01029327 N/A
Curcumin C3 complex Psoriasis Completed NCT00235625 Phase 2
Curcuminoids Oral lichen planus Completed NCT00525421 Phase 2
Standardized turmeric root extract Cystic fibrosis Completed NCT00219882 Phase 1
Carbohydrate drink Nutrition processes Completed NCT00799630 N/A

3.4 Histone methyltransferases

Histone methylation has been shown to play a key function in the regulation of gene-expression patterns and DNA repair; this kind of post-transcriptional (epigenetically controlled) modification can affect lysine (K) or arginine (R) residues of histone tails (Greer and Shi, 2012). In contrast to histone acetylation, histone methylation does not alter the charge of the histone tail, but influences the basicity, hydrophobicity of histones and their affinity to certain proteins such as transcription factors (Rice and Allis, 2001). A methyl group is relatively small and its addition to lysine or arginine residues does not neutralize their charge, and it is therefore unlikely that methylation alone will significantly affect the chromatin structure (Bannister and Kouzarides, 2011). Lysine side chains may be mono-, di- or tri-methylated, whereas the arginine side chain may be mono-methylated or (symmetrically or asymmetrically) di-methylated (Smith and Denu, 2009).

On the basis of target residue for methylation, histone methyltransferases (HMTase) can be grouped into two different enzymatic classes: lysine methyltransferases and arginine methyltransferases.

Arginine methylation of histones H3 (Arg2, 17, 26) and H4 (Arg3) promotes transcriptional activation and is mediated by the family of protein arginine methyltransferases (PRMTs), including the co-activators PRMT1 and CARM1 (PRMT4). In contrast, a more diverse set of histone lysine methyltransferases has been identified, all but one of which contain a conserved catalytic SET domain originally identified in the Drosophila Su[var]3–9, enhancer of zeste, and Trithorax proteins. Lysine methylation has been implicated in both transcriptional activation (H3 Lys4, 36, 79) and silencing (H3 Lys9, 27, H4 Lys20).

At present, there are many well-known methylation sites on histones. Taking into consideration all three possible methylation states of lysine and arginine, an enormous number of methylation states of histones exist. This might explain the difficulty in studying the methylation pattern on histones.

Conversely, due to the complex pattern of histone methylation, it may be possible to interfere with this enzyme in a promising manner.

Although attempts to interfere with DNA methylation (e.g., by DNMTIs) and histone deacetylation (e.g., by HDACIs) have received the bulk of attention, recent efforts have begun to focus on pharmacologic disruption of other epigenetic regulatory processes. Histone methylation represents one such target, but to date studies on specific HMTi classes are far from the clinic.

3.4.1 Histone methyltransferase inhibitors

The first applied inhibitor used as an anticancer drug was S-adenosylmethionine (SAM) and its analogs (e.g., SAH). However, these compounds target not only HMTs but also other enzymatic classes using AdoMet as methyl-donor (such as DNMTs). Their use is therefore limited by low specificity (Spannhoff et al., 2009a).

As a lysine methyltransferase, chaetocin, a fungal mycotoxin, has been reported to act against G9a at low concentration without inhibition of other KMT enzymes (such as EZH2 or SET7/9) (Copeland et al., 2009; Greiner et al., 2005; Spannhoff et al., 2009b). Inhibition mediated by chaetocin is competitive against the co-substrate SAM. Chaetocin potently induces cellular oxidative stress, selectively killing cancer cells and rapidly proliferating primary cells (Isham et al., 2007). The effects of chaetocin on oxidative stress are at least in part due to its capacity to act as a competitive and selective substrate for theoredoxin reductase-1 (Km = 4.6 μM) (Tibodeau et al., 2009).

Another specific inhibitor of G9a is BIX-01294 (and its derivative BIX-01338, both hydrochloride hydrates), which is effective in vitro at a concentration of 2.7 μM with no effects on SUV39H1 and PRMT1 (Kubicek et al., 2007; Shi et al., 2008). Cell lines treated with BIX-01294 showed a reduction in histone H3 lysine 9 (H3K9) dimethylation, while the mono- or tri-methyl levels were unaffected. Other lysine methylation sites such as H3K27 or H4K20 were not altered. Based on a kinetic inhibition model, BIX-01294 showed an uncompetitive pattern compared to the co-substrate SAM, suggesting that it only binds to G9a complexed with SAM. BIX01294 has been used in combination with the calcium channel activator BayK8644 to facilitate the generation of induced pluripotent stem cells from somatic cells in vitro.

UNC0224 is another potent and relatively selective G9a HMTi, exhibiting an IC50 value of 15 nM. Isothermal titration calorimetry revealed that UNC0224 binds to G9a with a Kd value of 29 nM. UNC0224 also inhibits G9a-like protein (GLP), a closely related H3K9 HMTase, with assay-dependent IC50 values of 20–58 nM, but is over 1000-fold selective against SET7/9 (a H3K4 HMTase) and SET8 (a H4K20 HMTase) (Liu et al., 2009).

The lysine methyltransferase EZH2 (KMT6), part of the polycomb repressive complex 2, catalyzes trimethylation of lysine 27 on histone H3 and is involved in proliferation and aggressive cell growth associated with neoplastic cells. Given its importance in cell proliferation and homeostasis maintenance, intense efforts have been directed toward discovering specific EZH2 inhibitors (Simon and Lange, 2008). 3-Deazaneplanocin A (DZnep) is a cyclopentenyl analog of 3-deazaadenosine, originally synthesized as an inhibitor of S-adenosyl-l-homocysteine hydrolase (Tseng et al., 1989). It has been shown to deplete EZH2 levels and to inhibit trimethylation of lysine 27 on histone H3 in cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells in a dose-dependent manner (0.2–1 μM) (Fiskus et al., 2009). DZnep treatment of cultured human AML cells induces increased expression of the cell-cycle regulators p21, p27, and FBXO32, leading to cell cycle arrest and apoptosis. When used in combination with the pan-HDACi Panobinostat (10 mg/kg), the antileukemic effects of DZnep (1 mg/kg) are synergistically enhanced in mice implanted with AML cells.

The arginine methyltransferase enzyme AMI-1 (and subsequently its derivatives AMI-2 to AMI-6) is the first and to date best-known inhibitor. It is cell-permeable, symmetrical sulfonated urea compound that acts as a potent, specific and non-AdoMet (SAM)-competitive inhibitor of protein arginine N-methyltransferases (PRMTs; IC50 = 8.81 μM for PRMT1 and 3.03 μM for yeast-RMT1p) with minimal effect on lysine methyltransferases. It inhibits nuclear receptor reporter gene activation in MCF-7 cells, and HIV-1 RT polymerase (IC50 = 5 μM).

In summary, although many of these inhibitors are able to reduce or abolish HMT activity in vitro or in cell-based assays, they are still at pre-clinical stage due to the low specificity and toxicity observed in different cell lines.

3.5 Histone demethylase enzymes and their inhibitors

Two families of histone demethylating enzymes (HDs) have recently been discovered. Lysine-specific demethylase 1 (LSD1) is a flavin-dependent monoamine oxidase which can demethylate mono- and di-methylated lysines, specifically histone 3, lysines 4 and 9 (H3K4 and H3K9) (Forneris et al., 2005). Jumonji domain-containing (JmjC) histone demethylases are able to demethylate mono-, di-, or tri-methylated lysines. Two specific JmjC HDs are PHF8 and JHDM1D.

LSD1 shares similar catalytic sites with monoamine oxidases (MAO) A and B, the inhibition of which is used clinically to treat depression, anxiety, and Parkinson's disease (Lee et al., 2006).

2-PCPA (Tranylcypromine) is an inhibitor of LSD1 with an IC50 value of 20.7 μM and a Ki value of 242.7 μM, which effectively inhibits histone demethylation in vivo. Although not as selective, 2-PCPA also irreversibly inhibits MAO A and MAO B with IC50 values of 2.3 and 0.95 μM and Ki values of 101.9 and 16 μM, respectively (Schmidt and McCafferty, 2007). Given these collateral effects, Tranylcypromine has not yet entered clinical trials.

4 DNA methyltransferases enzymes and their inhibitors

DNA methylation refers to a covalent modification of the cytosine base (C), localized at 5′ of a guanidine base (G) in a CpG dinucleotide (Das and Singal, 2004; Robertson, 2001; Robertson and Wolffe, 2000). DNA methylation is involved in the control of gene expression, regulation of parental imprinting and stabilization of X chromosome inactivation as well as maintenance of genome integrity. It is also implicated in the development of immune system, cellular reprogramming, brain function and behavior (Di Croce et al., 2002).

The transfer of methyl groups from SAM to cytosine in CpGs is catalyzed by a family of enzymes called DNA methyltransferase (DNMTs) (Iyer et al., 2011; Jurkowska et al., 2011; Peedicayil, 2012; Xu et al., 2010). In mammals, three DNMTs have so far been identified, including two “de novo” methyltransferases (DNMT3A and DNMT3B) and “maintenance” methyltransferase (DNMT1), generally the most abundant and active of the three (Goll and Bestor, 2005). The protein DNMT2 can also be found in mammalian cells. Although the structure of DNMT2 is very similar to other DNMTs, its role is less understood (Schaefer and Lyko, 2010). It has been reported that DNMT2 does not methylate DNA but instead methylates aspartic acid transfer RNA (tRNAAsp) (Jurkowski and Jeltsch, 2011). Recent evidence suggests that DNMT2 activity is not limited to tRNAAsp and that DNMT2 represents a non-canonical enzyme of the DNMT family (Schaefer et al., 2010). Given the critical role of DNMTs, intense interest has focused on developing drugs able to interfere with aberrant DNMT activities, and using them to correct epigenetic defects such as tumor suppression gene (TSG) silencing (Rajendran et al., 2011).

DNMTs modulators represent a useful tool in epigenetic therapies. Several epi-drugs interfering with DNMT activity are currently in pre-clinical and clinical trials (Foulks et al., 2012; McGovern et al., 2012). Most of these trials have involved various types of cancer, such as solid and hematological tumors (Chaib et al., 2011; Fandy, 2009; Ren et al., 2011; Song et al., 2011). Currently, however, the main challenge in using epigenetic modulators for therapy, especially for interfering with DNMT enzymes is their specificity (Veeck and Esteller, 2010).

It is well known that abnormal patterns of DNA methylation are often displayed in human neoplasms (Chin et al., 2011; Fonseca et al., 2012; Lokk et al., 2012; Peedicayil, 2012). A combination of regional promoter hypermethylation with the concomitant silencing of important genes involved in cell death, surveillance and proliferation is always present. However, demethylation per se is not sufficient to induce gene expression, as it is regulated by a combination of different epi-mutations (Choo, 2011; Eglen and Reisine, 2011). DNA methylation and histone modifications are tightly correlated because inactive chromatin is enriched with hypermethylated DNA and active chromatin is associated with hypomethylated DNA. In contrast, genomic hypomethylation mediates tumorigenesis (via chromosomal instability) and supports the metastatic process (Hatziapostolou and Iliopoulos, 2011; Kulis and Esteller, 2010; Plitta et al., 2011; Watanabe and Maekawa, 2010). Consequently, induction of global DNA hypomethylation using DNMTi may help speed tumor progression from cancer cells surviving DNA demethylation therapy (Guz et al., 2010; Villa et al., 2004; Wild and Flanagan, 2010).

Moreover, the interrelation between drugs targeting chromatin and those targeting DNA methylation could be utilized therapeutically by combining different epigenetic drugs against different epigenetic effectors to increase the efficacy of a monotherapy.

Several clinical trials testing different DNMTi are terminated and ongoing.

4.1 Nucleoside analogs

This class of DNMTi includes nucleoside analogs, which are phosphorylated by cellular kinases. Once incorporated into DNA, they form a covalent bond with the DNMT trapping the enzyme and making it unavailable for further methylation, thus resulting in demethylation of replicating nascent DNA (Szyf, 2009). Two agents have been developed clinically: 5-aza-2′-deoxycytidine (decitabine) and 5-azacytidine (azacitidine or Vidaza). These agents have been tested in numerous trials (Goffin and Eisenhauer, 2002; Sorm et al., 1964) (Tables 9–12).

Table 9. Decitabine in clinical trials.
Drug Conditions Status NCT number Phases
Decitabine MDS|CML Completed NCT00067808 Phase 2
Decitabine Thalassemia Completed NCT00661726 Phase 2
Decitabine Lymphoma, intestinal neoplasms Completed NCT00089089 Phase 1
Decitabine AML Completed NCT00866073 Phase 2
Decitabine AML, MDS Completed NCT00760084 Phase 2
Decitabine MDS, CML Terminated NCT00113321 Phase 2
Decitabine Leukemia Completed NCT01378416 Phase 1
Decitabine CML Completed NCT01098084 Phase 2
Decitabine AML Completed NCT00398983 Phase 2|3
Decitabine MDS Completed NCT00043381 Phase 3
Decitabine AML Completed NCT00538876 Phase 1
Decitabine MDS Terminated NCT00282399 Phase 1|2
Decitabine CML Completed NCT00042003 Phase 2
Decitabine CML Completed NCT00042016 Phase 2
Decitabine CML Completed NCT00041990 Phase 2
Decitabine MDS Completed NCT00260065 Phase 2
Decitabine MDS Completed NCT01041846 N/A
Decitabine MDS Completed NCT00796003 Phase 1
Decitabine AML Completed NCT00358644 Phase 2
Decitabine Leukemia, MDS Completed NCT00003361 Phase 2
Decitabine Leukemia, MDS Completed NCT00049582 Phase 1
Decitabine MDS, secondary myelofibrosis Terminated NCT00630994 Phase 2
Decitabine Bladder cancer, breast cancer, melanoma Completed NCT00030615 Phase 1
Decitabine Esophageal, lung cancer, malignant mesothelioma Completed NCT00019825 Phase 1
Decitabine Leukemia Completed NCT00042796 Phase 1
Decitabine MDS, leukemia, lymphoma, MM Completed NCT00002980 Phase 1
Table 10. Decitabine in combinated clinical trials.
Drug Conditions Status NCT number Phases
Decitabine + vorinostat Myeloproliferative disorders, leukemia Completed NCT00357708 Phase 1
Decitabine + interferon alfa-2b Renal cell carcinoma Terminated NCT00561912 Phase 2
Decitabine + filgrastim + cyclosporine Leukemia, MDS Completed NCT00002832 Phase 1|2
Decitabine + VPA Leukemia, MDS Completed NCT00075010 Phase 1|2
Decitabine + cytarabine or supportive care AML Completed NCT00260832 Phase 3
Decitabine + carboplatin Fallopian tube, ovarian, peritoneal cavity cancer Terminated NCT00748527 Phase 2
Decitabine + azacitidine MDS Terminated NCT01011283 Phase 4
Decitabine + arsenic trioxide + ascorbic acid MDS Completed NCT00621023 Phase 2
Decitabine + PEG-interferon alfa-2b Unspecified adult solid tumor Completed NCT00701298 Phase 1
Decitabine + pegylated interferon-alfa 2b Cancer Terminated NCT00886457 Phase 1
Decitabine + chemotherapy AML Terminated NCT00943553 Phase 2
Decitabine + VPA Lymphoma Completed NCT00109824 Phase 1
Decitabine + romidepsin SCC|mesothelioma|NSCLC Completed NCT00037817 Phase 1
Decitabine + vorinostat Leukemia|MDS Completed NCT00479232 Phase 1
Decitabine + filgrastim + pegfilgrastim + cyclophosphamide + doxorubicin hydrochloride Neuroblastoma, childhood solid tumor Completed NCT00075634 Phase 1
Decitabine + imatinib mesylate Leukemia Completed NCT00054431 Phase 2
Decitabine + romidepsin Leukemia, MDS Completed NCT00114257 Phase 1
Decitabine + filgrastim + busulfan + cyclophosphamide + cyclosporine + methotrexate + methylprednisolon + tacrolimus Leukemia Completed NCT00002831 Phase 1|2
Decitabine + AMG 531 (romiplostim) + azacitidine MDS, thrombocytopenia Completed NCT00321711 Phase 2
Decitabine deferoxamine + deferiprone + arginine + sildenafil Hematologic diseases, osteoporosis, hypertension Completed NCT00000623 N/A
Table 11. Azacitidine in clinical trials.
Drug Conditions Status NCT number Phases
Azacitidine MDS|AML Completed NCT00795548 Phase 2
Azacitidine Ovarian cancer Terminated NCT00842582 Phase 1
Azacitidine MDS|leukemia Completed NCT00350818 Phase 1
Azacitidine Myelofibrosis Completed NCT00569660 Phase 2
Azacitidine MDSs Completed NCT00118287 Phase 1|2
Azacitidine Leukemia Completed NCT00739388 Phase 2
Azacitidine MDSs Completed NCT01186939 Phase 3
Azacitidine AML|MDSs Terminated NCT00446303 Phase 2
Azacitidine MDSs Completed NCT00102687 Phase 2
Azacitidine MDSs Completed NCT00071799 Phase 3
Azacitidine for injectable suspension Prostate cancer Completed NCT00384839 Phase 2
Azacitidine Beta thalassemia Completed NCT00005934 Phase 2
Azacitidine CMPD, secondary myelofibrosis Terminated NCT00381693 Phase 2
Table 12. Azacitidine in combinated clinical trials.
Drug Conditions Status NCT number Phases
Azacitidine + VPA + ATRA AML|MDS Completed NCT00339196 Phase 2
Azacitidine + VPA + ATRA MDSs Completed NCT00439673 Phase 2
Azacitidine + Ara-C AML, MDS, leukemia Completed NCT00569010 Phase 1|2
Azacitidine + sodium phenylbutyrate Leukemia, MDSs Completed NCT00004871 Phase 1
Azacitidine + arsenic trioxide Leukemia, MDSs Terminated NCT00234000 Phase 1
Azacitidine + sodium phenylbutyrate Lymphoma, small intestine cancer Completed NCT00005639 Phase 1
Azacitidine + entinostat Colorectal cancer Completed NCT01105377 Phase 2
Azacitidine + arsenic trioxide Leukemia, MDSs Completed NCT00118196 Phase 2
Azacitidine + amifostine trihydrate MDSs Completed NCT00005598 Phase 2
Azacitidine + sodium phenylbutyrate Hematologic and solid tumors Completed NCT00006019 Phase 2
Azacitidine + liothyronine sodium + radiation: iodine I 131 Head and neck cancer Completed NCT00004062 Phase 1
Azacitidine + sodium phenylbutyrate Thalassemia major Terminated NCT00007072 Phase 2
Azacitidine + AMG 531 (romiplostim) + decitabine MDS, thrombocytopenia Completed NCT00321711 Phase 2
Azacitadine and hematopoietic growth factors Leukemia, MDSs Terminated NCT00398047 N/A
Intravenous azacitidine MDSs Completed NCT00384956 Phase 2
Azacitidine AML, MDS Completed NCT00422890 Phase 3
Azacitidine + VPA + ATRA MDS, AML Completed NCT01575691 Phase 1
Azacitidine + MGCD0103 AML, MDS Terminated NCT00666497 Phase 2
Azacitidine + cisplatin Squamous cell carcinoma Terminated NCT00443261 Phase 1
Azacitidine + entinostat Lung cancer Suspended NCT00387465 Phase 1|2
Azacitidine + erythropoietin MDSs Terminated NCT00379912 Phase 2
Azacitidine + VPA + ATRA MDS, AML Completed NCT00326170 Phase 2
Azacitidine + midostaurin Hematologic cancers Suspended NCT01093573 Phase 1|2
Azacitidine + VPA Advanced cancers Completed NCT00496444 Phase 1
Azacitidine + Ara-C + VPA AML, MDS, leukemia Completed NCT00382590 Phase 2
Azacitidine, erlotinib Advanced solid tumor malignancies Completed NCT00996515 Phase 1
Azacitidine, decitabine MDSs Terminated NCT01011283 Phase 4
Azacitidine, MGCD0103 Hodgkin and non-Hodgkin lymphoma Terminated NCT00543582 Phase 2
Azacitidine, entinostat Leukemia, MDSs Completed NCT00101179 Phase 1

Decitabine is incorporated into DNA, while azacitidine is incorporated preferentially into RNA (Leone et al., 2002). Demethylation by decitabine has been shown to allow re-expression of silenced genes and cellular differentiation. Additionally, incorporation of these agents into RNA causes ribosomal disassembly, defective tRNA function and inhibited protein production. However, azacitidine exhibits greater cytotoxicity during S-phase, supporting the greater importance of its DNA effects. Both these drugs are inactivated via deamination by cytidine deaminase (Galmarini et al., 2001) and have been approved by the FDA for the treatment of myelodysplastic syndromes (Kadia et al., 2011; Marks, 2012).

To date, decitabine and azacitidine have been studied as single drug treatment (Tables 9 and 11) for many malignancies, though the best results have been obtained for MDS and various types of leukemia.

These DNMTis have been in clinical trial for hematological diseases, such as MDS and leukemias (Chen et al., 2012; Garcia-Manero, 2012; Keating, 2012; Kimura et al., 2012; Oki et al., 2012; Platzbecker et al., 2012; Ritchie, 2012), thalassemia (Mabaera et al., 2008; Olivieri et al., 2011; Rose et al., 2011; Saunthararajah et al., 2003) and solid tumors, such as prostate (Gravina et al., 2011; Shabbeer et al., 2012), colon (Al-Salihi et al., 2011; Hagemann et al., 2011; Ikehata et al., 2012; Yang et al., 2012), bladder (Shang et al., 2008), breast (Mirza et al., 2010), melanoma (Alcazar et al., 2012; Liu et al., 2011), esophageal (Dong et al., 2012; Liu et al., 2005; Meng et al., 2012; Schrump et al., 2006), lung cancers (Kaminskyy et al., 2011; Wu and Hu, 2011).

The most promising results have been obtained with combination therapy, especially with HDACi (Tables 10 and 12).

4.2 Small molecules

Blocking the enzymatic activity of DNMTs by using small molecule inhibitors is another strategy to achieve gene demethylation (Datta et al., 2009). Hydralazine and procainamide are FDA approved for the treatment of hypertension and cardiac arrhythmia, respectively (Peng et al., 2010). Recently, their ability to inhibit DNMT activity has been discovered and is associated with direct binding to CpG-rich sequences (Amatori et al., 2010). More precisely, these molecules act as partial competitive inhibitors of DNMT1, decreasing the affinity of DNMT for its substrates (DNA and S-adenosyl-l-methionine), reducing the processivity of the enzyme and favoring the dissociation of DNMT1 from hemimethylated DNA. No data are currently available for their use in clinical trails as DNMT inhibitors; however, they are in pre-clinical studies.

The antihypertensive (Singh et al., 2009) hydralazine was tested as a DNMT inhibitor due to its capability to induce (as a side effect) a lupus-like syndrome known to be related to disorders associated with DNA methylation (Candelaria et al., 2011; Lu et al., 2005). Although its mechanism of action is still under investigation, some evidence indicates that hydralazine, similar to procaine and procainamide, binds to CpG-rich sequences and interferes with translocation of DNMTs along the DNA strand. A phase 1 study has shown that hydralazine is able to induce re-expression of various tumor suppressor genes, including p16 and RAR-β, in cervical cancer patients, even at lower doses than those considered safe for the treatment of cardiovascular disorders. On the basis of promising data arising from phase 1, hydralazine is currently under phase 2 and 3 investigations. Table 1 reports three clinical trials in which hydralazine is used in combination with magnesium valproate against solid tumors (discussed in “ Short-chain fatty acid”).

A major drawback of these drugs is the high concentrations required for their demethylating activity, which can elicit undesired toxic effects if administered clinically.

Rational design of DNMTi that interact noncovalently with the active catalytic site of DNMTs, utilizing a three-dimensional model of the human DNMT1 catalytic pocket, is a sound alternative approach to silence the DNA methylation machinery. RG108 [2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl)propanoic acid] is the first rationally designed DNMT1i that demonstrates demethylating activity both in vivo and in vitro (Braun et al., 2010; Brueckner et al., 2005; Chestnut et al., 2011; Schirrmacher et al., 2006; Suzuki et al., 2010).

This molecule was found to induce the re-expression of different hypermethylation-silenced genes, such as p16 and the putative tumor suppressor genes SRFP1 and TIMP-3, in colon cancer cells. Despite its encouraging activity, RG108 is still in pre-clinical phase.

4.3 Natural molecules

The use of natural products in cancer chemoprevention is currently receiving much attention.

Psammaplins, for example, are bisulfide bromotyrosines derived from a marine sponge and are able to inhibit both DNMT1 and HDAC in vitro. Although psammaplin A administration at low doses was found to exert a strong cytotoxic effect in human tumor cell lines and to limit tumor cell growth in a A549 lung xenograft mouse model, DNMT inhibition was not followed by DNA demethylation and re-expression of tumor suppressor genes, suggesting that an intracellular target different from DNMT1 is responsible for the cytotoxic effect of the molecule (Baud et al., 2012).

Tea polyphenols are strong antioxidants and tea preparations demonstrate inhibitory activity against carcinogenesis. (−)-Epigallocatechin-3-gallate (EGCG), the major polyphenol from green tea, is a potent inhibitor of catechol-O-methyltransferase activity (COMT) (Fang et al., 2007; Lee et al., 2005; Nandakumar et al., 2011). The structural similarity between DNMTs and COMT suggests possible inhibition of DNMTs by EGCG. EGCG inhibits DNMT activity in a dose-dependent manner and induces re-expression of hypermethylated genes such as CDKN2A, RARbeta and MGMT (Nandakumar et al., 2011). In many pre-clinical studies, EGCG exhibits a very strong DNMT inhibitory action and this natural molecule is able to induce cell death and apoptosis in many cancer types (Gu et al., 2009).

Interestingly, other dietary catechol-containing polyphenols, such as different tea catechins (catechin, epicatechin) and bioflavonoids (quercetin, genistein, fisetin), were also found to inhibit DNMT activity in vitro through mechanisms different to that of EGCG (Fang et al., 2007; Lee et al., 2005). The results obtained from the use of natural compounds are intriguing, especially considering their ease of use and low costs. However, further investigation is necessary to establish their real efficacy as DNMTi, as well as to evaluate the toxicity induced by their administration at pharmacological doses.

A major concern associated with the use of natural products is product standardization. Multiple sources can provide extracts with different activities and therefore create discrepancies in their reported demethylating activity.

4.4 Antisense oligonucleotide inhibitors of DNMTs

Antisense oligonucleotides are short, defined sequences of nucleotides that are complementary to mRNAs. They hybridize mRNAs, making them inactive and thereby blocking translation. Antisense oligonucleotides that are complementary to mRNAs for human DNMT1 are undergoing pre-clinical (Yan et al., 2003) as well as clinical trials (Davis et al., 2003).

The idea to target directly DNMT1 enzyme arises from the observation that, excluding some rare exceptions, tumor cells generally show increased expression levels of DNMTs.

The most interesting isotypic-specific DNMT1 inhibitor tested in clinical trials is MG98 (Amato et al., 2012; Patutina et al., 2009; Winquist et al., 2006), a second-generation antisense oligonucleotide that specifically targets DNMT1 mRNA. This agent eliminates the expression of DNMT1 protein resulting in the inhibition of DNA replication, triggering of damage response, and induction of TSGs. The immediate blockage of replication by DNMT1 knockdown dramatically limits demethylation induced by DNMT1 inhibition, thus avoiding the potential deleterious impact of global demethylation. The main issue with antisense oligonucleotides is delivery to solid tumors (Davis et al., 2003; Klisovic et al., 2008; Plummer et al., 2009).

Some clinical trials utilizing MG98 were stopped because of lack of objective response in the last phase 2 trials in metastatic renal cancer. Nevertheless, this strategy carries great promise. A new trial has been completed (Stewart et al., 2003) in which pharmacodynamic evaluations were performed to explore and validate the biological mechanisms of MG98 in solid tumors (Clinical trial identifier: NCT00003890).

Interesting results are provided by miR-29b, a microRNA (miRNA) that directly targets DNMT3A and DNMT3B expression. MiR-29b induces a decrease in methylation levels and induces the re-expression of hypermethylated TSGs, FHIT and WWOX in lung cancer cells, as well as of p15 and ER in AML cells. Although these studies are all still at pre-clinical level, the above findings support the possibility of miRNA-based approaches.

5 Non-coding RNAs

MicroRNAs (miRNAs) are short RNA molecules, 19–25 nucleotides long, that bind to complementary sequences in the 3′ UTR of multiple target mRNAs, usually resulting in their silencing. miRNAs were recently identified as key players in regulating gene expression by inhibiting translation and/or triggering degradation of their target mRNAs (Bartel, 2004). miRNAs target ∼60% of all genes, are abundantly present in all human cells and are each able to repress hundreds of targets. Most miRNAs are transcribed by RNA polymerase II and subsequently processed by multiple maturation steps resulting in mature double-stranded miRNA duplexes. miRNAs are implicated in a wide range of basic biological processes, including development, differentiation, apoptosis and proliferation (Bartel, 2004; Harfe, 2005) and their misregulation is linked to the development of diseases in humans and other organisms. Researchers worldwide have validated the theory of “miRNA replacement therapy”, which involves introducing synthetic miRNAs or miRNA mimetics into diseased tissues in an attempt to restore normal proliferation, apoptosis, cell cycle, and other cellular functions that have been affected by the misregulation of one or more miRNAs (Raver-Shapira et al., 2007).

Over the last 5 years, a particularly important role for miRNAs in cancer pathogenesis has emerged. Many tumor types are characterized by globally abnormal miRNA expression patterns. Up to now, 23 studies have been completed (Table 13), and describe the use of miRNAs in cancer therapy. miRNA expression profiles are highly informative for tumor classification, prognosis, and response to therapy. Moreover, recent results have documented a functional contribution of specific miRNAs to cellular transformation and tumorigenesis. The pioneering groups of specialized pharmaceutical companies have initiated studies on creating viable therapeutic candidates with miRNA inhibitors and miRNA mimetics in diverse fields such as cancer, cardiovascular diseases, neurological disorders, and viral infections.

Table 13. miRNAs in clinical trials.
Drug Conditions Status NCT number Phases
Observational study Sepsis Completed NCT00862290 N/A
Observational study Cutaneous melanoma Completed NCT01444560 N/A
Observational study Aspirin-exacerbated respiratory disease Completed NCT01631773 N/A
Observational study Periodontal disease Completed NCT01399034 N/A
Observational study Basal cell carcinoma Completed NCT01498250 N/A
Observational study Cutaneous squamous cell carcinoma Completed NCT01500954 N/A
Observational study Malignant melanoma Completed NCT00862914 N/A
Observational study Skin cancer Completed NCT00849914 N/A
Observational study Leukemia Completed NCT01057199 N/A
Observational study Barrett's esophagus, esophagea, adenocarcinoma Completed NCT00909350 N/A
Observational study Cutaneous malignant melanoma Completed NCT01482260 N/A
Observational study Cancer of the skin Terminated NCT01143311 N/A
Observational study Melanoma Completed NCT00536029 N/A
Observational study Skin cancer Completed NCT01345760 N/A
Observational study The study focused on reducing tobacco smoke exposure for child Completed NCT01317628 N/A
Observational study Muscle anabolism Completed NCT00812396 Phase 1
Observational study Hepatitis C Completed NCT00688012 Phase 1
Observational study Lung cancer Completed NCT00897234 N/A
Observational study Influence of genotype of drug Completed NCT01503658 Phase 4
Observational study Awaiting organ transplant|infection in solid organ transplant recipients Completed NCT01558037 N/A
Observational study Melanoma (skin) Completed NCT01216787 Phase 2
Observational study Neuroblastoma Completed NCT01058798 N/A
Observational study Recurrent calcic urolithiasis Completed NCT01022060 Phase 3

6 Summary and concluding remarks

The present review highlights the enormous impact that small molecules such as HDACi and DNMTi have and will continue to have in the treatment of human diseases, especially cancer. Their key importance is due to the differentiation- and apoptosis-inducing activity of this new class of anticancer drugs. By targeting histone (chromatin) modulators, epi-drugs activate a complex transcriptional reprogramming, exemplified by reactivation of TSGs and repression of oncogenes.

Widespread changes in post-translational modifications of histones and DNA determine diseases and represent marks that play a crucial role in chromatin packaging and gene expression. The aberrant recruitment of HDACs and/or DNMTs or the activation of miRNAs in normal cells are involved in a number of pathologies, primarily cancer. It has been reported that HDACis/HMTis or DNMTis induce strong re-expression of TSGs and generally induce transcription-regulating activity on various genes, cell cycle inhibitory activity and apoptosis. Hence, their importance in epigenetic therapy. The vast number of clinical trials underlines the promising use of these drugs in diagnostic therapeutics of human diseases. However, their application is more effective in combination therapy. Trials related to combination therapies are based on the recognition that certain combinations of compounds combine more effectively with each other, improve tolerance, require lower dosages of each agent, and/or reduce side effects caused by at least one of the compounds in the combination. Although many anti-cancer therapies exist, there is a need to develop therapeutics that are safe and effective, circumvent resistance to hormonal and other therapies, do not cause the onset of other pathologies, and extend the disease-free survival of patients.

Cells that have developed mutations within the drug-binding pocket display a growth advantage in the presence of the drug, eventually leading to disease progression. Current clinical strategies aimed at combining these molecularly targeted drugs with standard chemotherapeutics, radiation, or other targeted agents will lead to novel strategies aimed at improving the overall response rate and increasing the number of complete remissions.

A good synergistic effect is obtained by the combination of drugs that inhibit DNMTs and HDACs.

In recent years, we have been facing the new concept of “personalized therapy”, which takes into consideration individual differences between patients, and represents an attempt to ethically and medically improve cost performance of medication treatment by administering a pharmaceutical agent to patients after verification in advance of the probability of its effect, thereby enhancing efficacy as well as avoiding toxicity of the agent, and reducing inappropriate use of drugs. In cancer treatment, the development of a method for predicting the efficacy of anti-cancer agents is highly desirable as it may be an important way to bridge the gap between basic research and clinical application.

The clinical benefits of epi-therapy are currently being investigated in several human diseases but better results could undoubtedly be obtained with the development of new-generation epi-drugs. It is therefore essential to identify a characteristic profile for successful candidates as epi-drugs.


This work was supported by EU: Blueprint (contract no. 282510), ATLAS (contract no. 221952); Epigenomics Flagship Project EPIGEN (MIUR-CNR); the Italian Association for Cancer Research (AIRC no. 11812); Italian Ministry of University and Research (PRIN_2009PX2T2E_004; PON002782; PON0101227). We thank Catherine Fisher for linguistic editing of the manuscript. The authors declare that they have no conflict of interest.