|Year : 2016 | Volume
| Issue : 1 | Page : 5-10
Epigenetics meets hematology
Ali Z Galal1, Sabry A Shoieb1, Mohammad A Abdelhafez1, Mohammad S Elhawwary2
1 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Kafr El-Sheikh Institute of Hepatology, Kafr El-Sheikh, Egypt
|Date of Submission||12-Nov-2014|
|Date of Acceptance||18-Jan-2015|
|Date of Web Publication||18-Mar-2016|
Mohammad S Elhawwary
MBBCh, Elgomhorai District, Elmahalla Elkobra City, 33713 Elgharbia Governorate
Source of Support: None, Conflict of Interest: None
The aim of the study was to review the concept of epigenetics and its role in the evolution and treatment of blood disorders.
Data were obtained from Medline databases (PubMed, Medscape, Science Direct) and from materials available on the Internet from 2002 to 2014.
The initial search presented 90 articles, of which 30 met the inclusion criteria. The articles studied the role of epigenetics in the pathogenesis and treatment of hematological diseases.
If the studies did not fulfill the inclusion criteria, they were excluded. Study quality assessment included whether ethical approval had been obtained, eligibility criteria had been specified, appropriate controls had been established, adequate information was available, and assessment measures had been defined.
Each study was reviewed independently; the obtained data were translated into the language of the researcher and have been presented in sections throughout the article.
In total 30 potentially relevant publications were included: 29 were human studies and one was an animal study. The studies define epigenetics as changes in gene expression without changes in the DNA itself. Epigenetic regulation was achieved by DNA methylation, histone modification, and microRNA interference. Deregulations in epigenetic mechanisms present an important pathway toward the development of hematological disorders. DNA-demethylating and histone-deacetylating agents are the first era of drugs directed at treating epigenetic deregulations with significant success rates.
Unlike genetics, the reversible nature of epigenetics makes them highly attractive targets for cancer therapies. DNA-demethylating and histone-deacetylating agents are the first drugs directed at treating epigenetic deregulations. Understanding epigenetic mechanisms will be helpful in introducing new lines of treatment.
Keywords: Epigenomics, genetics, hematology
|How to cite this article:|
Galal AZ, Shoieb SA, Abdelhafez MA, Elhawwary MS. Epigenetics meets hematology. Menoufia Med J 2016;29:5-10
| Introduction|| |
In multicellular organisms, different gene expression patterns determine the fates of cells, causing them to differentiate into various cell types. Therefore, it is critical to precisely coordinate the gene expression pattern based on cell types during developmental processes. Furthermore, gene expression patterns need to be maintained throughout the life span once established. Each cell appears to 'memorize' the genetic information to be expressed and precisely passes this memory on to its daughter cells after cell division. This process is referred to as 'epigenetic cellular memory' .
The classical view defines epigenetics as heritable changes that affect gene expression without altering the DNA sequence. Epigenetic regulation of gene expression is facilitated through different mechanisms such as DNA methylation, histone modifications, and RNA-associated silencing by small noncoding RNAs. All these mechanisms are crucial for normal development, differentiation, and tissue-specific gene expression. These three systems interact and stabilize one another and can initiate and sustain epigenetic silencing, thus determining heritable changes in gene expression. Alterations in one or more of these systems lead to inappropriate target gene expression or silencing that results in epigenetic regulation of human diseases, including blood disease .
The chromatin structure is modified and altered in several layers. First of all, DNA itself is methylated, and this event mostly occurs at the cytosine in cytosine-phosphate guanine (CpG)-rich regions. DNA methylation at promoter regions generally occludes the binding of transcription factors or recruits methyl-DNA-binding proteins, leading to the inactivation of gene expression, with few exceptions in which DNA methylation can be involved in preventing gene repression .
Two different classes of DNA methyltransferases (DNMTs) are responsible for establishing and maintaining DNA methylation. DNMT1 maintains DNA methylation through its substrate preference for hemimethylated DNA at CpG regions. DNMT3 family members DNMT3A, DNMT3B, and DNMT3L are involved in establishing de-novo DNA methylation patterns, although DNMT3L is catalytically inactive and might cause gene repression independent of DNA methylation .
Ten eleven translocation (TET) family proteins were identified to oxidize 5-methylcytosine to 5-hydroxymethylcytosine, eventually leading to the removal of the methyl group from methylcytosine .
The fundamental unit of chromatin is the nucleosome, which is made up of a core of eight histones (two each of H2A, H2B, H3, and H4) around which a double strand of DNA (147 nucleotides long) is wrapped in 1.75 super helical turns. The amino-terminal tails of all eight core histones protrude through the DNA and undergo a variety of post-translational covalent modifications, such as acetylation, methylation, ubiquitylation, sumoylation, and phosphorylation, on specific residues. In the last few years, it has been demonstrated that acetylation and methylation are important epigenetic mechanisms involved in regulating key cellular processes, such as gene transcription, DNA replication, and DNA repair .
Histone acetylation occurs on lysine residues present in the tails of histone proteins H2B, H3, and H4, and signals for transcription activation, whereas hypoacetylated histone proteins are found in transcriptionally inactive regions. Acetylation is a dynamic process controlled by two families of enzymes, histone acetyltransferases and histone deacetylases (HDACs), both of which include multiple enzyme classes whose expression and activity are finely regulated .
Unlike acetylation, histone methylation can be associated with either activation or repression of transcription, depending on the particular methylated lysine or arginine and on the degree of methylation (monomethylation, dimethylation, and trimethylation). Histone methylation is not a permanent histone modification, but rather a more dynamic process regulated by two classes of enzymes: histone methyltransferases and histone demethylases .
MicroRNAs (miRNAs) are RNAs that are 18-23 nucleotides in length and function as post-transcriptional regulators. They regulate messenger RNA translation by binding to complementary sequences that are cut or repressed. Many miRNAs are transcribed from intergenic regions or from introns of protein-coding genes and, sometimes, they are expressed at the same time that the protein gene is transcribed. Just a few miRNAs have been located in exons of protein-coding genes. Of all these miRNAs, the intergenic miRNAs are the only ones that have their own gene promoter and regulatory region .
Another mechanism by which miRNAs affect gene expression is by histone modification and DNA methylation of promoter sites. This mechanism occurs thanks to the RNA-induced transcriptional silencing complex. This protein complex binds to miRNAs to perform post-translational modification of histone tails, such as methylation of H3K9 to form heterochromatin, and to cause transcriptional repression .
| Materials and methods|| |
We reviewed papers on the role of epigenetics in hematology from Medline databases (PubMed, Medscape, Science Direct) and also from materials available on the Internet. We used genetics/epigenomics/hematology and epigenetic mechanisms/blood disorders as searching terms. In addition, we examined references from the specialist databases and reference lists in relevant publications and published reports. The search was performed in the electronic databases from 2002 to 2014.
All of the studies were independently assessed for inclusion. They were included if they fulfilled the following criteria.
Inclusion criteria of the published studies:
Published in English language,
Published in peer-reviewed journals,
Focused on the role of epigenetics in hematology,
Discussed epigenetic mechanisms and its role in the evolution and treatment of blood disorders,
If a study had several publications on certain aspects we used the latest publication giving the most relevant data.
If the studies did not fulfill the above criteria, they were excluded - for example, reports without peer-review and studies not focused on the role of epigenetics in hematology.
The analyzed publications were evaluated according to evidence-based medicine criteria using the classification of the US Preventive Services Task Force.
US Preventive Services Task Force:
- Level I: Evidence obtained from at least one properly designed randomized controlled trial.
- Level II-1: Evidence obtained from well-designed controlled trials without randomization.
- Level II-2: Evidence obtained from well-designed cohort or case-control analytic studies, preferably from more than one center or research group.
- Level II-3: Evidence obtained from multiple time series with or without the intervention. Significant results in uncontrolled trials might also be regarded as this type of evidence.
- Level III: Opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees.
The quality of all studies was assessed. Important factors included study design, attainment of ethical approval, evidence of a power calculation, specified eligibility criteria, appropriate controls, adequate information, and specified assessment measures. It was expected that confounding factors would be reported and controlled for and appropriate data analysis made in addition to an explanation of missing data.
Each study was reviewed independently; the obtained data were translated into the language of the researcher and have been presented in sections throughout the article.
| Results|| |
Study selection and characteristics
In total, 90 potentially relevant publications were identified; 70 articles were excluded as they did not meet our inclusion criteria. A total of 30 studies were included in the review as they were deemed eligible because they fulfilled the inclusion criteria. Of these 30 articles included in this review, 29 were human studies and one was an animal study. The majority of the studies discussed epigenetic mechanisms: how deregulation in epigenetic control gives rise to hematological diseases and how this knowledge is used in the treatment of blood disorders. The studies were analyzed with respect to the study design using the classification of the US Preventive Services Task Force.
Epigenetic regulators involved in blood diseases
From all known epigenetic regulators, only a few that are relevant to blood disorders have been identified to date. Of these, this review will focus on isocitrate dehydrogenase (IDH), TET2, and DNMT3A. Mutations in these genes seem to substantially affect the epigenetic modifications of somatic cells.
DNA methyltransferase 3A
Inactivating mutations of DNMT3A have been detected in about 20% of all investigated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients; patients with a DNMT3A mutation have a significantly poorer outcome compared with those without . A recent study demonstrated that the loss of DNMT3A causes a defect in the silencing of genes necessary for hematopoietic stem cell renewal. Of interest, DNMT3B mutations have not been observed in malignant cells. This is presumably because the two DNMTs have different functions in human cells .
Ten eleven translocation
TET1 was first identified as a mixed lineage leukemia fusion partner in two independent AML patients. Later, TET2 mutations (missense, frameshift, and nonsense) were frequently identified in up to 20% of patients with MDS, myeloproliferative neoplasia, and AML. The homozygous knockout of the TET2 gene resulted in the expansion of hematopoietic stem cells and the rapid onset of myeloid malignancies in about one-third of the knockout cohort .
Mutations in IDH1 and IDH2 are also implicated in cancer progression. Mutants IDH1 and IDH2 were initially identified in glioma , but recurrent mutations have also been characterized in MDS and AML. Under normal conditions, IDH1 and IDH2 are part of the citric acid cycle and act to catalyze the production of a-ketoglutarate from isocitrate. When mutations occur in either enzyme, an aberrant metabolite known as 2-hydroxyglutarate is produced, inhibiting the activity of a-ketoglutarate-dependent enzymes, which include the TET proteins and the Jumonji (JmjC) family of histone demethylases . Expression of mutant IDH1/2 or a deficiency of TET2 in normal hematopoietic cells leads to an expansion of stem cell function and a differentiation blockade consistent with a proleukemogenic effect .
Epigenetic treatment of blood disorders
Epigenetic treatment of blood is based on findings that tumor cells display a deregulated histone acetylation pattern and promoter hypermethylation of distinct tumor suppressor genes. Therefore, small-molecule drugs designed as histone deacetylase inhibitors (HDACi) and DNA-demethylating agents were considered and thoroughly tested for treatment .
There are two main US Food and Drug Administration (FDA)-approved DNA hypomethylating agents, 5-azacitidine (5-aza) and decitabine (5-aza-deoxycitidine), which have proven efficacious in treating human tumors. Both drugs covalently trap the DNMT enzymes to cytosine residues, preventing them from completing the enzymatic reaction. These compounds act generally at the CpG dinucleotide but do not show any specificity for particular DNA loci . They have been perhaps most successful against hematologic malignancies, specifically MDS and AML. 5-aza was shown to have a significant response rate in patients with MDS, along with reduced risk of leukemic transformation and increased survival, which helped lead to its FDA approval in 2004 . A large-scale clinical study known as AZA-001 helped establish 5-aza as the preferred treatment for patients with high-risk MDS. Fewer data exist regarding the usage of these compounds in treating lower-risk MDS .
Histone deacetylase inhibitors
HDACi are another class of drugs that have become more prominent in the treatment of hematopoietic diseases. These inhibitors promote the retention of acetyl groups on the tails of histone proteins, which allows for a more active, open chromatin conformation. The first HDACi, vorinostat, received FDA approval in 2006 for the treatment of cutaneous T-cell lymphoma . Other HDACi, such as sodium phenylbutyrate, have existed for a number of years, and have given rise to second-generation compounds. These include entinostat and panobinostat, and have moderate effects in treating MDS, AML, and acute lymphocytic leukemia when used alone .
Although single-agent therapy has proven efficacious, mostly for hypomethylating agents, combinatorial studies have been conducted to enhance the effectiveness of these drugs. In the case of HDACi, induced expression of highly methylated genes is generally not detected when these compounds are administered alone. However, administration of a hypomethylating agent in combination with an HDACi in MDS and AML has been shown to promote the re-expression of epigenetically silenced genes, along with major clinical responses . In contrast, another study showed that administration of 5-aza and entinostat did not show a correlation between clinical response and reversal of DNA methylation or gene expression of tumor suppressor genes .
Even though these therapies are supposedly targeting the epigenome, further studies are still required to pinpoint how these drugs interact, as it is still highly possible that other mechanisms, such as DNA damage, apoptosis, or immune response, are accountable for the responses observed in patients .
| Discussion|| |
Taking a closer look at clinical trials with hematopoietic diseases treated with epigenetic drugs in the stated definition (HDACi and DNA-demethylating agents), a complex picture emerges. For MDS treatment alone, more than 1200 studies have been initiated with 58 completed and evaluated to date. Administered epigenetic drugs included 5-aza or decitabine alone or in double/triple combinations with compounds like romiplostim. HDACi are rarely utilized for MDS; a phase II study administering vorinostat alone was terminated because of fulfillment of the futility criteria (NCT00486720). Similarly, a comparative phase I study using vorinostat combined with decitabine yielded an objective response rate of only 7.1% and disease progression in refractory/relapsed as well as untreated AML and MDS patients (NCT00479232). However, in two mouse models, administration of vorinostat after allogenic bone marrow transplantation led to reduced acute mortality because of graft-versus-host disease. By contrast, a stable graft-versus-leukemia activity was observed in addition to a reduction in proinflammatory cytokines .
The efficacy of 5-aza treatment alone in MDS was highlighted in a randomized phase III study, showing a remarkably improved survival of up to 9 months (24.46 vs. 15.02 months; P = 0.0001), a delay in disease progression (median 14.13 vs. 8.82 months; P = ·0466), and a more sustained hematological improvement in comparison with best supportive care and intense induction chemotherapy with daunorubicin. Therefore, 5-aza was shown to be the first employable option for treatment in elderly MDS patients (>55 years of age) who are ineligible for allogenic stem cell transplantation (allo-SCT). Despite the undeniably impressive clinical improvement, it should be kept in mind that a considerable number of patients died, despite 5-aza treatment, within 42 months (82 vs. 113 patients), indicating that further improvement is required. A current clinical trial of this patient group is investigating whether 5-aza treatment might be superior to allo-SCT in general (NCT01404741), because treatment failure due to relapse is a major challenge after transplantation .
To underline this notion, a combination of 5-aza with donor lymphocyte infusions induced remission in 23% of AML-relapsed patients after allo-SCT (NCT00795548). Furthermore, post-transplantation treatment with 5-aza increased the number of immunomodulatory T-regulatory cells and cytotoxic CD8(+) T-cell response against several tumor antigens, leading to an improved graft-versus-leukemia effect without increased graft-versus-host disease. Single decitabine treatment was described in a randomized phase III study, which showed a benefit of progression-free survival and delayed AML transformation; however, overall survival was not improved. Although preclinical experiments showed that romidepsin had an effect, at least toward RUNX1/RUNX1T1 AML cells, the available clinical data for romidepsin in AML/MDS did not substantiate these positive results in patients .
Although aberrant DNA methylation is likewise a common event during the pathogenesis of chronic lymphocytic leukemia (CLL), a phase I trial showed no observable benefit for decitabine in CLL/NHL patients . Regarding acute lymphocytic leukemia, several phase I/II trials performed during the mid-90s aimed to investigate the antileukemic potential of decitabine and fazarabine (arabinosyl-5-azacytidine), the latter showing no benefit in treatment . The same holds true for the treatment of multiple myeloma. Although there are encouraging data from myeloma cell lines, results from clinical trials are less promising. Two phase I studies and a case report show modest beneficial outcomes with the use of vorinostat, partially in combination with bortezomib in relapsed/refractory and advanced multiple myeloma .
Unfortunately, to date, it would seem that the vast amount of in-vitro data produced to elucidate the impact of epigenetic drugs cannot fully be translated into clinical practice. This may be due to the lack of specificity of the treatment caused by the pleiotropic role of epigenetic modifications and modifiers.
| Conclusion|| |
Epigenetics is the change in gene expression without change in the DNA itself. Epigenetic mechanisms are achieved by adding chemical groups covalently attached to DNA and histones, making chromatin more or less accessible to the transcription machinery. Deregulations in epigenetic players like TET1/2, DMNT3A, and IDH1/2 mutations present an important pathway in the development of hematological malignancy. The reversible nature of epigenetics makes them highly attractive targets for cancer therapies. Histone-deacetylating drugs and DNA-demethylating drugs are the first era of epigenetic therapy. Although these drugs have been successfully used in some blood disorders, their exact mechanism of action remains unclear. Further studies are required to provide new classes of epigenetic drugs.
| Acknowledgements|| |
Conflicts of interest
| References|| |
Kang C, Song JJ, Lee J, Kim MY. Epigenetics: an emerging player in gastric cancer. World J Gastroenterol 2014; 20
Moss TJ, Wallrath LL. Connections between epigenetic gene silencing and human disease. Mutat Res 2007; 618
Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat Rev Genet 2013; 14
Denis H, Ndlovu MN, Fuks F. Regulation of mammalian DNA methyltransferases: a route to new mechanisms. EMBO Rep 2011; 12
Pastor WA, Aravind L, Rao A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat Rev Mol Cell Biol 2013; 14
Bernstein BE, Meissner A, Lander ES. The mammalian epigenome. Cell 2007; 128
Wang X, Hayes JJ. Acetylation mimics within individual core histone tail domains indicate distinct roles in regulating the stability of higher-order chromatin structure. Mol Cell Biol 2008; 28
Cloos PA, Christensen J, Agger K, Helin K. Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease. Genes Dev 2008; 22
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116
Scaria V, Hariharan M, Maiti S, Pillai B, Brahmachari SK. Host-virus interaction: a new role for microRNAs. Retrovirology 2006; 3
Yan H, Parsons DW, Jin G, Mclendon R, Rasheed BA, Yuan W, et al.
IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009; 360
Beaulieu N, Morin S, Chute IC, Robert MF, Nguyen H, Macleod AR. An essential role for DNA methyltransferase DNMT3B in cancer cell survival. J Biol Chem 2002; 277
Li Z, Cai X, Cai CL, Wang J, Zhang W, Petersen BE et al.
Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. Blood 2011; 118
Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH et al.
Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell 2011; 19
Quivoron C, Couronne L, Della Valle V, Lopez CK, Plo I, Wagner-Ballon O, et al.
TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell 2011; 20
Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007; 8
Kihslinger JE, Godley LA. The use of hypomethylating agents in the treatment of hematologic malignancies. Leuk Lymphoma 2007; 48
Silverman LR, Demakos EP, Peterson BL, Kornblith AB, Holland JC, Odchimar-Reissig R, et al.
Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 2002; 20
Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A, et al.
Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 2009; 10
Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R. FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist 2007; 12
Dimicoli S, Jabbour E, Borthakur G, Kadia T, Estrov Z, Yang H, et al.
Phase II study of the histone deacetylase inhibitor panobinostat (LBH589) in patients with low or intermediate-1 risk myelodysplastic syndrome. Am J Hematol 2012; 87
Gore SD. New ways to use DNA methyltransferase inhibitors for the treatment of myelodysplastic syndrome. Hematology Am Soc Hematol Educ Program 2011; 2011
Fandy TE, Herman JG, Kerns P, Jiemjit A, Sugar EA, Choi SH, et al.
Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies. Blood 2009; 114
Popovic R, Shah MY, Licht JD. Epigenetic therapy of hematological malignancies: where are we now? Ther Adv Hematol 2013; 4
Reddy P, Maeda Y, Hotary K, Liu C, Reznikov LL, Dinarello CA, et al.
Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect. Proc Natl Acad Sci USA 2004; 101
Spyridonidis A, Labopin M, Schmid C, Volin L, Yakoub-Agha I, Stadler M, et al.
Outcomes and prognostic factors of adults with acute lymphoblastic leukemia who relapse after allogeneic hematopoietic cell transplantation. An analysis on behalf of the Acute Leukemia Working Party of EBMT. Leukemia 2012; 26
Goodyear OC, Dennis M, Jilani NY, Loke J, Siddique S, Ryan G, et al.
Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML). Blood 2012; 119
Blum KA, Liu Z, Lucas DM, Chen P, Xie Z, Baiocchi R, et al.
Phase I trial of low dose decitabine targeting DNA hypermethylation in patients with chronic lymphocytic leukaemia and non-Hodgkin lymphoma: dose-limiting myelosuppression without evidence of DNA hypomethylation. Br J Haematol 2010; 150
Advani AS, Gibson SE, Douglas E, Jin T, Zhao X, Kalaycio M, et al
. Histone H4 acetylation by immunohistochemistry and prognosis in newly diagnosed adult acute lymphoblastic leukemia (ALL) patients. BMC Cancer 2010; 10
Mazumder A, Vesole DH, Jagannath S. Vorinostat plus bortezomib for the treatment of relapsed/refractory multiple myeloma: a case series illustrating utility in clinical practice. Clin Lymphoma Myeloma Leuk 2010; 10