Epigenetics

EpigeneticsTime

Introduction

Cancer represents a spectrum of diseases that share clinical, pathologic, genetic and epigenetic features. The tenets of oncogene activation concomitant with inactivation of a tumour suppressor gene remain central to the theme of oncogenesis; however, knowledge of epigenetic regulation of these two events, as well as of events before and after the process of malignant transformation, is contributing greatly to our understanding of cancer.
Distinct from direct mutational changes in the DNA sequence, epigenetic regulation of transcription occurs through a variety of mechanisms that include:

  1. Direct methylation of DNA
  2. Chemical modifications of histone proteins
  3. Chromatin Remodeling

Recognizing both genetic and epigenetic pathways has led to a better understanding of oncogenesis and is improving the classification and diagnosis of cancer. Unravelling the mechanisms of epigenetic regulation has resulted in the identification of novel targets and medications for cancer treatment.

How Epigenetics Affects Genetics

The term “epigenetics” was originally coined by Conrad Waddington to describe heritable changes in a cellular phenotype that were independent of alterations in the DNA sequence. Despite decades of debate and research, a consensus definition of epigenetics remains both contentious and ambiguous. Epigenetics is most commonly used to describe chromatin-based events that regulate DNA-templated processes, and this will be the definition we use in this review.
While epigenetics and genetics can cooperate in cancer initiation and progression, the interconnectedness between of these two processes is becoming increasingly apparent with the realization that several epigenetic modifiers are mutated in human cancers. Some examples of genetic mutations of epigenetic modifiers are shown in Table 1. The mutation of epigenetic modifiers presumably leads to profound epigenetic changes, including aberrant DNA methylation, histone modifications, and nucleosome positioning, although this remains to be demonstrated. These epigenetic alterations can lead to abnormal gene expression and genomic instability, which may predispose to cancer.

Gene Function Tumor Type Alteration
DNA methylation DNMT1 DNA methyltransferase Colorectal, non-small cell lung, pancreatic, gastric, breast cancer Mutation (Kanai et al., 2003)
Overexpression (Wu et al., 2007)
DNMT3A DNA methyltransferase MDS, AML Mutation (Ley et al., 2010, Yamashita et al., 2010 and Yan et al., 2011)
DNMT3B DNA methyltransferase ICF syndrome, SNPs in breast and lung adenoma Mutation (Wijmenga et al., 2000)
Mutation (Shen et al., 2002)
MBD1/2 Methyl binding protein Lung and breast cancer Mutation (Sansom et al., 2007)
TET1 5′methylcytosine hydroxylase AML Chromosome translocation (De Carvalho et al., 2010 and Wu and Zhang, 2010)
TET2 5′methylcytosine hydroxylase MDS, myeloid malignancies (AML), gliomas Mutation/silencing (Tan and Manley, 2009)
IDH1/2 Isocitrate dehydrogenase Glioma, AML Mutation (Figueroa et al., 2010, Lu et al., 2012 and Turcan et al., 2012)
AID 5′cytidine deaminase CML Aberrant expression (De Carvalho et al., 2010)
Histone modification MLL1/2/3 Histone methyltransferase H3K4 Bladder TCC, ALL and AML, non-Hodgkin lymphoma, B cell lymphoma, prostate (primary) Translocation, mutation, aberrant expression (Gui et al., 2011 and Morin et al., 2011)
BRD4 Bromodomain containing 4 Nuclear protein in testis, midline carcinoma, breast, colon, and AML Translocation (fusion protein), aberrant expression (Filippakopoulos et al., 2010 and Zuber et al., 2011)
EZH2 Histone methyltransferase H3K27 Breast, prostate, bladder, colon, pancreas, liver, gastric, uterine tumors, melanoma, lymphoma, myeloma, and Ewing’s sarcoma Mutation, aberrant expression (Chase and Cross, 2011 and Tsang and Cheng, 2011)
ASXL Enhancer of trithorax and polycomb group (EAP) Additional sex combs like 1 MDS and AML, Bohring-Opitz syndrome Mutation (Gelsi-Boyer et al., 2012 and Hoischen et al., 2011)
BMI-1 PRC1 subunit Ovarian, mantle cell lymphomas and Merkel cell carcinomas Overexpression (Jiang et al., 2009 and Lukacs et al., 2010)
G9a Histone methyltransferase H3K9 HCC, cervical, uterine, ovarian, and breast cancer Aberrant expression (Varier and Timmers, 2011)
PRMT1/5 Protein arginine methyltransferase Breast/gastric Aberrant expression (Miremadi et al., 2007)
LSD1 Histone demethylase H3K4/H3K9 Prostate Mutation (Rotili and Mai, 2011)
UTX (KDM6A) Histone demethylase H3K27 Bladder, breast, kidney, lung, pancreas, esophagus, colon, uterus, brain Mutation (Rotili and Mai, 2011)
JARID1B/C Histone demethylase H3K4/H3K9 Testicular and breast, RCCC Overexpression (Rotili and Mai, 2011)
EP300 Histone deacetyltransferase Breast, colorectal, pancreatic cancer Mutation (Miremadi et al., 2007)
CREBBP Histone acetyltransferase Gastric and colorectal, epithelial, ovarian, lung, esophageal cancer Mutation, overexpression (Miremadi et al., 2007)
PCAF Histone acetyltransferase Epithelial Mutation (Miremadi et al., 2007)
HDAC2 Histone deacetyltransferase Colonic, gastric, endometrial cancer Mutation (Ropero et al., 2006)
SIRT1, HDAC5/7A Histone deacetyltransferase Breast, colorectal, prostate cancer Mutation, aberrant expression (Miremadi et al., 2007)
Chromatin remodeling SNF5 (SMARCB1, INI1) BAF subunit Kidney malignant rhabdoid tumors, atypical rhabdoid/teratoid tumors (extra-renal), epithelioid sarcomas, small cell hepatoblastomas, extraskeletal myxoid chondrosarcomas, and undifferentiated sarcomas Mutation, silencing, loss of expression (Wilson and Roberts, 2011)
BRG1 (SMARCA4) ATPase of BAF Lung, rhabdoid, medulloblastoma Mutation, low expression (Wilson and Roberts, 2011)
BRM (SMARCA2) ATPase of BAF Prostate, basal cell carcinoma Mutation, low expression (de Zwaan and Haass, 2010 and Sun et al., 2007)
ARID1A (BAF250A) BAF subunit Ovarian clear cell carcinomas, 30% of endometrioid carcinomas, endometrial carcinomas Mutation, genomic rearrangement, low expression (Guan et al., 2011 and Jones et al., 2010)
ARID2 (BAF200) PBAF subunit Primary pancreatic adenocarcinomas Mutation (Li et al., 2011)
BRD7 PBAF subunit Bladder TCC Mutation (Drost et al., 2010)
PBRM1 (BAF180) PBAF subunit Breast tumors Mutation (Varela et al., 2011)
SRCAP ATPase of SWR1 Prostate Aberrant expression (Balakrishnan et al., 2007)
P400/Tip60 ATPase of SWR1, acetylase of SWR1 Colon, lymphomas, head-and-neck, breast Mutation, aberrant expression (Mattera et al., 2009)
CHD4/5 ATPase of NURD Colorectal and gastric cancer, ovarian, prostate, neuroblastoma Mutation (Bagchi et al., 2007, Kim et al., 2011 and Wang et al., 2011a)
CHD7 ATP-dependent helicase Gastric and colorectal Mutation (Wessels et al., 2010)

 Table 1.  Table 1. Epigenetic Modifiers in Cancer

MDS – myelodysplastic syndromes; AML – acute myeloid leukemia; TCC – transitional cell carcinoma; RCCC – renal clear cell carcinoma.

DNA Methylation Machinery

DNA methylation in mammals occurs predominantly at CpG dinucleotides, and methylation of CpG islands acts as a relatively stable gene silencing mechanism. DNA methylation is regulated by a family of DNA methyltransferases (DNMTs). DNMT3A and DNMT3B, which are expressed throughout the cell cycle, establish new DNA methylation patterns early in development. During replication, the original DNA methylation pattern is maintained largely by DNMT1 activity and is therefore responsible for the maintenance of methylation patterns during cell division, with some participation by DNMT3A and DNMT3B.
Mutations of DNMT1, 3A & 3B have been shown to be present in numerous solid and haematological cancers. In addition to the various mutations, DNMT1, DNMT3A, and DNMT3B are often overexpressed in various cancers and possibly contribute to ectopic hypermethylation. Additionally, mutations in Methyl-binding domain (MBD) proteins (e.g. MeCP2, MBD1, MBD2, and MBD4) have been shown to increase the risk of certain solid tumours.
Demethylation can occur through two processes: active and passive. Passive DNA demethylation occurs when maintenance DNA methylation is impaired during DNA replication, resulting in loss of methylation of the newly synthesized DNA strand. In contrast, active DNA demethylation is dependent on the ability of enzymes to hydroxylate (TET proteins) followed by deamination (AID/APOBEC proteins) and can occur independent of DNA replication. Three TET family members (TET1, TET2, and TET3) have been reported so far, and each protein seems to have a distinct function in different cellular contexts with mutations been found in several haematological diseases.
Considering that DNMTs/MBD proteins and enzymes involved in DNA demethylation contribute directly to the level of DNA methylation but also to nucleosome occupancy patterns, the alteration of these machineries in cancer development could be broader than previously realized.

Histone Modification Machinery

Nucleosomes, which are the basic building blocks of chromatin, contain DNA wrapped around histones. Histones are regulators of chromatin dynamics either by changing chromatic structure by altering electrostatic charge or providing protein recognition sites by specific modifications. Histone modifications at specific residues characterize genomic regulatory regions.
These histone modification patterns are regulated by enzymes include:

  1. Histone acetyltransferases (HATs) and deacetylases (HDACs), which introduce and remove acetyl groups, respectively.
  2. Histone methyltransferases (HMTs) introduce methyl groups
  3. Histone demethylases (HDMs), on the other hand, remove methyl groups.

During tumorigenesis, cells undergo global changes in histone modifications and in the distribution of histone variants which may affect the recruitment of transcription factors (TFs) and often components of the transcription machinery, thereby contributing to aberrant gene expression.

Histone acetyltransferases (HAT) & Histone deacetylases (HDAC)

Mutations of HATS have been observed in solid and haematological cancers, whereas HDACs have been implicated in cancer due to their aberrant binding and consequent silencing of tumor suppressor genes. As a result, HDAC inhibitors have been developed as anti-cancer drugs, and Screening for mutations that confer resistance to HDAC inhibitors may improve the efficacy.
In addition to chromatin modifying enzymes, chromatin binding proteins or so-called epigenetic “readers”, such as the bromodomain proteins (e.g. Brd4) which read lysine acetylation marks, can also play an important during tumorigenesis.

Histone methyltransferase (HMT)

HMTs involve methylation of arginine and lysine residues on histones or nonhistone proteins such as TFs regulate chromatin structure and therefore gene expression. Mutations in HMTs have been linked with mixed linkage leukaemia (MLL) and in addition, alternative splicing and mutations in MLL1, MLL2, and MLL3 genes have been identified in bladder, breast, and pancreatic cancers and in glioblastoma.
The Polycomb group (PcG) of repressor proteins controls the accessibility of gene regulatory elements to the transcription machinery. This group is crucial for early development and often becomes deregulated in cancer. EZH2, together with SUZ12 and EED, form the polycomb repressive complex 2, which methylates H3K27. Overexpression of EZH2 has been reported in several cancers such as prostate, breast, lung, and bladder and seems to result in an increase in H3K27me3 and EZH2 mutations have been reported in lymphoma and myeloid neoplasm. The EZH2 mutations in myeloid neoplasms are associated with poor prognosis, and the mutations frequently result in loss of function of HMT. Although the mechanism of action of EZH2 in cancer is not yet clear, it appears to play a role in growth control.

Histone demethylases (HDM)

Two distinct classes of HDMs have been defined based on their mechanism of action. Lysine-specific histone demethylase 1 (LSD1), lysine-specific demethylase 6A (KDM6A/UTX), and jumonji C-domain containing proteins (JARID1A-D) have all been implicated in tumorigensis. Mutations in LSD1 (prostate cancer) and KDM6A/UTX (various cancers including bladder, breast, kidney, and colon) have been reported. Reintroduction of KDM6A/UTX in the UTX mutant cancer cells results in the slowing of proliferation, suggesting that genetic mutations of these enzymes reinforce the epigenetic deregulation in cancers.
The exact mechanism by which these histone modifying enzymes affect tumorigenesis remains to be elucidated; altered expression of histone modifiers caused by mutations may disrupt whole epigenetic regulation mechanisms and result in aberrant gene expression patterns. Indeed, the disruption of histone modifications has been linked to all the hallmarks of cancer, and it is important to be aware that a precise balance between the enzymes that write, read, and erase histone marks is crucial in preventing tumorigenesis.

Chromatin Remodelling Complexes

Chromatin remodeling is the dynamic modification of chromatin architecture to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression. Such remodeling is principally carried out by:

  1. Covalent histone modifications by specific enzymes, i.e., histone acetyltransferases (HATs), deacetylases, methyltransferases, and kinases
  2. ATP-dependent chromatin remodeling complexes which either move, eject or restructure nucleosomes.

Besides actively regulating gene expression, dynamic remodeling of chromatin imparts an epigenetic regulatory role in several key biological processes, e.g., DNA replication and repair; apoptosis; chromosome segregation as well as development and pluripotency. Aberrations in chromatin remodeling proteins are found to be associated with human diseases, including cancer. Targeting chromatin remodeling pathways is currently evolving as a major therapeutic strategy in the treatment of several cancers.
Nucleosome occupancy is a key mechanism for gene expression, and it has been known for some time that chromatin remodelers are responsible for regulating this process. ATP dependent chromatin remodelers are generally divided into four main families:

  1. switch/sucrose non-fermenting (SWI/SNF)
  2. imitation SWI
  3. inositol requiring 80
  4. nucleosome remodeling and deacetylation chromatin helicase DNA binding (NURD/Mi2/CHD) complexes.

Although the ATPase domains are highly similar, the distinct chromatin interacting domains carry out specific roles and can be selectively targeted. These ATPase dependent remodelers act in the context of multisubunit complexes and have dual roles as activators and repressors of gene expression. The importance of chromatin remodeling machines is becoming apparent with the realization that many of them are mutated in several types of cancer.
In addition to SWI/SNF complexes, mutations of other ATP dependent chromatin remodelers are beginning to be identified in several cancers. Despite emerging evidence that closely connects these ATPase remodelers in tumorigenesis, the direct causality and/or mechanism still remains to be explicated.

Epigenetic drugs in clinical use and trials

Tumours that display global epigenomic alterations might also benefit from therapies that restore these global patterns. Some of the modifying enzymes that put in place the mark are mutated in disease and can be targeted by specific molecules, representing the first examples of the development of personalized medicine therapies that combine genomic and epigenomic knowledge. Considering the dynamic and reversible nature of epigenetic marks, they represent an attractive target for personalized therapy. However, treatment with currently approved epigenetic drugs (DNA methyl transferase (DNMT) and histone deacetylase (HDAC) inhibitors) is rather broad, and yet to be defined epigenetic cancer subtypes might respond differently. Here, high-resolution profiling might improve the treatment efficiency by guiding the therapy decision. Currently approved epigenetic treatments are described below.

Epigenetic drugs in clinical use [4]

To date, five epigenetic drugs have been approved by the US Food and Drug Administration (FDA):

  • 2 DNA methyltransferase (DNMT) inhibitors.  (i) 5-azacytidine (5-aza-CR; manufactured by Celgene as Vidaza) was approved in 2004 specifically to inhibit DNA methylation. Two years later its variant 5-aza-2′-deoxycytidine was approved (5-aza-CdR; manufactured by Eisai as Dacogen). Both were approved for the treatment of higher-risk myelodysplastic syndrome (MDS). (ii) S110, which is a dinucleotide containing 5-aza-CdR and is suggested to have enhanced stability and efficiency, completes the list of DNMT inhibitors that are in use in the clinical setting.
  • 3 histone deacetylase (HDAC) inhibitors. (i) Vorinostat (Merck, known as Zolinza) — an FDA-approved HDAC inhibitor for the treatment of cutaneous T cell lymphoma (CTCL) — was also confirmed to induce complete response or haematological improvement in AML patients. (ii) Romidepsin (Celgene, known as Istodax)  revealed remarkable efficacies in the treatment of cutaneous T cell lymphoma (CTCL) patients. (iii) Panobinostat (Novartis, known as Farydak) was recently approved (February 2015) by the FDA for treatment of  Multiple Myeloma. An additional HDAC inhibitors —  CI-994 (Pfizer, known as Tacedinaline) — is currently being tested in clinical phase III trials for the treatment of non-small-cell lung cancer (NSCLC).

A new generation of targeted epigenetic drugs

Owing to an increased resolution of genomic and epigenetic profiling methods, targeted epigenetic treatments, such as the specific inhibition of enzymes by small molecules, are being developed, adding to the drug arsenal to improve drug performance further while reducing toxicity to healthy tissue. Specific treatments that target epigenetic modifiers are the subject of current investigation, and the most recent examples are briefly considered in the following section.

Bromodomain and extra-terminal domains (BETs) are adaptor molecules that are involved in chromatin-dependent signal transduction, resulting in transcriptional initiation and elongation. The bromodomain family member BRD4 has been shown to be involved in gene fusions in squamous carcinomas and leukaemia. The small molecule JQ1 has been established as a potent inhibitor of BRD4, and JQ1 treatment showed strong antiproliferative effects in cell line and xenograft models that harboured BRD4 fusion. Chromosomal translocation involving mixed lineage leukaemia (MLL), which is a histone lysine methyl transferase, is an initiator of aggressive forms of leukaemia with extremely poor prognosis. MLL fusion partners, especially members of the super elongation complex (SEC), such as AF4, AF6, AF9, AF10 and ENL, have been identified as interaction partners with BET proteins. Using the small-molecule inhibitor of the BET family I-BET151 causes displacement of BRD3–BRD4 and SEC from chromatin, followed by inhibition of co-activator function and subsequent repression of key oncogenes, such as MYC, B cell CLL/lymphoma 2 (BCL2) and cyclin-dependent kinase 6 (CDK6).

Fusion products of translocated MLL genes also initiate aberrant recruitment of DOT1L, which is a histone methyltransferase that is involved in histone H3 lysine 79 (H3K79) methylation, to MLL target promoters, resulting in mislocalized enzymatic activity. In patients harbouring the translocated MLL genes, altered DOT1L activity functions as a downstream event associated with the activation of leukaemogenic genes such as homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Recently, Daigle and co-workers developed the small molecule, EPZ004777, which specifically inhibits the H3K79 methylation activity of DOT1L. EPZ004777 treatment results in selective killing of leukaemia cells that harbour the MLL translocation in mouse xenograft models.

 

References
1. Riley LB et al. Handbook of Epigenetics: The new molecular and medical genetics, 2011 Chapter 32 Pages 521 – 534 <read more>
2. You JSY, et al. Cancer Genetics and Epigenetics: Two sides of the same coin?. Cancer Cell Volume 22, issue 1, 10 July 2012, Pages 9 – 20. <read more>
3. Dawson MA, et al. Cancer Epigenetics: From Mechanism to Therapy. Cell Volume 150, issue 1 6 July 2012, pages 12 – 27. <read more>
4. Heyn H et al. DNA methylation profiling in the clinic: applications and challenges. Nature Reviews Genetics 13, October 2012 pages 679 – 692. <read more>