Imprinted Genes in Myeloid Lineage Commitment in Normal and Malignant Hematopoiesis

REVIEW Imprinted genes in myeloid lineage commitment in normal and malignant hematopoiesis

L Benetatos1 and G Vartholomatos2

Genomic imprinting is characterized by the parent-of-origin monoallelic expression of several diploid genes because of epigenetic regulation. Imprinted genes (IGs) are key factors in development, supporting the ability of a genotype to produce phenotypes in response to environmental stimuli. IGs are highly expressed during prenatal stages but are downregulated after birth. They also affect aspects of life other than growth such as cognition, behavior, adaption to novel environments, social dominance and memory consolidation. Deregulated genomic imprinting leads to developmental disorders and is associated with solid and blood cancer as well. Several data have been published highlighting the involvement of IGs in as early as the very small embryonic-like stem cells stage and further during myeloid lineage commitment in normal and malignant hematopoiesis. Therefore, we have assembled the current knowledge on the topic, based mainly on recent ﬁndings, trying not to focus on a particular cluster but rather to have a global view of several different IGs in hematopoiesis.

Leukemia (2015) 29, 1233–1242; doi:10.1038/leu.2015.47

GENOMIC IMPRINTING: GENERAL CONCEPTS The current number of IGs in mouse is approximately 150 Genomic imprinting is an epigenetic phenomenon that restricts (www.mousebook.org), while fewer (~95) have been reported in the expression of a gene to a single parental chromosome as a humans (http:www.geneimprint.com-site-genes-by-status.Imprinted. consequence of parental inheritance and not of sex.1 It is not Homo+sapiens). IGs are located in clusters of 3–12 genes spread widespread among all eukaryocytic organisms but is observed in over 20 kb to 3.7 Mb of DNA in length although some single IGs 8 flowering plants, in placental and in marsupials mammals.2 are spread throughout the genome. Each cluster contains Imprinted genes (IGs) exhibit intra- and interspecies differential protein-coding RNAs, long non-coding RNAs, microRNAs, small 2 expression pattern highlighting the flexibility and adaptation of nucleolar RNAs and a cis-acting ICR. Regulation of genomic these genes.3 Three theories that attempt to explain the existence imprinting is achieved through the control of the cluster by the long non-coding RNAs or through the insulating action of the and evolution of genomic imprinting have been proposed: the 8–12 kinship theory, the sexual antagonism theory and the maternal– CCCTC-binding factor (CTCF). Novel data highlight a non- offspring coadaptation theory. All these theories have the canonical regulatory mechanism, which should be further studied potential to predict which of the two alleles is expressed and in the context of disease. According to such model loss of insulin fi which is silenced, and might predict the taxonomic distribution of receptor and Igf1r in mice induces a signi cant decrease in the expression of multiple IGs like Igf2, H19, Dlk1 and Cdkn1c genomic imprinting. They also have the ability to predict tissue 13 specificity of genomic imprinting.4 The life cycle of genomic regardless of the parent-of-origin expression status. imprinting is characterized by the establishment, the maintenance and the erasure. Imprint establishment occurs in male and female gametogenesis, and is mediated by the regulatory action of DNA BIOLOGIC ASPECTS OF IGS methylation through the action of DNA methyltransferases 3A/B Genomic imprinting has a role in regulating placental develop- (DNMT3A/B). Subsequently, imprinting maintenance in the pre- ment and fetal growth, neonatal feeding, body temperature implantation embryo protects it from genome-wide epigenetic maintenance, metabolism regulation, infant and maternal beha- modifications. The process of maintenance is mediated by the vior toward optimal maternal care, sleep regulation, adult action of DNMT1, of ZFP57, of the primordial germ cell-7–H3K9me neurogenesis, stem cell biology, and quiescence of the recently complex, of the Polycomb repressive (PRC2) proteins and of the discovered but controversial, very small embryonic-like stem cells methyl-CpG-binding protein MBD3.5 Finally, genomic imprinting is (VSELs).1,14,15 VSELs are important as they have been considered erased during early germ cell development through passive or the most primitive murine bone marrow (BM) residing cell stem TET1-induced demethylation at the imprinting control regions cell population, sharing some characteristics with the long-term (ICRs) (or differentially methylated region (DMR)), which that way hematopoietic stem cells (HSCs).16 VSELs seem to erase imprinting are protected from reprogramming6,7 (Figure 1). The presence of on distinct DMRs for some IGs in manner similar to primordial ICR at only one chromosome ensures that free diffusion of germ cells. In contrast to the maintenance of genomic imprinting silencing factors through the nucleus to reach the active gene in somatic cells throughout the body, VSELs exhibit IGs methyla- copy will not occur.2 tion profile that leads to low expression of growth-promoting IGs

1Blood Bank, Preveza General Hospital, Preveza, Greece and 2Molecular Biology Laboratory, Ioannina University Hospital, Ioannina, Greece. Correspondence: Dr L Benetatos, Blood Bank, Preveza General Hospital, Preveza 48100, Greece. E-mail: [email protected] or [email protected] Received 21 December 2014; revised 27 January 2015; accepted 16 February 2015; accepted article preview online 23 February 2015; advance online publication, 27 March 2015 Imprinted genes in hematopoiesis L Benetatos and G Vartholomatos 1234

Figure 1. The life cycle of genomic imprinting is schematically depicted. Imprint establishment occurs in male and female gametes, imprinting maintenance occurs after fertilization in the pre-implantation embryo and is maintained in the embryo, whereas erasure of genomic imprinting occurs during early germ cell development. Proteins involved in each step are also described.

and high expression of growth-inhibitory genes.14,17 These genes Several IGs have been implicated in various different cancer might originate from epiblast/migrating primordial germ cell-like subtypes,26–30 whereas LOI of tumor suppressors and oncogenes cells and aberrantly methylated DMRs of several imprinted loci provides the first step toward tumor formation by conferring including Igf2-H19 could affect VSELs expansion keeping them in a cellular immortality (Figure 2). quiescent state.17 IGs have been implicated in a hereditary pattern of breast Deregulated imprinting during the embryonic life leads to cancer predisposition. It has been reported that lobular breast developmental disorders with characteristic phenotypes such as cancer female patients were significantly associated with having a the Beckwith–Wiedemann syndrome, Angelman syndrome, Sil- father with cancer. This phenomenon, although not experimen- ver–Russell syndrome, Prader–Willi syndrome and the uniparental tally demonstrated, has been attributed in part to genomic disomy syndrome.8 Furthermore, increased incidence of such rare imprinting where the paternally expressed genes lead to imprinting disorders associated with assisted reproductive tech- predisposition of cancer in fathers and their daughters.31 nology (ART) has been reported. Multiple loci were more likely to Interestingly, in a large study was shown that single-nucleotide be affected in the patients born after ART than in those born after polymorphisms associations with breast cancer and basal cell natural conception. The onset mechanism of these diseases is carcinoma exhibit parental origin-specific associations and these caused by uniparental disomy, duplications, deletions and variants are located in the 11p15 and 7q32 imprinted loci. The aberrant DNA methylation. Perhaps the process of ART (hormone breast cancer paternally associated single-nucleotide polymorph- stimulation, in vitro culturing, cryopreservation and the timing of ism probably acts through the IGF2-H19 locus in a still unidentified embryo transfer) exposes the developing imprintome to many pathway.32 external influences that affect the proper establishment and maintenance of genomic imprints.18 IGs such as these located in the Dlk1-Dio3 cluster have been IGS IN HEMATOPOIESIS used as markers able to distinguish embryonic stem cells (ESCs)– Developmental hematopoiesis is an evolutionarily conserved equivalent induced pluripotent stem cells (iPSCs) but the debate is process that begins in embryonic life during which blood cells still open.19 Most recently, it was shown that loss of imprinting are generated in at least three distinct waves..33 Although novel (LOI) of the DLK1-DIO3 cluster occurs at similar frequencies in interesting views regarding the hierarchical model of hematopoi- nuclear transfer (NT)-ESCs and iPSCs during reprogramming in a esis have been reported, according to the current model of method-independent manner resulting in highly similar cell types lineage determination human hematopoiesis is organized begin- suggesting that imprinting defects in reprogrammed cells ning with the HSC followed by progenitor cells, and differentiated represent the somatic origin of the cells.20 Although the above- cells. However, it was recently demonstrated that HSCs and mentioned study showed that imprinting defects are not strictly lymphocytes share many similarities, including a conserved related to the reprogramming method, it can be assumed that mechanism of long-term quiescence interrupted by proliferative similar to ART, in iPSCs generation the timing and the stimuli.34,35 Hematopoiesis is under the control of transcription reprogramming conditions (transcription factors and chemical factors and several other signaling pathways. Deregulation of such compounds) are important for determining an imprinted domain regulatory mechanisms leads to the generation of the leukemic as a marker of ESCs–iPSCs equivalence. stem cells (LSCs) resulting in malignant phenotypes including The effect of IGs in development is gene dosage dependent. acute myeloid leukemia (AML) and myelodysplastic syndromes Experiments in mice show that abnormal Dlk1 gene dosage is (MDS).36–40 associated with overgrowth, intrauterine and perinatal embryonic Several IGs have been found to be involved in both fetal and death. Aberrant gene dosage probably leads to unbalanced adult hematopoiesis. Their expression follows the principle of high proliferation and differentiation with a compromise between expression during prenatal life with decreasing rates after birth, prenatal size and developmental maturity.21–23 Similarly, improper that is, high expression in HSCs and decreased expression in gene dosage is been observed in Beckwith–Wiedemann syn- differentiated counterparts. This notion arises from data showing drome, Silver–Russell syndrome and Prader–Willi syndrome in that IGs including Dlk1, Ndn, Igf2, Gnas, Cdkn1c, Wt1, G19 and humans.24 The deregulated balance between cellular differentia- Meg3 are differentially expressed in HSCs and in multipotent tion and proliferation as a consequence of altered IG dosage may progenitors.41,42 Furthermore, quantitative analysis of 36 IGs in also be related to tumorigenesis and malignant transformation.25 adult human peripheral leukocytes showed that 21 genes were

Leukemia (2015) 1233 – 1242 © 2015 Macmillan Publishers Limited Imprinted genes in hematopoiesis L Benetatos and G Vartholomatos 1235 while ST-HSCs activity was Igf2 independent.49,50 Similarly, Dlk1 maintains HSCs in an undifferentiated state and is responsible for the proper expansion of HSCs and/or progenitors. The Dlk1 contribution in fetal liver hematopoiesis probably requires the synergistic action of Igf2, as observed in mouse bimaternal embryos.51,52 Recent findings revealed that Slc22a3 and Meg3 were upregulated in hematopoietic stem-progenitor cells (HSPCs) exerting stabilizing function in multipotency, whereas knockdown showed a shift toward the differentiated progeny.53,54 Increased H19 methylation of the DMR located in an enhancer region downstream of H19 gene during the transition from HSCs to multipotent progenitors provides an explanation for the release of HSCs out of quiescence associated with loss of self-renewal.41 Conditional deletion of the maternal H19-DMR leads to Igf2 upregulation, increased translation of Igf1r and subsequent loss of Foxo3-mediated inhibition of HSCs cell cycle activity. Hence, H19 exclusively has the ability to regulate adult HSCs quiescence and total BM cellularity.46,47 However, not all IGs seem to be effective in regulating HSCs quiescence. Loss of CDKN1C promotes HSCs exhaustion as a consequence of p53 upregulation or increased RB phosphorylation in late G1 phase of the cell cycle, and causes cell cycle entry via regulation of the nuclear import of Hsc70-Cyclin D complex. On the contrary, Ndn despite its negative cell cycle regulatory function restricted to the regeneration phase of hematopoiesis, it has negligible effect on the apoptotic control of HSCs.55–58 DNMT1, which is essential for maintaining genomic imprinting, was recently shown to be paternally expressed in the human placenta and therefore is itself an IG.59 Yet, we still do not know if DNMT1 exhibits imprinting properties in physiological and pathological hematopoiesis. Dnmt1 seems dispensable during the hematopoietic process, but probably is required for pre- sumptive self-renewal divisions of HSCs, as well as differentiation. This is deduced by the findings in Dnmt1 mutants, which HSCs Figure 2. Aberrantly expressed IGs have been associated with an failed to generate mature myeloid cells but promoted the increased stem cell identity, reduced differentiating ability and expansion of myelo-erythroid lineage and the cycling of myeloid oncogenic potential. LOI of either a single IG or an entire imprinted 60,61 cluster in addition with founding mutations and epigenetic events cells. DNMT1 could also affect hematopoiesis indirectly might promote carcinogenesis. Relaxation of imprinting, LOI and through its action on BM mesenchymal stem cells. These cells upregulation of the normally expressed allele might lead to altered have the ability to form BM tissue in vivo exhibiting a unique DNA gene dosage, which is related to tumorigenesis.23 Blue and red methylation signature. They support human hematopoiesis, by genes represent paternally and maternally expressed IGs, respec- regulating self-renewal, differentiation, survival, migration of HSCs, tively. Black lollipops represent methylated imprinting control promote engraftment of HSCs, while composing a sanctuary for elements (ICE) and secondary DMRs. White lollipops represent the survival of LSCs.62,63 DNMT1 was upregulated in early-passage unmethylated ICE and secondary DMRs, whereas gray lollipop mesenchymal stem cells compared with late-passage mesenchy- represents epigenetic changes in the ICE leading to relaxation or LOI mal stem cells, whereas it interacts with the RB IG composing a RB- of a single gene reactivating the expression of the normally silent 64 allele. c-JUN-DNMT1 axis. As mesenchymal stem cells have a sustaining role in hematopoiesis, it would be interesting to verify whether undetectable in adult blood.43 Interestingly, some IGs, such as LOI of IGs or deregulation of the axis might lead to abnormal IGF2, might exhibit biallelic expression in BM and monoallelic in hematopoiesis. peripheral blood suggesting an imprint modulation during development.44 There are data that support the notion that IGs IGS IN MYELOID MALIGNANCIES are essential for the expansion of HSCs. Several IGs were Most myeloid malignancies stem cell reside in the HSCs preferentially expressed in long-term HSCs mainly associated with compartment suppressing the coexisting normal HSCs, or growth restriction, including H19, Cdkn1c, Ndn, Rb, Meg3, Peg3 and inhibit the production of normal differentiated hematopoietic GRB10. In contrast, Ascl2, Peg12, Sfmbt2, Pon3, Atp10a and Osbpl5 cells by blocking their differentiation at the HSCs–progenitor were expressed preferentially in short-term (ST)-HSCs and multi- transition.65,66 That malignant transformation of HSCs and potent progenitors and were associated with growth promotion committed progenitor cells is the result of the presence of driver and increased metabolism. These data suggest that different IGs mutations and the later accumulation of genetic and epigenetic may have different roles during the earliest phases of hemato- events.67,68 It was recently demonstrated that normal hemato- 45,46 poietic development. These findings extend those reporting poietic progenitors lymphoid-primed multipotential progenitors that only three members of the IG network, which is involved in and/or granulocyte macrophage progenitors as the sub- the regulation of embryonic growth and differentiation, probably population of origin for AML LSCs in the majority of cases, which display a regulatory function in HSCs: Ndn, Cdkn1c, H19.47,48 Igf2 through genetic or epigenetic events gained self-renewal ability has been considered a novel growth factor for HSCs, is highly that normally does not characterize normal hematopoietic expressed in fetal liver and also expressed by the CD3+ supportive progenitors.69 cells essential for the expansion of the HSC pool. Moreover, adult As mentioned, IGs exert a role in normal hematopoiesis. There murine long-term HSCs were able to bind Igf2 on their surface, are several data that highlight their involvement also in malignant

© 2015 Macmillan Publishers Limited Leukemia (2015) 1233 – 1242 Imprinted genes in hematopoiesis L Benetatos and G Vartholomatos 1236 pathogenesis of chronic myeloid leukemia can be assumed.76 Another putative mechanism through which the imprinted cluster affects malignant transformation implicates CTCF. CTCF is known to be involved in normal hematopoietic development but much less is known for its role in leukemia. Regulation of IGF2-H19 cluster falls within the CTCF insulator model. Therefore, a possible involvement of that factor through the IGF2-H19 cluster was evaluated in AML. It was shown in mice that CTCF, through the action of the chromatin remodeling factor Smarca5, binds to the Igf2-H19 ICR in AML blasts. Hence, the interaction between CTCF and Smarca5 epigenetic factor deregulates the transcription of the imprinted cluster in AML.77

DLK1-MEG3 CLUSTER DLK1 and MEG3 are located on human chromosome 14 and on mouse chromosome 12. They are part of a large cluster comprising coding genes, long non-coding RNAs, small nucleolar RNAs, pseudogenes and the largest known cluster of microRNAs. Figure 3. Schematic representation of interactions between IGs and In mouse, the particular cluster also contains three DMRs: the other factors involved in normal and malignant hematopoiesis. Dlk1-DMR at the 3′ end of the Dlk1 gene, the intergenic-DMR (IG- miR-29b suppresses the expression of the Sp1/NF-κB pathway, DMR) located ~ 15- kb upstream of Meg3 and the postfertilization which is able to enhance DNMT1 expression promoting hyper- derived Meg3-DMR located at the gene promoter.19 Methylation methylation of target genes. In AML, downregulation of miR-29b abnormalities in this cluster have been observed in IG-DMR and enhances the action of Sp1/NF-κΒ axis leading to increased expression of DNMT1 promoting gene hypermethylation. Similarly, MEG3-DMR as well. IGs of the DLK1-MEG3 cluster are deregulated in AML upregulation of nucleolin (NCL) enhances NF-κB activity in different myeloid malignancies. It was shown that DLK1 promoting DNMT1 overexpression. DNMT1 is also able to interact was upregulated in CD34+ cells in MDS patients slowing the with non-coding RNA—extra-coding CEBPA in this case—increasing progression of cells through G0–G1 into S phase inhibiting cell CEBPA methylation. In normal HSCs, CDKN1C maintains cytoplasmic differentiation. Furthermore, DLK1 was established as a member of localization of Hsc70/Cyclin D1 complex thus not permitting RB a set of genes that could discriminate primary myelofibrosis (PMF) phosphorylation and maintaining HSCs quiescence. CDKN1C defi- CD34+ cells among the other myeloproliferative neoplasms ciency promotes the nuclear import of Hsc70/Cyclin D1 stimulating (MPNs) and might contribute to the abnormal CD34+ cell HSCs cell cycle entry. The CTCF/Smarca5 complex binds to the ICE of 78-80 IGF2/H19 cluster enhancing the expression of H19 and at the same proliferation and differentiation. Dlk1 expression was induced time inhibits the expression of IGF2 in AML. In chronic myelogenous in response to the overexpression of the oncogene Meis1 in a leukemia (CML), the BCR–ABL fusion gene via the upregulation of Nup98-HoxD13 (ND13) murine leukemia model. Its overexpression Erk1/2 kinases inhibits CDKN1C expression, whereas, through the was dependent on both ND13 and Meis1, which cooperated enhanced expression of MYC oncogene upregulates the expression disrupting the genomic imprinting of Dlk1 via DNA hypermethyla- of H19, which in turn via STAT5 upregulates the expression of the tion of the IG-DMR and not through hypermethylation of the Dlk1 BCL-XL antiapoptotic protein. promoter. Although Meis1 overexpression led to an increase of IG- DMR methylation, it did not directly bind to the IG-DMR and an myelopoiesis (Figure 3). Their deregulated expression might be indirect mode of action through the Dnmts could not be associated with mutations, altered gene dosage and LOI, or as excluded.81 downstream targets in complex networks their expression might MEG3-DMR hypermethylation was observed in MDS and AML also be modulated. patients especially in the refractory anemia with excess blasts-I subcategory and was associated with a significantly reduced IGF2-H19 CLUSTER overall survival in AML and a trend for reduced overall survival in MDS.82 A more in depth analysis revealed a region upstream DLK1, The involvement of the particular cluster in the pathogenesis of which exhibited a methylation pattern with cis-acting insulator myeloid malignancies has been well known. LOI of the IGF2-H19 properties that might control DLK1 imprinting in AML.83 MEG3 cluster has been observed in high percentage in MDS and AML also showed hypermethylation at several CpG dinucleotides patients.70–72 LOI of IGF2-H19 enhanced the proliferation of 73 especially in the region located within the gene. However, no granulocytes, macrophages and erythroid cells. In addition, in inverse correlation was found between DLK1 and MEG3 expression polycythemia vera was suggested that H19 can facilitate commit- levels in AML patients. These data suggested that at least in AML ment. In fact, H19 loss could potentially delay commitment itself DLK1 transcription is not controlled by MEG3 as in mouse or prolong the maturation of committed precursors, leading embryonic stem cells.9,83 LOI because of aberrant hypermethyla- to an increase of precursors and mature cells characteristic tion of the MEG3-DMR exclusively was also observed in acute 74 of polycythemia vera. Furthermore, IGF2-H19 LOI was observed promyelocytic leukemia primary samples and in transgenic mice in patients with accelerated phase-blast phase chronic myeloid proposing a role of the cluster acute promyelocytic leukemia leukemia because of biallelic IGF2 expression in a H19 pathogenesis.84 DNA methylation at the CTCF-binding sites in methylation-independent manner.75 Moreover, H19 expression promyelocytic leukemia-retinoic acid receptor alpha transgenic seems to be BCR–ABL dependent and cooperates with the fusion mouse model was also observed leading to an overexpression of gene to promote tumor growth. This effect can be attributed to the hosted microRNAs. Consistent with the previous findings, the interaction of H19 with other factors. H19 depletion inhibited Meg3 was hypermethylated at the promoter and the gene body STAT5 phosphorylation decreasing Bcl-xL activity in leukemic cells. including all seven CTCF-binding sites abrogating its insulating Furthermore, in cell lines BCR–ABL disruption led to c-Myc activity. Aberrant methylation at the locus was considered decreased expression together with decreased H19 levels. characteristic of the acute promyelocytic leukemia cells and did 82–84 Hence, a putative BCR–ABL–MYC–H19–STAT5–BCL-XL axis in the not occur during normal myeloid differentiation.

Leukemia (2015) 1233 – 1242 © 2015 Macmillan Publishers Limited Imprinted genes in hematopoiesis L Benetatos and G Vartholomatos 1237 However, in PMF, MEG3 exhibits a variable expression levels; it of gene dosage by genomic imprinting at closely linked genes has might be up- or downregulated.85 Perhaps, similar to the findings implications for the understanding of the effect of epigenetic by Khoury et al.83 methylation and expression profiles depend on marks in myeloid cancer genesis.98 the region of the gene that has been studied. Very probably there should be other mechanisms than methylation that increase MEG3 expression in myeloid malignancies. Although hypermethy- WT1 lation and transcriptional silencing of MEG3 is characteristic of WT1 is a paternally expressed IG in humans and exhibits either several tumors,86 there are probably other upstream effectors that tumor-suppressor or oncogenic potential in a tissue-specific should be considered in the context of myelopoliferative manner. WT1 has an essential role in the normal development neoplasms. of the urogenital system, and it is mutated in a subset of patients with Wilm's tumors. In embryo, it is expressed in urogenital tissues, whereas in adult life it is involved in hematopoiesis where it is CDKN1C expressed mainly in the CD34+ fraction.99 It has the ability to CDKN1C is an IG expressed from the maternal allele and encodes suppress the transcription of hematopoietic-related proteins an inhibitor of several G1 cyclin-CDK complexes. It is a negative including macrophage colony-stimulating factor, transforming regulator of cell proliferation and possesses tumor-suppressor growth factor beta and RARa. WT1 protein is overexpressed in properties. Cdkn1c has the ability to maintain quiescence of long- leukemic blasts compared with normal CD34+ marrow cells.100 term HSCs via the interaction with the Mecom complex locus or The WT1 locus at 11p13 expresses two imprinted transcripts: the with Evi1 given that mice deficient for Mecom shown a shift non-coding antisense RNA WT1-AS and the alternate coding toward cycling HSCs.87,88 CDKN1C affects cell cycle arrest in CD34+ transcript alternative WT1 (AWT1).101 In myeloid cell lines, WT1, the hematopoietic cells through direct transcriptional upregulation by WT1-AS non-coding transcript and the AWT1 transcript are transforming growth factor beta. This pathway through which detected suggesting a coregulatory mechanism sharing a cis transforming growth factor beta mediates its cytostatic effects on regulatory element. AWT1 promoter hypermethylation is asso- human hematopoietic cells suggests an explanation for the ciated with a prognostic potential discriminating relapse after HSC frequent silencing of CDKN1C expression. Notably, transforming transplantation and for monitoring minimal residual disease.102 growth factor beta binds specifically to CDKN1C promoter without Similarly, WT1 expression could be used in the setting of affecting the expression of the other co-IGs in the locus.89 transplantation of ex vivo purging with chemotherapy before a There was no evidence of CDKN1C promoter hypermethylation second peripheral stem cell collection.103 WT1 is mutated in in AML and MDS suggesting that the specific epigenetic mark is almost 10% of cytogenetically normal AML cases and less in MDS, not the mechanism through which CDKN1C loses its tumor- MPN-MDS cases and PMF, while conferring worse prognosis. suppressor function.90–92 WT1 mutations may represent cooperating events able to interact In addition, a functional link between BCR–ABL and CDKN1C has with initiating mutations, or its deregulated expression might also been identified. Upregulation of the extracellular signal- share common regulatory mechanisms with other IGs.68,85,104–107 regulated kinase Erk1–Erk2 pathway by BCR–ABL might be WT1 is frequently overexpressed in a subset of LSCs in a mutation- involved in the downregulation of CDKN1C expression. Therefore, independent pattern but the exact mechanism is still identified. it was proposed that CDKN1C might be an effector of the anti- Nevertheless, WT1 overexpression per se does not confer a growth proliferative activity of imatinib and second-generation drugs advantage of HSCs.108 Therefore, several putative pathways used to treat chronic myeloid leukemia. In fact, these drugs through which WT1 affects leukemogenesis have been explored. enhance cell cycle arrest via the accumulation of CDKN1C.93 For example, it is shown that Wt1 overexpression in cooperation with AML1–ETO fusion gene is able to induce AML in mice, or it might interact with other IGs such as SNRPN and SNURF.109,110 In L3MBTL1 AML and chronic myeloid leukemia-deriving cells, WT1 interacts L3MBTL1 located on chromosome 20 is a PRC2 member that with HSP90, which stabilizes WT1 and protects it from functions as a transcriptional repressor, which can be directly proteasome-dependent degradation.111 Mutated forms of WT1 recruited to the TEL gene. It is expressed in CD34+ HSCs, in more in CD34+ cord blood cells block myeloid differentiation because of differentiated hematopoietic lineages, and in mesenchymal upregulation of EZH2 and consequent repression of PRC2 target cells.94 L3MBTL1 depletion causes replicative stress, DNA breaks, genes, which are involved in myeloid differentiation. It is also DNA damage response and genomic instability in cancer cells. It demonstrated that WT1 mutation induced DNA hypermethylation inhibits cell proliferation causing a G2-M arrest in cell lines and because of concurrent overexpression of DNMT3A on PRC2 target might contribute to the pathogenesis of 20q malignancies.95,96 genes. Although an EZH2 recruitment of DNMT3A to target genes L3MBTL1 knockdown accelerated erythroid differentiation of is not demonstrated in that study, a causal link and further leukemic cells and normal HSPCs as well, without affecting any therapeutic implications between WT1 mutations and the of the other known genes involved in the specific process. Hence, epigenetic machinery are revealed.112 Most recent findings it was suggested that L3MBTL1 might contribute to the show that AML patients with WT1 inactivating mutations pathogenesis of del20q disorders and especially polycythemia exhibit reduced 5-hydroxymethylcytosine levels in a manner vera.97 similar to the IDH1–IDH2–TET2 mutated AML. The reduced 5- Coordinated silencing of L3MBTL1 and SGK2 maintained hydroxymethylcytosine levels could be attributed to the physical megakaryopoiesis and enhanced erythropoiesis increasing pro- interaction between TET2-TET3 and WT1 in a IDH1/2–TET2/3–WT1 liferation of early erythroblasts without providing a selective pathway. The interaction between TET2 and WT1 might be advantage of HSPCs in del20q cell lines. This cooperation disrupted in the case of mutations of either factor leading to the enhanced MYC transcription, through the regulation of nucleoso- inability of TET2 to exert its function. In fact, WT1 directly binds mal compaction or nucleosomal positioning. These data provide and recruits TET2 to WT1-target genes activating their expression evidence of a link between an acquired cancer-associated genetic and through that interplay TET2 is able to inhibit the proliferation lesion (del20q) and an imprinted cluster. However, these findings of AML cells in a WT1-dependent pattern.113,114 contradict those reported by Bench et al. who observed that WT1 represents a member of a set of eight genes (CD9, GAS2, L3MBTL1 transcripts level were similar in cells from patients with DLK1, CDH1, WT1, NFE2, HMGA2 and CXCR4) with the discrimina- and without del20q, but they have might consider contaminated tory potential of PMF CD34+ cells. Notably, these genes are normal undeleted cells in their assays. The coordinate regulation aberrantly regulated also in PMF granulocytes that could

© 2015 Macmillan Publishers Limited Leukemia (2015) 1233 – 1242 Imprinted genes in hematopoiesis L Benetatos and G Vartholomatos 1238 differentiate from control polycythemia vera and essential genomic region is frequently deleted in AML and MDS-MPN. thrombocythemia granulocytes. Therefore, they can be used for Aberrant promoter hypermethylation and transcriptional repres- differential diagnosis purposes especially in the cases between sion of the NNAT locus is a frequent event observed in peripheral essential thrombocythemia and PMF. However, further evaluation blood leukocytes from AML patients.125 of the diagnostic potential of the gene set should be performed Knockdown experiments of Slc22a3 show that there is a greater before it could be widely applicable.78 proportion of myeloid cells generated from Slc22a3 knocked- down HSCs. These findings suggest that decreased expression of the specific gene might contribute to the myeloid directed DNMT1 differentiation of HSCs.54 DNMT1 was only recently reported as an IG in the placenta. It is In F9 promyelocytic leukemia-retinoic acid receptor alpha not known yet if it exhibits imprinting properties in normal and/or teratocarcinoma cells Mest and Slc38a4 IGs expression is malignant hematopoiesis. It would also be of interest to evaluate decreased and minimally changed respectively recapitulating the existence of autoregulatory loop in the maintenance of the RARa null phenotype. Probably, RARa regulates genomic genomic imprinting. Data suggest that its expression follows the imprinting in mammals although there is no clear experimental pattern of the IGs. DNMT1 is highly expressed in HSCs where demonstration. The fact that RARa is involved in hematopoiesis methylation is most relevant and loses importance after lineage made the authors assume that the regulation of both IGs by RARa commitment and is indispensable for cell autonomous survival of might occur during HSCs differentiation in an epigenetic HSCs.115 DNMT1 is at least indirectly involved in leukemogenesis manner.126 via two mechanisms. First, downregulation of miR-29b alters the expression of Sp1, which in turn increases DNMT1 expression, and second through the enhanced activity of the nucleolin–NF-κB– PERSPECTIVES DNMT1 axis, hence promoting hypermethylation of target IGs are highly expressed in HSCs with decreasing expression genes.116,117 Moreover, in the K562 cell line is shown that DNMT1 during the transition to committed and fully differentiated cells. interacts with a nuclear non-polyadenylated ncRNA in the CEBPA Elegant work by Goardon et al.69 demonstrated that LSCs originate locus named extra-coding CEBPA. This DNMT1–RNA interaction from the lymphoid-primed multipotential progenitors and gran- modulates genomic methylation of CEBPA, and moreover, is not ulocyte macrophage progenitors subfractions. Although no IGs restricted only to CEBPA but is rather a global effect and occurs in were identified among the gene expression signature that multiple gene loci. These findings might find clinical application characterizes LSCs,127 it has not been explored whether aberrant with demethylating strategies used in myeloid malignancies.118 In genomic imprinting at lymphoid-primed multipotential progenitor a MLL-AF9 AML Dnmt1 knockout murine model is shown that and granulocyte macrophage progenitor stage confers cells newly Dnmt1 is selectively maintained in LSCs suggesting that it could acquired self-renewal ability converting them to LSCs. Yet, in the initiate or promote the survival of the malignant stem cells sparing era of high-throughput massive sequencing and single-cell normal HSCs. The fact that LSCs and HSCs are Dnmt1 dependent analysis, the presence of aberrantly expressed IGs in LSCs cannot together with the finding that Dnmt1 haploinsufficiency was be excluded. LSCs share properties with normal HSCs including sufficient to delay the progression of MLL-AF9 induced AML might quiescence, self-renewal ability and resistance to apoptosis.128 argue in favor of an imprint-like activity of the particular They are chemoresistant difficult to eradicate composing a methyltransferase as no mutations of the Dnmt1 are reservoir for eventual disease relapse.129 According to Sharma observed.119 The findings of this study contradicted those et al.130 lung cancer stem cell chemoresistance was attributed to reported by Garzon et al.116 on DNMT1 expression in AML but it the upregulation of IGF1R and subsequent upregulation of the should be taken in consideration in that study mononuclear cells JARID1A demethylase, which in turn interacted with RB and HOX from patients with high blasts count were examined whereas, in genes affecting tumor progression. Based on these findings and the study by Townbridge et al.119 purified LSCs were evaluated. the data reported by Boucher et al.,13 a similar putative network/ loop comprising IGF1R-IGs-epigenetic machinery should be explored in LSCs chemoresistence. OTHER IGS According to a recently proposed leukemogeneisis model, SNRPN exhibits promoter hypermethylation in MDS and AML aging HSCs acquire somatic mutations, which are not sufficient to patients but without significant association with clinical features.82 generate leukemia but promote epigenetic deregulation.131 However, in myeloid leukemia cell lines SNRPN is shown to Cancerous tissue can also be considered prematurely aged tissue maintain its monolallelic expression and its asynchronous replica- in line with their DNA methylation profile.131 Moreover, as DNMT1- tion, which are properties of the IGs suggesting that its imprinting deficient HSCs harbored an increase of the myeloid compartment, status is not deregulated in myeloid malignancies.120 it has been suggested that myeloid regulators are preferentially Sequence data analysis of a large sample from The Cancer methylated in HSCs. DNA methylation helps regulate age- Genome Atlas reveals blood-specific recurrent mutations of GNAS dependent functional changes observed with aging where HSCs in AML. It is assumed that these mutations may confer advantages lose their functionality.132 Nevertheless, modulation of the IGF2- to affected HSPCs, resulting in enhanced cell renewal and/or H19 cluster might inhibit HSCs aging process preventing clonal expansion. The analysis shows that GNAS is also considered leukemogenesis.28 Hence, IGs and genomic imprinting should as the initiating events for MPN, MDS and/or AML.121 GNAS is also be evaluated in the context of aged HSCs and leukemogenesis, mutated in low percentage in MDS patients.122,123 and probably through an editing mechanism such as CRISPR/Cas9 IRAIN is a new intragenic imprinted long non-coding RNA could model HSCs conversion to a premalignant status. transcribed in an antisense orientation of IGF1R. It exhibits a IGs have been shown to affect cycling of murine hepatic stellate bimodal pattern of expression as it is downregulated in high-risk cells through a Wnt-b-catenin pathway.133 Recently, it was shown AML patients, whereas it is highly expressed in low-risk patients, in that b-catenin mutations in osteoblasts promote the clonal a pattern similar to Igf2r-Airn in mice. IRAIN interacts directly with expansion of HSCs and are associated with somatic mutations in IGF1R enhancer and promoter and is also involved in the myeloid progenitors leading to the appearance of AML.134 Hence, formation of an intrachromosomal loop between the IGF1R a deregulated pathway comprising IGs-Wnt-b-catenin in HSPCs promoter and enhancer.124 worths to be explored. The human protein-coding neuronatin gene NNAT located on The cancer cell of origin is considered as the normal cell chromosome 20q11.2-q12 is paternally imprinted. This particular because of genetic events that has the potential to initiate the

Leukemia (2015) 1233 – 1242 © 2015 Macmillan Publishers Limited Imprinted genes in hematopoiesis L Benetatos and G Vartholomatos 1239 genomic imprinting in a manner similar to primordial germ cells they are quiescent.35 After their deposition in adult tissues during development, they are prone to stimuli that can lead to a proliferative state where genomic imprinting is recovered.14 That change in genomic imprinting early at this stage might promote cell reprogramming and therefore could affect the proliferation and/or differentiation profile of the cells. Furthermore, if cell reprogramming occurs it could be attributed to the aberrant DNMT3A expression observed in VSELs, which via hypermethylation of DMRs promotes the abnormal expression of genes that enhance proliferation such as IGF2.14 The resulting abnormal methylation profile might prevent the normal transition of VSELs to HSCs and further differentiation. Therefore, VSELs harboring abnormal genomic imprinting might represent the preleukemic clone in AML (Figure 4). However, considering that VSELs are not yet universally accepted, further research is required in order to verify such assumption. As already shown LOI and cancer have been causally linked,30 and as genomic imprinting is one of the first genetic events that occur in stem cells soon after fertilization, we can hypothesize that abnormal genomic imprinting might represent such prime transforming event reprogramming HSCs and subsequently leading to cascade of events. So, what if aberrant genomic imprinting precedes founding mutations in preleukemic HSCs.

CONFLICT OF INTEREST The authors declare no conﬂict of interest.

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