Epigenomics of leukemia: from mechanisms to therapeutic applications

Epigenomics of leukemia: from mechanisms to therapeutic applications

Leukemia is a malignant neoplasm of blood-forming tissues characterized by an unrestrained proliferation of abnormal blood cells. Clinically and pathologically, leukemia is subdivided into a variety of large groups. The first division is between lymphocytic and myeloid lineages, according to which kind of blood cell is affected. In addition, the diseases are classified as acute, which denotes a rapidly progressing evolution of the disease with a predominance of highly imma-ture cells, or chronic, signifying slowly progress-ing disease with greater numbers of relatively more mature cells. Combining these two classi-fications provides a total of four main categories: chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) [1].

Chronic lymphocytic leukemia is the most fre-quent leukemia in adults in the Western world, with the highest incidence in the elderly popula-tion. These type of leukemic cells are found in the blood and bone marrow, and may crowd out other blood cells in the bone marrow, resulting in a shortage of red blood cells and platelets. This pathology is characterized by an accumulation of predominantly nondividing malignant CD5+

B cells blocked at the G0/G1 phase of the cell cycle and resistant to apoptosis owing, mostly, to an overexpression of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein. Independently of cytogenetic abnormalities, CLLs are clinically separated into two groups: an indolent form

that does not necessitate treatment and a more aggressive form requiring therapeutic interven-tion. Prognosis is correlated with several different markers, especially the presence of the 70-kDa z-associated protein (ZAP-70) and unmutated immunoglobulin-variable heavy chain (IgVH) genes, which are correlated to poor progno-sis [2,3]. In addition, several cytogenetic aberra-tions have been demonstrated to be independent predictors of disease progression and survival such as the deletion of a region located in chro-mosome 13(del13q14)and found in more than half of CLL patients [4]. Noteworthy is the hemi-zygous p53 inactivation (i.e., mutation or dele-tion), which is found in approximately 10% of patients with CLL and is considered to be the most significant marker associated with poor prognosis [5].

Chronic myeloid leukemia is characterized by the pathological increase of proliferation of vari-ous blood cells, predominantly myeloid cells in peripheral blood, and their precursors in the bone marrow, resulting in their accumulation in the blood. In the Western world, the median age of onset is 50–60 years. The Philadelphia chromo-some, a characteristic chromosomal translocation (breakpoint cluster region-Abelson [BCR–ABL]; t[9;22][q34;q11]) encoding a product, which is the target of tyrosine kinase inhibitors, occurs in over 90% of CML cases. The disease course is tri-phasic, starting with an early phase, also known as chronic phase (CP) disease. Then, le ukemia stem cells can acquire additional genetic defects

Leukemogenesis is a multistep process in which successive transformational events enhance the ability of a clonal population arising from hematopoietic progenitor cells to proliferate, differentiate and survive. Clinically and pathologically, leukemia is subdivided into four main categories: chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia and acute myeloid leukemia. Leukemia has been previously considered only as a genetic disease. However, in recent years, significant advances have been made in the elucidation of the leukemogenesis-associated processes. Thus, we have come to understand that epigenetic alterations including DNA methylation, histone modifications and miRNA are involved in the permanent changes of gene expression controlling the leukemia phenotype. In this article, we will focus on the epigenetic defects associated with leukemia and their implications as biomarkers for diagnostic, prognostic and therapeutic applications.

KEYWORDS: cancer prevention n DNA methylation n epigenetic anticancer therapy n epigenetics n histone modifications n leukemia n miRNA

Cristina Florean1*, Michael Schnekenburger1*, Cindy Grandjenette1, Mario Dicato1 & Marc Diederich†1

1Laboratoire de Biologie Moléculaire et Cellulaire de Cancer, Hôpital Kirchberg, 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourg †Author for correspondence: Tel.: +352 2468 4040 Fax: +352 2468 4060 [email protected] *Authors contributed equally

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that cause disease progression from CP to a more advanced phase and ultimately ending in a usually fatal terminal phase called blast crisis (BC) [6,7].

Acute lymphocytic leukemia is the most com-mon leukemia subtype observed in children (approximately 75%) and it is also the most fre-quent malignancy diagnosed in childhood; how-ever, it is a relatively uncommon cancer in adults. In ALL, early B- and T-lymphocyte progenitors become genetically altered and subsequently undergo dysregulated proliferation, survival and clonal expansion resulting in the accumulation of leukemic lymphoblasts in the bone marrow and various extramedullary sites. The most common cytogenic abnormalities observed in ALL are t(9;22)/BCR–ABL, t(4;11)/MLL–AF4, t(12;21)TEL–AML1 and t(1;19) E2A–PBX t ranslocations/fusion genes [8].

Acute myeloid leukemia arises from the clonal expansion of hematopoietic precursors that have lost their competence to advance through ter-minal differentiation leading to their accumula-tion in the bone marrow and peripheral blood. AML is the most common acute leukemia in adults. The majority of AML cases are asso-ciated with nonrandom chromosomal translo-cations. Over 700 recurrent aberrations have been described associated to the disease pheno-types. The four most common AML-associated translocations include t(15;17)/PML–RAR, t(8;21)/AML1–ETO, Inv(16)/core binding fac-tor (CBF )b–MYH11, 11q23 and mixed lineage leukemia (MLL)-fusion protein. The MLL gene was identified to fuse with over 50 protein cod-ing genes resulting in oncofusion proteins that act as potent proto-oncogenes [9].

Leukemia has traditionally been considered as the consequence of genetic alterations, includ-ing several nonrandom cytogenetic abnormalities described above, leading to irreversible defects of critical gene functions such as proliferation, differentiation, apoptosis and gene transcrip-tion associated to leukemogenesis. However, in the past few years, there is increasing evidence demonstrating that the neoplastic phenotype and the differential biologic behavior of tumor cells, including leukemia, could be explained by epi-genetic alterations [10–12]. Epigenetics is defined as the heritable changes in the patterns of gene expression that occur without a change in the pri-mary DNA sequence. There are three main inter-connected mechanisms of epigenetic regulation, which play a fundamental role in the interpreta-tion of genetic i nformation: DNA m ethylation, histone modifications and ncRNAs.

Aberrant epigenetic modifications associated with the pathogenesis of leukemia offer the promise of novel biomarkers for early cancer detection, diagnosis, prognosis and prediction of treatment response. Moreover, the poten-tial reversibility of epigenetic alterations rep-resents a potential target of novel therapeutic approaches [13].

In this article, we will describe each major epi-genetic mechanism and then focus on their role in the development of leukemia, with an empha-sis on how these alterations can p otentially be used from a clinical point of view.

DNA methylation in leukemia n DNA methylation & cancer

In mammalian cells, DNA methylation occurs almost exclusively at the 5´-position of the pyrimidine ring of the cytosine residues within the context of palindromic CpG dinucleotide (CG) sequences through the addition of a methyl moiety to form 5-methylcytosines. The reaction is catalyzed by a family of enzymes called DNA methyltransferases (DNMTs) that use S-adenosyl-methionine (AdoMet) as the methyl donor. The catalytic isoforms of the DNMT family are classified into de novo DNMTs, DNMT3A and DNMT3B, with activ-ity for both the unmethylated and hemimethyl-ated DNA, and function independently of repli-cation and the ‘maintenance methyltransferase’, DNMT1, which demonstrates at least a tenfold preference for hemimethylated DNA and func-tions during DNA replication. The combined function of DNMTs results in the generation and maintenance of the heritable DNA meth-ylation patterns observed in the mammalian genome [14–16].

Assuming a statistically random distribution, CGs are the only dinucleotides under-represented in the human genome (estimated at ~1%) owing to spontaneous hydrolytic deamination of 5-methylcytosines, which is responsible for the C-to-T transition. Curiously, remaining CGs are unequally distributed across the genome with long stretches of DNA without or with only few CGs and short stretches of DNA where CGs are found clustered. These CG-rich regions called CpG islands (CGIs) are regions of greater than 200 bp with a C+G content of at least 55% and a ratio of observed/statistically expected CG fre-quencies of greater than 0.6. CGIs are present in 60% of all genes, where they are usually identi-fied near the promoter and exogenic regions [16]. These CGIs are found generally unmethylated in normal differentiated cells although some of them

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(~6%) become methylated in a tissue-specific manner [17], whereas CGs located in intergenic regions are found methylated. DNA methylation is usually associated with transcriptional silenc-ing, therefore playing an essential role in physi-ological functions such as gene-dosage reduction, as observed in X-chromosome inactivation in females [18] and monoallelic expression related to genomic parental imprinting [19]. Moreover, a significant amount of deeply methylated CGs are found along retrotransposons, mainly long interspersed nucleotide elements (LINE), short interspersed elements and endo genous retrovi-ruses, which are genomic repetitive elements that make up approximately 40% of the mammalian genome [20]. Therefore, this DNA methylation acts as the guardian of our genome to insure its stability by preventing translocation-mediated gene disruption associated with chromosomal instability [14].

Methylated CGs are recognized by proteins that bind specifically to methylated DNA, the so-called methyl-CpG-binding domain proteins, which allow the recruitment of multiprotein complexes containing chromatin- remodeling proteins as well as histone-modifying enzymes such as histone deacetylases (HDACs) and histone methyltransferases (HMTs) targeting histone H3 on lysine 9 or 27 (see below and Figure 1). Together these factors are involved in DNA methylation-associated gene repression by increasing chromatin compaction and prevent-ing transcription factor recruitment. By contrast, unmethylated CGIs are usually associated with a loose chromatin structure allowing transcription factor recruitment and promoting gene expres-sion. This permissive chromatin state is actively maintained by histone-modifying enzymes such as histone acetyltransferases (HATs) and HMTs targeting histone H3 on lysine 4 (see below and Figure 1) as well as coactivator complexes [21,22].

Besides its physiological role in normal cells, DNA methylation plays a critical role in can-cer cells, where promoter CGI hypermethyl-ation inactivates various tumor suppressor genes (TSGs). The list of epimutation hotspots leading to gene silencing is growing and currently includes genes involved in a large variety of signalization pathways and cellular processes [15,23].

Interestingly, aberrant methylation can be critically involved in the early steps of tumori-genesis (initiation) and methylation patterns appear to be specific to both cancer type and tumor stage [15,23]. Thus, the pattern of methyla-tion can be considered as a biomarker for diag-nosis, prognosis, prediction of therapy response

and monitor response to therapy. Finally, induc-tion of hypomethylation of these promoter- associated CGIs can lead to gene re-expression and t herefore be of great clinical value.

n Aberrant DNA methylation in leukemiaLeukemia, as any other cancer, displays aberrant methylation profiles. Table 1 provides a list of TSGs found methylated in various leukemia subtypes. Besides their role as biomarkers for diagnosis, these TSGs were associated with prognosis and prediction of treatment response. Indeed, hyper-methylated in cancer 1 (HIC1) hypermethylation has been observed in late-stage AML patients who relapsed after a chemotherapy-induced complete remission [24]. A hypermethylation in the pro-moter region of homeobox (HOX)A4 is correlated with poor prognosis, and the WIT1 gene has been described to correlate with a chemo resistant phe-notype in AML [25,26]. In addition, AML patients exhibit a highly hetero genous pattern of cyclin-dependent kinase inhibition (CDKN )2B (p15) methylation [27]. The methylation of CDKN2B (p15) may play a role in disease progression as well as in leukemogenesis [28].

Several groups have investigated stage-specific DNA methylation events in CML patients, in order to better understand the molecular mechanisms responsible for cancer progression. An increase in the DNA methylation levels of the ABL1 [29], calcitonin 1 (CALC1) [30], HIC1 [24] and estrogen receptor [31] genes have all been described during progression from CP to BC. In particular, ABL1 promoter methylation of the BCR–ABL fusion gene is associated with repression of this TSG gene in CML and plays a role in the progression of CML from CP to BC [29]. HIC1 methylation is also increased during progression from CP to BC. Indeed, HIC1 is methylated in 50% of patients in CP and all patients in BC [24]. Moreover, a report demonstrated that Bcl-2-interacting mediator (BIM) expression is downregulated by promoter hypermethylation and is associated with imatinib resistance in CML patients. Therefore, the com-bination of imatinib with a demethylating agent may restore BIM expression in imatinib-resistant patients and could improve the response rate [32]. Finally, the HOXA4 gene is frequently found hypermethylated in CML and is correlated with poor p rognosis [25].

Promoter methylation of CDKN1A (p21) is highly c orrelated to transcriptional repression and reduced disease-free and overall survival in ALL patients. Thus, the methylation status of p21 could be an important factor in predicting the clinical

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Epigenomics of leukemia: from mechanisms to therapeutic applications Review

outcome of these patients [33]. The methylation status of the CALC1 gene has also been reported to be a strong prognostic indicator in ALL. Indeed, hypermethylation of CALC1 is associated with an unfavorable clinical outcome in ALL [34]. In addition, gene-specific ABL1 promoter methyla-tion has been described to be exclusively associated with the P210 isoform of BCR–ABL [35].

IgVH gene mutational status is well estab-lished as a powerful independent prognostic marker in CLL for identifying patients at high risk of disease progression. Few methylated genes have been correlated with IgVH mutation status. ZAP-70 is well established as a powerful independent prognostic marker in CLL that sur-rogates for mutational status [36]. Interestingly,

Figure 1. Epigenetic inactivation of tumor suppressor genes in leukemia. For footnote please see facing page.



ZAP-70 methylation status is strongly corre-lated with lack of ZAP70 expression and IgVH-mutated patients with good prognosis [37]. Twist homolog 2 (TWIST2, also known as Dermo-1) exhibits a differential methylation pattern rela-tive to the IgVH mutational status. Indeed, TWIST2 is predominantly methylated in CLL patients with mutated IgVH genes. By contrast, the majority of patients with unmutated IgVH, which is strongly associated with a poor progno-sis, do not exhibit TWIST2 promoter methyla-tion [38]. Furthermore, a recent study pointed out that BTG4 and CD38 are frequently hypermeth-ylated in CLL with a mutated IgVH gene [39]. By contrast, promoter hyper methylation-mediated silencing of the HOXA4 gene is correlated with a poor prognosis in unmutated IgVH CLLs [40]. Therefore, a panel of methylation-based mark-ers can be used in addition to routine prognos-tic factors in order to improve the CLL-patient risk stratification.

In conclusion, the association between the methylation level of critical genes and clinical outcome within major subgroups of leukemia patients suggests that methylation ana lysis needs to be explored as a method to improve s tratification of these patients.

A plethora of studies has emphasized that the transcriptional silencing of a number of TSGs has been associated with extensive de novo meth-ylation of their promoter regions. However, the initiating signal for aberrant DNA methylation during cancer and in particular hematologi-cal neoplasms remains unclear. Interestingly, DNMT1, DNMT3a and DNMT3b expression is increased in AML and CML patients [41]. Especially, AML cells with a hypermethylated CDKN2B (p15) gene tended to express higher levels of DNMT1 and DNMT3B. These results

suggest that DNMT over expression could play a role in CDKN2B methylation. By contrast, in CML, DNMT expression increases in con-cert with disease progression. In CP, CML cells do not demonstrate significant changes in the DNMT levels compared with normal bone marrow cells. However, CML cells in blastic transformation express higher levels of DNMTs. Therefore, rather than being involved in the onset or maintenance of the CP, the overex-pressed DNMTs could be associated with CML progression and BC [41]. In conclusion, DNMTs are considerably upregulated in leukemia cells in a leukemia type- and stage-specific manner and may play a significant role in leukemogenesis by inducing aberrant regional hypermethylation.

Furthermore, the PML–RAR fusion protein, a hallmark of acute promyelocytic leukemia (APL), could recruit DNMT1 and DNMT3A to the promoter of its target gene, RARb2, inducing methylation-mediated transcriptional repression. Although the authors focused on only one target promoter, this study has important implications for leukemogenesis [42].

In contrast to hypermethylation that leads to transcriptional repression of TSGs, hypometh-ylation results in derepression of few potential proto-oncogenes in leukemia. Indeed, the onco-gene T-cell leukemia/lymphoma 1 (TCL-1) is expressed in mature B-cell malignancies such as CLL [43]. Hypomethylation of the gene promoter may be implicated in TCL-1 activation in CLL patients [44]. Similarly, Bcl-2 overexpression is observed in CLL patients. The mechanisms lead-ing to Bcl-2 upregulation are not completely eluci-dated, but may be caused by hypomethylation in the promoter of this gene [45]. DNA hypomethyl-ation was also observed in the oncogenic GTPase H-RAS in CLL patients [46]. Furthermore,

Figure 1. Epigenetic inactivation of tumor suppressor genes in leukemia (please see facing page). In normal cells, CpG island promoters near the transcription start site of typical TSGs are mostly unmethylated. These promoters present an enrichment of active histone marks such as histone H3 and H4 acetylation and histone H3K4 methylation, which are actively maintained by HATs and HMTs, respectively. In addition, repressive histone marks (H3K9 and H3K27) are maintained unmethylated by specific HDMs. Together these marks promote euchromatin formation allowing the recruitment of transcription factor (TF) and CoA complexes associated to active gene transcription by RNA pol II complex. In cancer cells, the loss of TSG functions associated to aberrant gene silencing-mediated leukemogenesis may occur via chromatin condensation or miRNA-induced silencing (i.e., inhibition of translation). Chromatin condensation is promoted by an accumulation of DNMT-mediated DNA methylation in the promoter region, attracting methyl-binding proteins, which are recruited with other CoR complexes. In addition, increased activity of HDACs, HMTs targeting H3K9 and H3K27, and HDMs targeting H3K4 leads to loss of active markers such as histone H3 and H4 acetylation and histone H3K4 methylation, and accumulation of inactive markers such as H3K9 and H3K27 methylation. Together these marks promote heterochromatin formation, blocking the recruitment of TFs and RNA pol II complex. Although neoplastic cells are characterized by an overall miRNA downregulation often associated with miRNA promoter hypermethylation, some miRNAs are found specifically overexpressed in certain cancer subtypes. This increase leads to miRNA-mediated gene silencing that may occur either via mRNA degradation or by translational suppression. Thus, epigenetic loss of TSG functions implicated in the regulation of many biological processes including proliferation, differentiation and survival contributes to leukemogenesis. Epigenetic therapy targeting either miRNA synthesis (antagomirs) or enzymes involved in chromatin condensation has the potential to reverse this aberrant TSG silencing. CoA: Coactivator; CoR: Corepressor; DNMT: DNA methyltranferase; HAT: Histone acetyltransferase; HDAC: Histone deacetylase; HDM: Histone demethylase; HMT: Histone methyltransferase; MBD: Methyl-CpG-binding domain; TSG: Tumor suppressor gene.



Table 1. Genes with aberrant promoter methylation in leukemia.

Gene Locus Function Leukemia Methylation prevalence (%)

Comments Ref.

ABCB1 7q21 Transporter ALL 45 [194]

ALL 90–83 B vs T [195]

ABL1 9q34 Kinase ALL 33 [35]

CML 23–42 From CP to BC [196]

ADAM12 10q26 Protease CLL 72 [197]

ADCY5 3q13 Adenylyl cyclase ALL 68 [198]

AHR 7p15 Detoxification ALL 33 [199]

APC2 19p13 Wnt signaling CLL 77 [197]

ARHGAP20 11q23 Small G protein ALL 65/33 B vs T [200]

ATG16L2 11q13 Autophagy-related gene ALL 27/42 B vs T [200]

CML 69/63 CP vs BC [200]

BARHL2 1p22 Homeobox gene ALL 77/25 B vs T [200]

BIM 2q13 Apoptosis inductor CML 44 CP [32]

BMAL1 11p15 Circadian gene ALL 33 [201]

AML 19 [201]

BMP2 20p12 Bone and cartilage formation ALL 4/58 B vs T [200]

BNIP3 10q26 Apoptosis ALL 15 [202]

AML 17 [202]

BRCA1 17q21 DNA repair AML 29/75 B vs T [203]

BTG4 11q23 Cell cycle control (G1–S) CLL 47 [39]

CALC1 11p15 Calcium metabolism ALL 43–93 [204]

AML 71–95 [46]

CLL 100 [46]

CAST1 3p14 Organization of the cytomatrix ALL 86 [198]

CD10 3q21 Peptidase ALL 20 [194]

CD38 4p15 Cell adhesion CLL 58 [39]

CDC14B 9q22 Cell cycle ALL 44/33 B vs T [200]

CDH1 16q22 Cell adhesion ALL 37–54 [205]

AML 13–64 [46]

CLL 60 [46]

CRBP1 3q21–23 Retinol transport AML 45–50 [206]

CDKN2B (p15) 9p21 Cell cycle control (G1–S) ALL 23–50 [205]

AML 30–70 [46]

CLL 50 [46]

CDKN2A (p16) 9p21 Cell cycle control (G1–S) ALL 7/22 Adult vs childhood [207]

AML 25 [46]

CLL 20 [46]

CDKN1A (p21) 6p21 Cell cycle control (G1–S/G2–M) ALL 41 [33]

CYP1B1 2p22 Xenobiotic metabolism ALL 77/42 B vs T [200]

DAPK1 9q34 Cell death AML 61 [208]

CLL 100 [38]

DBC1 9q32 Putative TSG ALL 17 [209]

DCC 18q21 Putative TSG ALL 90/83 B vs T [195]

DCL-1 2q24 miRNA processing ALL 80/50 B vs T [195]

DDIT3 12q13 Growth arrest CML 66 [210]

DDX51 12q24 Helicase ALL 70/0 B vs T [195]

DKK3 11p15 Wnt signaling ALL 33 [211]

The table summarizes data regarding genes assessed for hypermethylation in AML, ALL (in either T or B-ALL, childhood or adult, Philadelphia chromosome-negative [Ph-] or -positive [Ph+]), CML (in CP or BC) and CLL patients. ALL: Acute lymphocytic leukemia; AML: Acute myeloid leukemia; BC: Blast crisis; CLL: Chronic lymphocytic leukemia; CML: Chronic myeloid leukemia; CP: Chronic phase; LDL: Low-density lipoprotein; TSG: Tumor suppressor gene.



Table 1. Genes with aberrant promoter methylation in leukemia (cont.).


Comments Ref.

DLC-1 8p22 Putative TSG CLL 69 [197]

DLEU7 13q14 Putative TSG CLL 58 Mainly in CD38 low [197]

DMRT2 9p24 Sex determination CLL 47 [197]

DUOX2 15q15 Oxidase CLL 37 [197]

DUSP4 8p12 Phosphatase ALL 25/25 B vs T [200]

EBF2 8p21 Transcription factor ALL 94/83 B vs T [200]

CML 12/52 CP vs BC [200]

EFNA5 5q21 Axon fasciculation ALL 76 [198]

ER 6q25 Hormone receptor ALL 92 [212]

AML 40–95 [46]

EYA2 20q13 Phosphatase ALL 32/25 B vs T [200]

FAT1 4q35 Cell adhesion ALL 65/25 B vs T [200]

FOXF2 6p25 Transcription factor ALL 29/25 B vs T [200]

GFPT2 5q34 Glucose transport ALL 23 [198]

GIPC2 1p31 Adhesion/growth factor signaling ALL 100 [198]

GNA14 9q21 G-protein-coupled receptor ALL 59 [198]

GPR123 10q26 G-protein-coupled receptor ALL 21/33 B vs T [200]

H-cadherin 16q23 Cell adhesion AML 26 [213]

Hck 20q11 Kinase ALL 20/6 ALL Ph-/ALL Ph+ [214]

HDPR1 14q23 Wnt signaling ALL 26 [211]

HIC1 17p13 Transcriptional repressor AML 10–90 [46]

CML 33 [46]

HLA-F 6p21 Immune response ALL 14/33 B vs T [200]

HOXA4 7p15 Homeobox gene ALL 69 Childhood [25]

AML 64 [25]

CLL 38 [25]

CML 59/91 CP vs BC [25]

HOXA5 7p15 Homeobox gene ALL 77 Childhood [25]

AML 59 [25]

CLL 59 [25]

CML 34/87 CP vs BC [25]

HOXA6 7p15 Homeobox gene ALL 19/13 Adult vs childhood [25]

CLL 34 [25]

HOXB7 7p15 Homeobox gene ALL 26 Childhood [25]

HOXC10 12q13 Homeobox gene CLL 79 Mainly in CD38 high

[197]

HSPA4L 4q28 Heat shock protein ALL 69 [198]

ID4 6p22 Inhibitor of DNA binding AML 65 [213]

KCNK2 1q41 Ion channel ALL 90/83 B vs T [195]

CLL 29 [197]

KNDC1 10q26 Kinase ALL 23/8 B vs T [200]

LHX2 9q33 Homeobox gene CLL 69 [197]

LRP1B 2q21 LDL receptor ALL 80/66 B vs T [195]

CLL 56 [197]

MAGI1 3p14 Kinase ALL 60 [198]

MGMT 10q26 DNA repair ALL 56 Childhood [207]

miR-10b 2q31 Translational repression ALL 25 [190]






Comments Ref.

miR-124-1 8p23 Translational repression ALL 49 [190]

miR-124-2 8q12 Translational repression ALL 46 [190]


miR-139 11q13 Translational repression CLL [215]

miR-196b 7p15 Translational repression ALL 25 [190]

miR-203 14q32 Translational repression ALL 27 [190]

AML 10 [216]

CLL 42 [216]

miR-34b/c Translational repression ALL 34 [190]

miR-582 5q12 Translational repression CLL [215]




MN1 22q12 Putative TSG ALL 85 [198]

MSX2 5q34 Homeobox gene ALL 98 [198]

MYO10 5p15 Myogenic factor ALL 16/50 B vs T [200]

MYO5B 18q21 Myogenic factor ALL 100 [198]

MYOD1 11p15 Myogenic factor ALL 17 [205]

NKX2-1 14q13 Homeobox gene ALL 63/42 B vs T [200]

NOPE 15q22 Immune response ALL 90/66 B vs T [195]

NR4A2 2q22 Nuclear receptor ALL 10/50 B vs T [200]

NRP2 2q33 Neuropilin family receptor CLL 45 [197]

OCLN 5q13 Tight junction ALL 76 [198]

p73 1p36 Cell cycle control (G1–S) AML 10 (10–37) [208]

PAX2 10q24 Transcription factor ALL 44/20 B vs T [200]

PAX6 11p13 Transcription factor ALL 67/50 B vs T [200]

PCDHGA12 5q31 Cell adhesion ALL 90/100 B vs T [195]

PCDHGB7 5q31 Cell adhesion CLL 100 [197]

PGR 11q22 Hormone receptor AML 71 [217]

POU3F3 2q12 Homeobox gene CLL 77 [197]

POU4F1 13q31 Homeobox gene ALL 43/42 B vs T [200]

PRDM12 9q33 Zn finger protein ALL 67/50 B vs T [200]

PTEN 10q23 Tumor suppressor ALL [218]

PTGS2 1q25 Inflammation ALL 92/50 B vs T [200]

PTPRG 3p21 Phosphatase CML 100/45 Diagnosis vs follow-up

[219]

RARB2 3p24 Hormone receptor AML 18–44 [208]

RASSF10 11p15 Ras signaling ALL 16/88 B vs T [220]

RASSF6 4q21 Ras signaling ALL 94/41 B vs T [220]

RLN2 9pter-q12 Hormone CLL 63 [197]

RPIB9 7q21 Binding protein ALL 90/83 B vs T [195]

RSPO1 1p34 Wnt signaling ALL 100 [198]

SALL1 16q12 Zn2+ finger protein ALL 100 [198]

SALL3 18q23 Zn2+ finger protein ALL 48/33 B vs T [200]

sFRP1 8p12 Wnt signaling ALL 38 [211]

AML 65 [213]

CML 10 [213]




overexpression of DNMT3B4, a splice variant of DNMT3B lacking enzymatic activity, has been found to correlate with LINE1 hypomethylation in CLL [47].

LINE1 promoter hypomethylation is an impor-tant feature in CML. Indeed, the sense and anti-sense transcriptions of the LINE1 retro transposon are activated by promoter hypomethylation, which increases from CP toward advanced phase, suggesting that it is important in the progres-sion of the disease [47]. Helical antigen (HAGE) hypomethylation in CML is also associated with overexpression of this gene. It was significantly more frequent in BC (46%) than in CP (22%) and associated with poor outcome [48]. In addi-tion, hypomethylation of the T-cell antigen CD7 promoter region associated to a higher expression, is an early prognostic marker in CMLs [49].

Hypomethylation of several other genes such as c-fms (tyrosine kinase), myeloperoxidase and T-cell receptor b has also been reported to occur in AML and demethylation of H-RAS

occurs in CML and AML [46]. In addition, mutations in DNMT3A have been described in 22.1% of patients with AML. These mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome [50]. DNMT3A mutation study could then be helpful to classify intermediate-risk AML. The presence of these mutations in AML patients is likely to imply that aberrant epigenetic regulation is critical for pathogen-esis. However, the exact mechanism by which DNMT3A may affect gene expression is still unknown [51]. Indeed, although a study has reported that DNMT3A mutation results in a loss of DNA methylation activity of >50% [52], an in vitro assay has demonstrated that patients with DNMT3A mutations did not present any altered patterns of methylation [50]. Furthermore, mutation in two isocitrate dehydrogenases, IDH1 and IDH2, have been identified in 15–30% of AML patients [53]. Remarkably, these mutations



Comments Ref.

sFRP2 4q31 Wnt signaling ALL 16 [211]

CLL 69 [197]

sFRP4 7p14 Wnt signaling ALL 21 [211]

sFRP5 10q24.1 Wnt signaling ALL 28 [211]

SHP1 12p13 Phosphatase AML 78 [213]

CML 29 [213]

SLC2A14 12p13 Glucose transport ALL 60/100 B vs T [195]

SOCS1 16p13 Cytokine signaling AML 40 (0–60) [208]

SSPN 12p11 Tumor suppressor ALL 62/33 B vs T [200]

TCF2 17q12 Transcription factor ALL 10/50 B vs T [200]

TERT 5p15 Catalytic subunit of telomerase CLL 47 [221]

TFAP2A 6pter–p22 Transcription factor ALL 67/46 B vs T [200]

CML 6/63 CP vs BC [200]

TFAP2C 20q13 Transcription factor ALL 86/83 B vs T [200]

THBS1 15q15 Cell adhesion ALL 25 [194]

THBS2 6q27 Cell adhesion ALL 91 [194]

TP53I11 11p11 p53 signaling ALL 15/25 B vs T [200]

TRPC4 13q13 Ion channel CML 18/50 CP vs BC [200]

TSHZ3 19q13 Homeobox gene ALL 31/0 B vs T [200]

TWIST2 2q37 Transcription factor CLL 40 [38]

UBE2C 20q13 Cell cycle control (G2–M) ALL 23/17 B vs T [200]

WIF1 12q14.3 Wnt signaling ALL 30 [211]

WIT-1 11p13 Transcription factor AML 37 [26]

ZAP-70 2q11 Kinase CLL 50 [222]

ZNF382 19q13 Zn2+ finger protein ALL 52 [198]




were found to be more frequent in AML patients with DNMT3A mutations [54]. Inactivating mutations of TET2 are also frequently found in AML. Interestingly, IDH1 and IDH2 mutations disrupt the function of TET2, which promotes demethylation by hydroxylating methyl cytosine groups. IDH1/2- and TET2-mutant AML cells display global hypermethylation and a gene-spe-cific methylation signature and then contribute to AML pathogenesis, highlighting the link between cancer metabolism and epigenetic con-bolism and epigenetic con-trol of gene expression in AML [53]. The HOX11 proto-oncogene is frequently activated in T-cell ALL. The expression of HOX11 in T-cell ALL was associated with extensive demethylation of the proximal HOX11 promoter whether or not translocation was involved [55].

There are many examples of cancer-associated gene promoter hypomethylation affecting gene expression in leukemia. Nevertheless, the poten-tial of hypomethylation markers in cancer diag-nosis, classification and prediction of treatment response has yet to be evaluated.

n Hypomethylating agents & leukemiaThe potential reversibility of DNA methyla-tion encouraged the development of pharma-cological inhibitors as anticancer therapeutics. 5-azacytidine (Vidaza®) and decitabine (5-aza-2 -́deoxycytidine; Dacogen®) are two classical demethylating agents that belong to a class of cytosine analogs. 5-azacytidine and decitabine were approved by the US FDA for treatment of patients with myelodysplastic syndromes in 2004 and 2006, respectively [56]. In addition, a recent prospective Phase II study has demonstrated the efficacy of maintenance 5-azacytidine for patients with high-risk myelodysplastic syndromes/AML after induction chemotherapy [57]. 5-azacytidine and decitabine are also currently undergoing clinical trials for leukemia in association or not with other epigenetic or conventional drugs [301].

Their exact mechanism in vivo has not been clearly elucidated. However, in vitro studies have demonstrated their ability to induce DNA hypo-methylation, re-expression of aberrantly silenced genes, cell cycle perturbation, induction of apop-tosis and differentiation [58–61]. More recently, decitabine has been demonstrated to induce senescence and autophagy in CML cells [62].

Recent clinical trials involving conventional chemotherapy in association with these demeth-ylating agents have demonstrated promising results for leukemia treatment. Further work with the HDAC inhibitors acting in related pathways may result in further advances in the

care of leukemia patients. However, more study is required in order to get a better understand-ing of the molecular mechanisms induced by 5-azacytidine and decitabine.

Histone code in leukemia n Covalent histone modifications

in leukemiaN-terminal tails of core histones are modified by phosphorylation, acetylation, methylation, ubiq-uitination, sumoylation and ADP-ribosylation. The most extensively studied modifications are acetylation of lysine and methylation of arginine and lysine. While acetylation occurs as a single addition, more than one methylation moiety can be present on the same residue (e.g., mono-, di-, and tri-methylation abbreviated me1, me2 and me3, respectively) [22].

Four big families of proteins regulate these covalent histone modifications. Acetyl groups are attached at the e-amino group of lysine residues by HATs and removed by HDACs. Methylation is performed on lysine and arginine residues by HMTs and removed by histone demethylases (HDMs). These families of proteins act in con-cert, and sometimes by physically associating in molecular complexes, establishing a specific pattern of histone modifications suitable to the cellular context, frequently referred to as the ‘histone code’. Their activity regulates chroma-tin accessibility to transcription factors and to other effector proteins, playing a major role in determining the transcriptional pattern of the cell (Figure 1) [21,63].

Aberrant expression of histone-modifying enzymes has been correlated to different forms of leukemia. Moreover, many leukemia-a ssociated fusion proteins are able to recruit epigenetic part-ners with chromatin-modifying activities that contribute to the oncogenic property of the chime-ric proteins. We will review here the most frequent alterations/dysfunctions of enzymes establishing the histone code, involved in leukemia transfor-mation and progression, and the recent progresses in targeting these enzymes for leukemia therapy.

Histone acetylation: HDACs/HATsAcetylation of histone tails has been demonstrated to correlate with open chromatin and gene expres-sion. By contrast, hypoacetylation is associated with condensed chromatin and repression of tran-scription. This effect is achieved mainly through the neutralization of lysine’s charge by the acetyl group, which leads to a diminished inter action between histones and DNA and allows the recruit-ment of protein factors to gene promoters [64,65]. In



addition, HATs and HDACs control the acetyla-tion of many nonhistone p roteins such as p53, HSP90 and a-tubulin [66].

Histone acetyltransferases are divided into three major families: the Gcn5-related N-acetyltransferase family, the CBP/p300 fam-ily and the MYST (for the founding members MOZ, Ybf2/Sas3, Sas2 and Tip60) family [22]. HDACs consist of 18 protein members, divided into four classes. Class I HDACs comprise HDAC-1, -2, -3 and -8. Class II is subdivided in class IIa (HDAC-4, -5, -7 and -9) and class IIb (HDAC-6 and -10). Class IV includes one member, HDAC11, which exhibits homology to class I and II HDACs. Class III is composed of seven members called sirtuins (SIRT1–7). Class I, II and IV act in the presence of Zn2+ as a co factor, while class III acts with a NAD+-dependent mechanism. Class I and II HDACs form macro-molecular complexes with various partners that co-operate in their repressing function. HDACs belonging to different classes can also interact and regulate one another in the same complex [67,68].

Histone acetyltransferases and HDAC activi-ties are exerted on various gene classes, associated for example to development, proliferation and differentiation. Therefore, a dysfunction or an imbalance in those enzymatic activities could be responsible for cancer-related events.

HDACs & leukemiaHistone deacetylases are clearly implicated in some forms of leukemia. The most studied case is a form of APL, in which a chromosomal rear-rangement of the retinoic acid receptor (RAR) gene leads to the formation of RAR fusion p roteins, with different partners involving in most cases the PML protein. The fusion protein PML–RAR induces leukemogenesis by disrupt-ing the normal epi genetic regulation of retinoic acid (RA) target genes. The presence of PML in the chimera protein induces oligomerization of RAR, leading to an abnormal interaction with the corepressive complex NCoR/SMRT, which recruits HDACs. In cells bearing PML–RAR, physiological amounts of RA are not able to dis-sociate the chimera protein from NCoR/SMRT to release the repressive complex. This sustained recruitment of HDACs maintains an aberrant repression of RA target genes, which in turn blocks hematopoietic differentiation, thus leading to malignancy. Therefore, therapy with all-trans retinoic acid was introduced with success for APL patients with PML–RAR fusion protein, restor-ing the differentiation of blasts into granulocytes with, in most cases, remission of the patient [69–71].

It has been proposed that aberrant recruitment of HDACs to target genes owing to oligomeriza-tion of a chimeric transcription factor could be a mechanism underlying a broader group of AMLs. Indeed, the non-APL leukemia fusion protein AML1–ETO was demonstrated to act with a similar aberrant HDAC-recruiting mechanism that leads to the block of hematopoietic differen-tiation [72]. Moreover, combination of RA with HDAC inhibitors in patients with RA-resistant APL (such as those bearing the PLZF–RAR chi-mera) has induced interesting clinical responses, even if some blasts tend to remain resistant to dif-ferentiation [73,74]. Altered distribution of HDAC1 in AML and consequent specific patterns of chro-matin modifications at hematopoietic genes could also be associated with the patient’s outcome, and thus be a tool to improve prognosis prediction [75].

A recent genome-wide binding ana lysis of PML–RAR added complexity to the network of epigenetic enzymes that are targeted by this chimeric protein. The list includes JMJD3 (a H3K27me3 demethylase), SETDB1, JMJD1A (targeting H3K9), HDAC-4 and -9, and the already described DNMT3A [42,76]. These data point out that PML–RAR could have an impact in the modulation of many epigenetic modifica-tions, and not simply histone acetylation. The complete mechanism of leukemogenesis in this type of leukemia is yet to be fully understood.

The implication of HDACs in leukemia is not limited to their aberrant recruitment by fusion proteins, as alterations in the expression of vari-ous HDAC isoforms have also been reported, and in some cases have been associated with the patient’s prognosis. The ana lysis of Bradbury and colleagues of HDAC mRNAs in ALL samples revealed the overexpression of HDAC6 and SIRT1, and the downregulation of HDAC5 [77]. The work of Moreno and colleagues presented an ana lysis of the mRNA levels of 12 different HDAC isoforms in childhood ALL. HDAC-2, -3 and -6 to -8 mRNAs were overexpressed in ALL compared with normal bone marrow samples. HDAC1 and HDAC4 levels were also found to be higher in T-cell ALL, whereas HDAC5 mRNA was higher in B-cell ALL. Starting form these data, the correlation of HDAC transcript levels with patient’s survival has been investigated, resulting in the association of overexpression of HDAC-3, -7 and -9 with poor prognosis [78].

H4 acetylation was recently suggested as a prognostic marker in ALL patients. Indeed, high levels of H4 acetylation were correlated with an increased overall survival, although the authors stated that the study has to be confirmed on a



greater number of patients and adding the ana lysis of H3 acetylation levels [79]. Moreover, different HDAC isoforms modulate different functions depending also on the tissue and the develop-mental stage of the cell; sometimes they partially overlap and/or co-operate. Presently, the role of every single HDAC isoforms on gene transcrip-tion is not fully understood, thus arguing for the necessity of deeper knowledge to obtain powerful prognostic tools.

HATs & leukemiaSome HATs are recurrent components of fusion oncoproteins generated by chromosomal rear-rangement in leukemia; CBP or p300 have been found fused to MLL, MOZ and MORF. Interestingly, MOZ–CBP, a fusion protein asso-ciated with AML carrying the t(8;16)(p11,p13) translocation, is composed of two proteins with acetylating activities: CBP and MOZ, the lat-ter belonging to the MYST family of acetyl-transferases [22]. The different fusion proteins described contribute to leukemic transformation most likely by a mechanism involving mistar-geted histone acetylation and thus aberrant acti-vation of gene expression [80,81]. Moreover, inac-tivating mutations within the HAT domain of CBP have been found in approximately 18% of relapsed AML cases in a recent study, suggesting that the impaired HAT activity could be linked to resistance to therapy in AML [82].

Histone methylation: HMTs/HDMsTwo families of proteins, HMTs and HDMs are responsible for the attachment and the removal of methyl marks to and from specific histone lysine and arginine residues. The HMT fam-ily comprises more than 40 proteins presently identified. Almost all of them present a con-served Su(var)3,9, Enhancer of zest, Trithorax (SET) domain in their structure, which bears the histone-methylating activity. Demethylases have been more recently discovered and are divided into two subclasses: the flavin- dependent monoamine oxidase family (including lysine-specific demethylase [LSD]1 and LSD2) and the big jumonji C (JmjC) domain protein family [22,83–85].

Lysine methylation of histones can both enhance or repress transcription, according to the specific residue involved and the degree of meth-ylation. Especially, H3K9me2/3, H3K27me2/3 and H4K20me3 are commonly associated with silent chromatin, whereas H3K4me2/3, H3K36me2, H3K79me3 and arginine meth-ylation are frequently associated with gene

transcription [63,86]. Similarly to HDACs/HATs, HMTs and HDMs regulate the expression of genes that are important for crucial cellular pro-cesses, and alterations in their functions may promote carcinogenesis [87,88].

While HMT alterations have already been relatively intensively studied for their implication in leukemia transformation, the understanding of the HDM family’s link with hematological cancer is still in its infancy.

HMTs & leukemiaAmong HMTs, the most studied in leukemia is the MLL protein, which belongs to the Trithorax group (Trx-G) of proteins. MLL methylates H3K4 and is important in the regulation of HOX genes. Chromosomal rearrangements of the MLL gene are frequently found in ALL and AML [89]. These chromosomal translocations give rise to different MLL fusion proteins.

Among the genes misregulated by MLL fusion proteins, the most relevant class is the HOXA family, a group of genes pivotal in devel-opment, which are frequently overexpressed in leukemia [87,89]. The MLL chimeric proteins are devoid of intrinsic methyltransferase activity, owing to loss of the MLL SET domain after rearrangement. The activation of HOXA genes is thus achieved by mechanisms other than simple H3K4 methylation. Indeed, the H3K79 methyl transferase human disruptor of telomeric silencing-1L (hDOT1L) is a pivotal element in leukemogenesis driven by MLL fusion pro-teins [90]. In leukemia expressing the MLL–AF10 chimera, hDOT1L is aberrantly recruited to HOX genes and this correlates with abundant H3K79 methylation and overexpression of these genes. Accordingly, in a genome-wide study of MLL–AF4-induced leukemia, Krivtsov and col-leagues found a significantly greater number of H3K79 methylated promoters than in normal B lymphocyte precursors. The H3K4me3 mark was still associated with HOXA genes in this study; this modification is normally linked to transcriptional priming, and could be carried out by the wild-type allele of MLL or by other methyltransferases. hDOT1L promotes H3K79 methylation on such primed genes, resulting in transcription [91]. Moreover, a recent study reported hDOT1L-mediated transformation through H3K79 methylation, even in forms of leukemia bearing chimeric proteins that do not directly recruit this methylase. These data sug-gest an oncogenic role for hDOT1L in leukemia, and raise the interest regarding its pharmacologi-cal modulation for therapy [92].



An association between nucleoporin 98 and the methyltransferase nuclear-receptor-binding SET domain containing protein 1 (NSD1) that methylates H3K36 and H4K20 was found in AML with the translocation t(5;11)(q35;p15.5). This association was reported to contribute to transformation via activation of the HOX genes. The sustained methylation at H3K36 of HOXA7 and HOXA9 appears to prevent the repressive activity of enhancer of zeste homolog 2 (EZH2) on these sites, impairing differentiation. In contrast to MLL-driven leukemia, the methyl-transferase activity of NSD1 is preserved in the chimera and it is directly responsible for activa-tion of HOXA genes [93,94]. A NSD3 rearrange-ment has also been reported in AML associated with t(8;11)(p11.2;p15) [95].

The H3K27 methyltransferase EZH2 is a HMT whose overexpression has been correlated with several solid malignancies. EZH2 is part of the Polycomb repressor complex 2 (PRC2) that controls H3K27 status and represses gene transcription in complex with HDACs and DNMTs [96,97]. Although EZH2 is responsible for HOXA gene silencing during development in hematopoietic cells, its role in leukemia is still unclear. Recently, a group demonstrated that overexpression of EZH2 in T-cell leukemia patient’s cells is associated with a bad progno-sis [98]. EZH2 could thus be a promising target for epigenetic therapy of leukemia forms that overexpress this protein and have high levels of methylated H3K27. However, inactivating EZH2 mutations were found in a number of myeloid malignancies, where catalytic activity on H3K27 was reduced or abolished [99]. This argues, in at least some subtypes of leukemia, for a tumor suppressor role of EZH2 and thus points to the need of deeper knowledge in order to act safely on this target.

In CLL, epigenetic silencing of RELB, a mem-ber of the NF-kB pathway, has been associated with therapy resistance of cancer cells in male patients, with a mechanism involving trimethyl-ation of H3K9 and in parallel, deacetylation of H3. The authors found that these specific pat-terns of histone modifications are related to the sex of the patients, and could be new biomarkers for the aggressiveness of the disease according to the gender [100].

HDMs & leukemiaAlthough the involvement of HDMs in cancer progression has already been established, not much data has been collected regarding their role in leukemia [101].

KDM2b, a H3K36me2-specific JmjC demeth-ylase, was recently found to be upregulated in human leukemia and to have a critical role in transformation of hematopoietic progenitors. It has been proposed that KDM2b acts as an onco-gene and that it is necessary for AML development and persistence in mice, through the induction of HOXa9/Meis1 genes [102]. KDM2b enzymatic activity might thus be a potential t arget for l eukemia treatment.

We believe that discoveries in the field of HDM involvement in leukemia will increase over the next years, allowing a broadening of the picture for possible therapeutic design against these tar-gets. In particular, the flavo-enzyme demethylase LSD1 appears to be another interesting target with an important role in differentiation and h ematopoiesis and being overexpressed in various cancers [103].

n Epigenetic therapies targeting histone modificationsAberrant activation or overexpression of histone-modifying enzymes leads to deregulation of gene transcription, that may involve as described, both the silencing of TSGs and activation of develop-mental genes that sustain cancer progression. The reversal of these aberrant conditions with small inhibitory molecules, targeting histone- modifying enzymes, is thus a promising strategy for the treatment of many types of cancers including leukemia [104–106]. Some molecules have already been demonstrated to restore a healthy transcrip-tional pattern in cancer cells, inducing cell death through many different mechanisms (e.g., cell cycle arrest, restoration of apoptotic pathways, induction of differentiation). Interestingly, these molecules usually preferentially affect cancer cells. We will describe here the most relevant of these promising compounds for leukemia therapies.

HDAC inhibitorsMany molecules with HDAC-inhibiting activities have been discovered in the last few years [107–109], some of which have entered clinical trials for solid tumors and hematological malignancies (Table 2) [110].

Some compounds of natural origin have been initially isolated for their ability of inhibiting deacetylation. Sodium butyrate and tricho-statin A are the prototypes of two families of inhibitors, chemically classified as short-chain fatty acids and hydroxamic acids, respectively. Sodium butyrate exhibits a short half-life that limits its therapeutic application; however, from its structure, other more promising molecules



have been developed, in particular, sodium phenylbutyrate [111] and AN-9 (Pivanex), which demonstrated selective toxicity for leukemia cells against healthy blood cells [112].

Another well-known short-chain fatty acid is valproic acid, a derivative of valeric acid com-monly used to treat epilepsy and which dem-onstrates selective inhibition of class I and IIa HDACs [113]. This compound induces differen-tiation in AML cells and increases responsive-ness of Philadelphia-positive and APL cell lines to cytarabine. However, many studies suggested that valproic acid affects hematopoiesis, induc-ing anemia in epileptic patients and impairing the erythroid differentiation while stimulating the myelo-monocytic pathway [114,115].

The group of hydroxamic acids harbors suberoyl anilide hydroxamic acid (SAHA, Vorinostat), a pan-HDAC inhibitor that has been approved by the FDA for the treat-ment of cutaneous T-cell lymphoma as well as Panobinostat (LBH589), and Belinostat (PXD101), all undergoing c linical investigation for leukemia [110].

Benzamides are another class of molecules that demonstrate specificity for class I HDACs. Among them, Entinostat (MS-275) was used together with 5-azacytidine to improve cyto-toxicity in AML and ALL and have entered clinical trials, together with mocetinostat (MGCD103) [116–118].We want to underlie the importance of finding new isoform-specific inhibitors of HDACs in leukemia treatment. Indeed, a recent paper reported that CLL cells are sensitized to TNF-related apoptosis-inducing ligand-induced apoptosis by specific HDAC1 and HDAC2 knockdown, but not HDAC3, HDAC6 and HDAC8 [119]. Moreover, as described, a dif-ferential expression of some HDAC isoforms has been reported in different types of ALL (i.e., B- versus T-cell) [78].

HAT inhibitorsOnly a few molecules have been brought to light as HAT inhibitors, which have not been extensively studied in leukemia [120]. Anacardic acid, garcinol and curcumin have been proven to be valid natural HAT inhibitors. The first synthetic compounds designed were coenzyme A (CoA)-conjugated peptide analogs (e.g., Lys-CoA, H3-CoA-20 and others). Subsequently, other compounds such as garcinol analogs, the g-butyrolactone MB-3, isothiazolones and quino-line derivatives have been reported to inhibit spe-cific HAT members and to be effective in block-ing proliferation of some solid tumor cell lines, although the mechanism of function is still not elucidated. A p300-specific molecule, the pyrazo-lone-furan C646, is able to suppress proliferation of melanoma and lung cancer cell lines, reinforc-ing the idea that HATs are promising targets for cancer therapy, including l eukemia [121,122].

HMT inhibitorsFew compounds with HMT-inhibiting activ-ity have presently been found. Some deriva-tives of AdoMet, the c ofactor of the methyla-tion reaction, were reported to be i nhibitors of HMTs, and of DNMTs as well. AzaAdoMet and Sinefungin belong to this group, from which recently other derivatives have been produced and tested in vitro [123]. A screening of natural compounds identified the fungal metabolite chaetocin, an alkaloid produced by Chaetronium minutum, as an inhibitor of SUV39H1 [124,125]. This compound was able to restore expression of methylated CDNK2B (p15) and E-cadherin genes, by demethylation of H3K9, in AML cell lines [126]. Several G9a inhibitors have been identified such as a small diazepin-quin-azolin-amine derivative, BIX-01294 and its derivative E72, the 2,4-diamino-7-aminoalk-oxy-quinazolines UNC0224 and its derivative

Table 2. Histone deacetylase inhibitors in clinical trials for leukemia.

Chemical class Name Target Study phase

Short-chain fatty acids Phenylbutyrate Pan-inhibitor Phase I/II

Valproic acid Class I/IIa HDAC Phase II

Hydroxamic acids Vorinostat (SAHA) Pan-inhibitor Phase I/II

Panobinostat (LBH589) Pan-inhibitor Phase I/II

Belinostat (PXD101) Pan-inhibitor Phase I/II

Cyclic tetrapeptides Depsipeptide (FK228) Class I HDAC Phase I/II

Benzamides Entinostat (MS-275) Class I HDAC Phase I

Mocetinostat (MGCD-0103) Class I/IV HDAC Phase IHDAC inhibitors of different chemical classes are reported, with their respective targets. More information on these trials can be found at [301]. HDAC: Histone deacetylase; SAHA: Suberoylanilide hydroxamic acid.



UNC0321; however, they have not been tested in leukemia models [127–131]. The activity of the PCR2 component EZH2 is inhibited by the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep), which depletes PCR2 complex levels, thus inhibiting histone H3K27 methylation, resulting in apoptosis [132]. A combined treatment study with DZNep and the HDAC inhibitor panobinostat resulted in selective apoptosis in AML cells [133]. All of these compounds appear promising, but are still rela-tively unexplored, and more data is needed in order to evaluate their efficacy in cancer and, in particular, leukemia.

HDM inhibitorsThe LSD family of demethylases is inhibited by the monoamine oxidase inhibitor 2-phenyl-cyclopropylamine, a compound used for the treat-ment of depression and neurological disorders. This molecule was the first to be found as being active against this class of enzymes; however, it displays very low potency against LSD with respect to monoamine oxidase inhibition [134,135].

Recently, more specific LSD inhibitors have been designed starting from a 2-phenyl-cyclopropylamine structure [136–139]. One of these derivatives has been tested on an APL cell line, demonstrating a strong synergy with RA in exerting growth inhibitory and differentiating effects [137]. Independently, a set of bisguanidine compounds has been d emonstrated to be effective in inhibiting LSD1 [140].

Concerning JmjC domain proteins, a series of active compounds has been provided by some initial studies. N-oxalylglycine, an analog of the cofactor in JmjC-demethylating reaction, 2-oxoglutarate, was used as a leading structure for generating new compounds, mainly target-ing a H3K9-specific methylase JmJd2 [141–144]. However, biological data regarding these com-pounds are still scant and more insights on the mechanisms of action of those targets in leukemo-genesis will most likely help the characterization and d evelopment of those drugs.

miRNA & leukemiaIn the past few years, an additional layer of epigenetic regulatory mechanisms, namely ncRNAs, emerged as a key player in gene regu-lation and in cancer development. The most studied small ncRNAs to date, miRNA (miR), are endogenous small RNAs of approximately 22 nucleotides that post-transcriptionally regulate mRNA expression levels in a sequence-specific manner. Imperfect base pairing of a miR with

complementary sequences located mainly in the 3 -́UTR or 5́ -UTR of the target transcripts leads to mRNA degradation or translational repres-sion [145–147]. This highly evolutionarily con-served RNA family represents approximately 800 miRs in the human genome, and could regulate the expression of more than half of all protein coding genes [148]. Therefore, miRs are crucial regulators of fundamental cell processes, such as development, differentiation, p roliferation, cell cycle, survival and death [149,150].

Emerging evidence suggests that deregula-tion of miR expression patterns is observed in tumorigenesis, suggesting that they might act as a novel class of oncogenes or TSGs [145–147]. Noteworthy, the first report demonstrating a causal link between miRs and cancer was estab-lished in leukemia by Calin and colleagues. They demonstrated a downregulation of two miRs targeting Bcl-2, miR-15a and miR-16-1, located in a region deleted in more than half of CLL patients [4]. In addition, aberrant miR expres-sion could predict clinical outcome [151]. Since these observations, there have been an increasing number of reports demonstrating a critical role of miRs in the pathogenesis and prognosis of leukemia. Those that have been most extensively studied and appear the most significant markers of leukemia based on experimental data from patients are summarized in Table 3 and/or are dis-cussed in this section regarding their diagnostic, prognostic or predictive values.

n miR in CLLSeveral reports have defined miR signatures accompanying CLL pathogenesis (Table 3).Interestingly, most dysregulated miRs are linked to chromosomal abnormalities r epresented by frequent deletions found in CLL patients [152]. The first report of miR downregulation through this mechanism is related to miR-15a/16-1 down-regulation as a consequence of del13(q14) [4], which is the most common cytogenetic abnor-mality observed in CLL patients and is usually associated with good prognosis [153]. This deletion was demonstrated to be associated with the over-expression of the antiapoptotic factor Bcl-2 [154]. In addition, a signature of 13 miRs with prognos-tic values was established since they are correlated with the status of the two negative CLL prog-nostic factors, ZAP-70 and unmutated IgVH; from which nine are upregulated (miR-15a, -195, -221, -23b, -155, -24-1, -146, -16-1 and -16-2) and four are downregulated (miR-223, -29a-2, -29b-2 and -29c) [155]. Furthermore, the same group later demonstrated that deletions of miR-29



Table 3. List of miRNAs presenting aberrant expression in leukemia based on experimental data from patients.

miRNA Gene localization Status in disease

Comments Ref.

Chronic lymphocytic leukemia

let-7a-1 9q22 - [223]

miR-106b 7q22 + Targets Itch to accumulate p73 [185]

miR-143/miR-145 5q32 - Targets ERK5 [224]

miR-150 19q13 + [157]

miR-155 21q21 + [155,223]

miR-15a/miR-16-1 13q14 - Targets Bcl-2 [4,155]

miR-17 13q31 - [163]

miR-181b-1 1q32 - Targets CD69 and TCL-1 [156,157,223]

miR-181a 9q33 - Targets TCL-1 [223]

miR-196a-1 17q21 - [165]

miR-21 17q23 + Targets Bcl-2 and PTEN [157]

miR-221/miR-222 Xp11 + Targets p27 [225]

miR-223 Xq12 - [166]

miR-29a/29b 7q32 - Targets Bcl-2, TCL-1 and MCL1 [156]

miR-29c 1q32 - [166]

miR-30d 8q24 - [223]

miR-34a 1p36 - Targets CDK6, CCND1, CDK4, CCNE2 and n-myc [165]

miR-34b/miR-34c 11q23 - TCL-1 [226]

miR-497 17p13 - [165]

miR-92a-1 13q31 - [157]

Chronic myeloid leukemia

miR-10A 17q21 - Targets USF2, BCR–ABL independent [227]

miR-142 17q22 + Upregulated only in CP [169]

miR-146a 5q34 - Downregulated only in CP [169]

miR-150 19q13 - BCR–ABL dependent [169,227]

miR-151 8 - BCR–ABL dependent [227]

miR-17–92 cluster 13q31 + Regulated by BCR–ABL, targets c-myc [167]

miR-199b 9q34 + Upregulated only in CP [169]

miR-203 14q32 - Targets ABL1 [168]

miR-96 7q32 + [227]

Acute lymphocytic leukemia

miR-125b-1 11q24 + [228–230]

miR-128b 3p22 + [231]

miR-17–92 cluster 6p22 + [231]

miR-181b-1 1q32 + Targets CD69 and TCL-1 [231]

miR-196b 7p15 - Targets c-myc [232]

miR-204 9q21 + [231]

mir-331 12q22 + [231]

Acute myeloid leukemia

let-7c 21q21 - [233]

let-7d 9q22 + [233]

miR-106b 7q22 - [234]

miR-107 10q23 - Targets NFIA [233]

miR-125a 19q13 - [234]

miR-126 9q34 - [172,234]

miR-130a 11q12 - [234]

-: Downregulated; +: Upregulated; APL: Acute promyelocytic leukemia; Bcl-2: B-cell lymphoma 2; BCR–ABL: Breakpoint cluster region-Abelson; CDKN18: Cyclin-dependent kinase inhibitor 18; CP: Chronic phase; MLL: Mixed lineage leukemia; TCL: T-cell leukemia/lymphoma.



and miR-181, which target the TCL-1 oncogene are correlated with the more aggressive form of the disease [156]. These data were supported by experimental data from Fulci et al., which dem-onstrates that miR-150, -223, -29b and -29c were overexpressed in patients with IgVH-mutated cells [157], and from a study on Chinese patients demonstrating a concomitant downregulation of miR-15a, -16-1, -29b, -181a and -181 band overexpression of Bcl-2 and TCL-1 [158].

Fludarabine is the most extensively studied purine nucleoside analog, which is often used as first- and second-line therapy of CLL. However, a significant fraction of patients are refractory or become resistant to fludarabine treatments leading to a poor prognosis for these patients. Noteworthy, Ricci et al. demonstrated that p53 gene deletion is the strongest cause of primary refractoriness associated with a poor survival in CLL [159]. Nonetheless, two recent studies pub-lished at the same time associate miR signatures to fludarabine-refractory CLL patients. The first study established that miR-181a and miR-221 were strongly upregulated in fludarabine-resistant patients, whereas miR-29a was downregulated in the same patients [160]. In the second study, they demonstrated that high expression of miR-148a, -222 and -21 was tightly associated to refrac-tory CLL patients [161]. Moreover, both studies revealed that the differential gene expression in

these two groups was mainly related to p53 signal-ing network. Indeed, activation of p53-responsive genes was only detected in fludarabine-responsive cases. These observations are consistent with pre-viously published data demonstrating defects in the p53 pathway of fludarabine-refractory CLL patients and presenting low miR-34a, -29c and -17-5p expression levels [162,163]. In addition, low levels of miR-34a correlate the presence of the inactive variant SNP309 of the negative regulator of the p53 tumor suppressor MDM2 [164].

Taken together, these data demonstrate that aberrant miR expression is associated with the clinical outcome of CLL patients. Noteworthy, Rossi and colleagues developed a promising score system termed 21FK score corresponding to a combined miR-21 quantitative real-time PCR, FISH and karyotype analyses capable to strat-ify the survival of CLL patients [165]. Similarly, Stamatopoulos and colleagues developed a score system based on miR-29c, miR-223, lipoprotein lipase and ZAP-70 expression, capable of dis-criminating between good and poor prognosis subgroups of CLL patients [166].

n miR in CML Only a few reports demonstrate a link between aberrant miR expression and CML pathogenesis (Table 3). Interestingly, it was demonstrated that the MYC- and BCR–ABL-regulated polycistronic

Table 3. List of miRNAs presenting aberrant expression in leukemia based on experimental data from patients (cont).

miRNA Gene localization Status in disease

Comments Ref.

Acute myeloid leukemia (cont.)

miR-142-3p/miR-142-5p 17q22 + [233]

miR-146 5q34 - [234]

miR-15a 13q14 + Targets Bcl-2 [233]

miR-15b 3q25 + Targets Bcl-2 [233]

miR-17–92 cluster 13q31 + Targets APP and RASSF2 [235–237]

miR-193a 17q11 - Targets KIT [238]

miR-196b 7p15 + Regulated by MLL fusion proteins [239]

miR-21 17q23 + Targets Bcl-2 and PTEN [171]

miR-221/22 Xp11 + Targets CDKN1B (p27) and KIT [174]

miR-223 Xq12 + Targets NFIA [233,240]

miR-23b 9q22 - [174]

miR-25 7q22 - [240]

mir-29b1 7q32 - Targets SP1, regulated by myc [241]

miR-342 14q32 - Targeted by PML–RAR in APL [233]

mir-34a 1p36 + [174]

miR-362-3p Xp11 + [240]

miR-93 7q22 - [234]

-: Downregulated; +: Upregulated; APL: Acute promyelocytic leukemia; Bcl-2: B-cell lymphoma 2; BCR–ABL: Breakpoint cluster region-Abelson; CDKN18: Cyclin-dependent kinase inhibitor 18; CP: Chronic phase; MLL: Mixed lineage leukemia; TCL: T-cell leukemia/lymphoma.



miR-17-92 clusters are upregulated in patients in early CP, but not in BC CML [167]. Accordingly, CML is associated with a downregulation of miR-203, owing to either genomic instability (i.e., deletion) or CpG methylation [168], which in turn leads to ABL1 upregulation. Interestingly, newly diagnosed CML patients treated for 14 days with imatinib demonstrated an increased expression of miR-150 and miR-146a, accompa-nied by a decreased expression of miR-142, -199b and -145, which reach levels comparable to nor-mal control values [169]. Finally, a study identified several miRs that may predict clinical resistance to imatinib. Only one was upregulated (miR-191) and 18 (miR-7, -23a, -26a, -29a, -29c, -30b, -30c, -100, -126*, -134, -141, -183, -196b, -199, -224, -326, -422b and -520a) were downregulated in imatinib-resistant patients [170].

n miR in acute leukemiaConcerning acute leukemia, the majority of stud-ies focused on AML, with only few miRs reported as being altered in ALL. Beyond the general miR signatures reported in Table 3, many groups have underlined the existence of peculiar miR expres-sion patterns in AML subtypes with particular chromosomal rearrangement or specific muta-tions. Jongen-Lavrencic and colleagues reported a set of miR signatures capable of distinguish-ing between t(15;17), t(8,21), Inv(16), NPM1, CEBPA and FLT3–ITD bearing AMLs [171]. Li and colleagues determined miR signatures capa-ble of discriminating between CBF, t(15;17) and MLL-rearrangements in AML [172]. Similarly, Garzon et al. reported a specific miR signature associated with mutated NPM1 in AML, with respect to forms that do not bear the mutation [173]. Interestingly, miR-221 and -222 were found to be upregulated in AML [174], but down-regulated in CBF AML, characterized by genetic mutations in subunits of the CBF. CBF fusion proteins induce miR-221 and -222 downregulation, which in turn determines the upregulation of the tyrosine kinase KIT [175]. Similarly, two reports described a downregulation of miR-34a in AML with CEBPA mutations, while an upregulation was reported in other AML samples[174,176,177]. Marcucci et al. conducted a study on AML patients with FLT3–ITD and wild-type NPM1, finding a miR signa-ture associated with the patient’s prognosis. High-risk AML displayed a downregulation of miR-181 family members [178]. Moreover, Debernardi et al. reported a correlation of miR-181a expression and AML morphological subtype [179]. Patients with EVI1-expressing AML demonstrated diminished levels of miR-124 that could be repressed by DNA

methylation, suggesting a direct role of the onco-protein EVI1 in the transcriptional regulation of this miR [180]. Further studies are needed to dis-cover the role of miR-124 in this form of AML. Remarkably, a set of m iRs has been reported to discriminate between ALL and AML, with miR-128a and -128b, being overexpressed, whereas let-7b and miR-22 were downregulated in ALL compared with AML. The ana lysis of these miRs presented a 97% accurate discrimination between the two leukemia forms [181]. Taken together, these studies offer a detailed overview of how miRs can be useful to discriminate between sub-types of acute leukemia, for both diagnostic and prognostic purposes.

miR expression could also be correlated with response to treatment. Indeed, Schotte and col-leagues identified specific miR signatures associ-ated to the childhood ALL subtypes [182]. More recently, they reported resistance to vincristine and daunorubicin to be linked to an overexpres-sion of miR-125b, -99a and -100 in pediatric ALL, confirming the potential of miR signatures in helping therapeutic decisions [183]. Treatment with all-trans retinoic acid in PML–RAR patients was also found to modulate the expres-sion of various miRs, among which miR-15a, -15b, -16-1, -223, -342, -107, let-7a-3, let-7c and let-7d were upregulated and miR-181b was d ownregulated [184].

n miR & therapyAs described above, miRs represent both TSG-like and proto-oncogene-like (oncomiR) activi-ties involved virtually in all cell processes. In addition, the use of miR-expression s ignatures appears to be valuable in diagnosis, risk stratifi-cation (prognosis) and prediction of potentially efficient therapies against leukemia, including the identification of subgroups of patients at high risk. Moreover, miRs represent appealing novel therapeutic targets for treatment of leukemia patients. Thus, the potential use of anti-miR molecules, so-called antagomirs, as a new class of therapeutic anticancer agents is under investiga-tion; however, this is currently limited to in vitro and mice models [148,185]. The only clinical trial attempting to apply antagomirs in humans is against Hepatitis C [186]. Nevertheless, some pre-clinical studies have reported promising anti-cancer activity of antagomirs in leukemia cell lines [187–189]. Finally, since DNA methylation-mediated miR silencing appears to be involved in leukemogenesis (Table 1), methylated miRs could represent a promising new t herapeutic t arget of hypomethylating therapies [190].



ConclusionIn the past few years, the amount of data impli-cating underlying epigenetic mechanisms in leu-kemogenesis has been growing exponentially, raising the field of epigenetic as an essential facet of modern anticancer research for the develop-ment of more effective therapies against leuke-mia. Indeed, it became evident that the deregu-lated expression of multiple genes participating in leukemia development can be regulated by altered epigenetic mechanisms including DNA methylation, histone modifications and miR expression, which seem to co-operate in regu-lating the biological behavior of leukemia cells

(Figures 1 & 2). Studying such mechanisms and the corresponding targeted genes (i.e., TSGs and proto-oncogenes) has increased our understand-ing of the molecular and genetic consequences of leukemia development and progression. In addi-tion, the prevalence of these epigenetic altera-tions in leukemia, represents potential markers for early detection, prognosis and prediction of response to therapy. Moreover, in combination to the already-existing genetic markers (i.e., leu-kemia-associated cytogenetic abnormalities), these marks may be used to improve disease classification and help to guide therapeutic intervention. Therefore, epi genetically silenced

Genetic aberrations(translocations): fusion proteins

Point mutations Point mutations

Genetic aberrations(deletions)

Altered histone-modifying enzyme activities

Aberrant histone(de)acetylating/(de)methylating

activity recruitment

Aberrant histone acetylation and methylation pattern

Aberrant DNAmethylation pattern

Aberrant miRNAexpression

Altered DNMTactivity

DNMT overexpression

Normal precursor cell

Leukemia

TSG silencingOncogene activationGenome instability

Figure 2. Epigenetic mechanisms and the causal genetic aberrations in leukemogenesis. Summary of how genetic and epigenetic factors are interconnected and co-operate in the leukemogenesis process. The complex interplay of members of the epigenetic machinery is here simplified for matter of clarity. In leukemia, genetic aberrations lead to the production of chimeric proteins that can bear or recruit aberrant epigenetic activities such as histone deacetylating or methylating activities. Moreover, in some forms of leukemia, inactivating mutations were found in histone-modifying enzymes. The combination of these genetic features with the frequently altered expression of epigenetic enzymes (e.g., histone deacetylases) gives a scenario in which the acetylation and methylation pattern on crucial chromatin loci is altered. A similar situation is present for DNA methyltransferases, which can be affected by point mutations or have an altered expression level, leading to a pro-oncogenic DNA methylation pattern. miRNAs can also be altered by DNA methylation and genetic aberrations, such as deletion, that abolish their silencing activity. Altogether, the genetic and epigenetic modifications contribute to genome instability, silencing of TSGs and activation of oncogenes, which ultimately lead to tumor formation and progression. DNMT: DNA methyltransferase; TSG: Tumor suppressor gene.



or activated cancer genes offer new targets for the use of epigenetic anticancer drugs. These drugs may be used alone or in combination with either additional epigenetic drugs targeting another layer of epigenetic regulation or classical chemotherapeutic drugs, and represent prom-ising novel therapeutic approaches and hopes for improved patient survival. Nevertheless, the complexity of epigenetics in leukemogenesis has many aspects left to unravel.

Future perspectiveSince disruption of epigenetic balance is associ-ated with the early steps of carcinogenesis, tar-geting epigenetic mechanism to prevent their alteration may have great interest. In this con-text, diet and environment-mediated epigenetic modulation could be used as a chemopreven-tion strategy against the development of virtu-ally all cancer types including leukemia as well as other epigenetic-related diseases [191–193]. In this regard, many dietary compounds have been found to modulate epigenetic components lead-ing to changes in gene expression (Table 4). A can-cer-preventive epigenetic diet is thus conceivable. Nevertheless, this strategy requires a thorough investigation of epigenetic signatures associated to cancer development and especially of the one associated with the early step of carcinogenesis. However, the identification of biomarkers of the disease is not sufficient. In addition, this approach requires, a careful ana lysis and a rig-orous control investigation of the impact of such dietary intervention since the modulation of the epigenetic landscape may engender adverse side effects such as promoting the expression

of proto-oncogenes. Nevertheless, we are con-vinced that in the future, it would be possible to make ‘epigenetic choices’ in everyday life that would be beneficial for our heath condition.

In order to improve biomarker identification, large-scale prospective data need to be generated before this information can be translated into clinical practice. In this context, data collection relies on efficient and reliable high-throughput, next-generation deep-sequencing technologies. The present technology is burgeoning and is still expensive; however, such techniques are promis-ing with notable increases in sequence reads per lane and read length, and we can be optimis-tic for the future. This technical field should generate a more robust database of e pigenetic signatures associated with a disease.

Finally, this type of data will increase without any doubt the number of potential therapeu-tic targets in the leukemia network and con-sequently increase therapeutic opportunities with both already existing and newly identified epigenetic drugs. Concomitantly, epigenetic biomarkers will be more and more importantly used for diagnostic prognostic and response prediction of leukemia to a therapy. Moreover, epigenetic biomarkers probably have potential for following up the efficiency of a designed therapy, going towards a personalized medical approach of leukemia, with the ultimate goal of curative intention.

In summary, despite these attractive scenarios, there is a long way to go from our current knowl-edge to the establishment of epigenetic signa-tures as chemopreventive or chemotherapeutic targets, and many studies are needed to decipher

Table 4. Epigenetic modulators available in dietary agents.

Dietary source Bioactive compounds Epigenetic targets Ref.

Apples Phloretin DNMTs [242]

Broccoli Isothiocyanates, sulforafane and 3,3-di-indolylmethane HDACs, miRNAs [243–247]

Cashew nuts Anacardic acid HATs [248–251]

Cinnamon Coumaric acid, cinnamic acid HDACs [252,253]

Citrus Quercetin DNMTs, HATs and SIRT1 [254–257]

Coffee Caffeic acid, chlorogenic acid DNMTs, HDACs [252,253,258]

Garcinia Garcinol, isogarcinol HATs [248,259]

Garlic Diallyl disulfide, allyl mercaptan and S-allylmercaptocysteine

HDACs [260–262]

Grapes Resveratrol SIRT1 [263–266]

Green tea Epigallocatechin gallate DNMTs, HATs, HMTs and miRNAs [267–270]

Soybean Genistein DNMTs, HDACs, HATs and miRNAs [243,271–273]

Tomatoes Lycopene DNMTs [274]

Turmeric Curcumin DNMTs, HDACs, HATs and miRNAs [275–280]

The list includes molecules from different eatable sources, targeting one or more epigenetic mechanisms. Enzymatic inhibition is the most frequent mechanism, but when present activators have also been listed. DNMT: DNA methyltransferase; HAT: Histone acetyltransferase; HDAC: Histone deacetylase; HMT: Histone methyltransferases; SIRT1: Sirtuin 1.



the complicated pathways in which epigenetic mechanisms are involved.

Financial & competing interests disclosureThis work was supported by the “Recherche Cancer et Sang” foundation, the “Recherches Scientifiques Luxembourg” association, by the “Een Häerz fir kriibskrank Kanner” association and by the Action LIONS “Vaincre le Cancer” a ssociation and by Télévie Luxembourg. Michael Schnekenburger is recipient of a Télévie Luxembourg

fellowship. Cristina Florean is supported by an AFR fellow-ship from the Ministry of Research, Luxembourg. Publication costs were covered by the Fonds National de la Recherche Luxembourg. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Executive summary

Leukemia � Leukemia is a malignant neoplasm of blood-forming tissues characterized by an unrestrained proliferation of abnormal blood cells.

Leukemia is subdivided into a variety of large groups: chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML).

Epigenetic modifications & leukemogenesis � Epigenetic modifications, including DNA methylation, histone modifications and miRNA, lead to the activation or repression of gene

expression. Aberrant epigenetic marks (landscapes) occur frequently in leukemia. � Leukemia cells are characterized by both an increase in the expression of the DNA methyltransferase enzymes and an aberrant

methylation profile. A genome-wide hypomethylation and a concomitant CpG island promoter hypermethylation-mediated tumor suppressor gene silencing characterize the latter.

� Many leukemia fusion proteins involve histone-modifying enzymes as components of the chimeric protein, implicating a disruption or a mistargeting of the corresponding epigenetic function. Histone-modifying enzymes may be also aberrantly recruited and sequestered by fusion proteins leading to gene deregulation. Altered expression of histone-modifying enzymes have also been reported in leukemia patients.

� miRNA downregulation (owing to recurring leukemia-associated chromosomal deletions) and upregulation are frequently associated with leukemogenesis.

Therapeutic opportunities � Epigenetic signatures associated with leukemogenesis represent useful biomarkers for diagnosis, prognosis, prediction of therapy

response and monitor response to therapy. � The field of epigenetic therapy is rapidly progressing, demonstrating interesting antileukemia activities. Besides the current use of

histone deacetylase inhibitors and hypomethylating agents in clinical trials, histone acetyltransferases, histone methyltransferases, histone demethylases and miRNAs seem to offer additional opportunities for epigenetic intervention.

� Many natural compounds found in nutrients are active against epigenetic targets. Therefore, a specific diet can be envisaged for chemoprevention purposes. However, this strategy requires a careful approach, owing to the underlying possible side effects.

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Epigenomics of leukemia: from mechanisms to therapeutic applications

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