Advances in Epigenetics and Epigenomics in Chronic Lymphocytic Leukemia

The development and progression of chronic lymphocytic leukemia (CLL), a highly heterogenous B cell malignancy, are influenced by both genetic and environmental factors. Environmental factors, including pharmacological interventions, can affect the epigenetic landscape of CLL and thereby determine the CLL phenotype, clonal evolution, and clinical outcome. In this review, we critically present the latest advances in the field of CLL epigenomics/epigenetics in order to provide a systematic overview of to-date achievements and highlight the potential of epigenomics approaches in light of novel treatment therapies. Recent technological advances have enabled broad and precise mapping of the CLL epigenome. The identification of CLL-specific DNA methylation patterns has allowed for accurate CLL subtype definition, a better understanding of clonal origin and evolution, and the discovery of reliable biomarkers. More recently, studies have started to unravel the prognostic, predictive, and therapeutic potential of mapping chromatin dynamics and histone modifications in CLL. Finally, analysis of non-coding RNA expression has indicated their contribution to disease pathogenesis and helped to define prognostic subsets in CLL. Overall, the potential of CLL epigenomics for predicting treatment response and resistance is mounting, especially with the advent of novel targeted CLL therapies.


Introduction
Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world, is viewed as a disease with immense heterogeneity at the clinical, cellular, and molecular levels.A multitude of studies have provided insight on how CLL clinical heterogeneity can be reflected on epigenetic signatures at the DNA methylation, histone modifications, and non-coding RNAs levels.Epigenetic patterns can alter as a response to changes in the (micro)environment, including antigenic stimulus, cross-talk with other cells, exposure to soluble factors at CLL niche, or as a result of pharmacological interventions.Investigating epigenetics and epigenomics can offer not only valuable insights into cell ontogeny and disease pathogenesis but into underlying mechanisms for clinical and molecular heterogeneity.Furthermore, identification of epigenetic signatures via epigenome-wide approaches can assist the shift towards a precision-medicine model for disease management [1,2].Previous research summaries have documented the key scientific approaches around the CLL epigenome [3,4].In this review, we highlight recent advances in CLL epigenetics and epigenomics with a focus on translational potential of key genes/regions, as biomarkers or drug targets (Fig. 1).

DNA Methylation Patterns Predict Cell of Origin, Clonal Evolution, and Clinical Outcome
Examining the normal DNA methylation patterns and understanding how this stable epigenetic mark works during development and ageing processes can provide valuable knowledge in terms of DNA methylation dynamics and their involvement at tumorgenesis.DNA methylation tends to increase with age at some CpG islands, particularly at Polycomb target genes and at promoters of tumor suppressor genes (TSG), while loss of DNA methylation occurs primarily in constitutiveheterochromatin repeat regions.There is a great deal of evidence that the DNA methylation seen in cancer and aging may stem from a small population of cells.It is therefore likely that the general de novo methylation seen in cancer already exists in a subpopulation of "normal" cells prior to their transformation by gene mutation [6,7].In line with this, it was shown that a higher degree of DNA methylation changes are more commonly found in IGHV-unmutated progressive cases of CLL [8,9].Mainly these changes affect PRC2 target genes and possibly involve developmental processes shared in common with the generation of normal memory B cells [9].
Although DNA methylation has been studied in CLL for almost two decades, only recently epigenetic studies have illuminated aspects of CLL ontogeny and clonal evolution [8].First, when Kulis et al. proposed a classification of CLL cases into 3 groups, based on their DNA methylation profiles.Groups were reported as naive B cell-like, memory B cell-like, and intermediate CLL (n-, m-, and i-CLL, respectively), and DNA methylation patterns were associated with disease outcome and putative normal counterparts [10].Subsequently, Martín-Subero's team expanded this work with quantitative DNA methylation assays in two independent CLL cohorts [8].An epigenetic signature of five CpGs (Table 1) located in promoter, gene body, and intergenic regions was identified as a robust biomarker for CLL classification in three distinct subgroups [10,32].
Intraclonal heterogeneity and clonal evolution was the major focus of Oakes and colleagues, in which DNA methylation was assessed by 450 K arrays and next-generation sequencing [33].It was shown that only a fraction of CLL cases with unmutated IGHV genes (U-CLL) and other poor prognostic markers are characterized by heterogeneity and clonal evolution at DNA methylation level and correlated with high subclonal genetic complexity.Their work suggests that the selection pressure at epigenetic level for high-risk CLL cases may be linked to the wide variation of driver mutations [33].
Further emphasis on CLL intraclonal heterogeneity and contribution of the DNA methylome to clonal evolution has been placed by a whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS) analysis on a large cohort of primary samples.Landau et al. showed that intraclonal diversification arises from stochastically disordered DNA methylation and was linked to lowlevel expression and poor clinical outcome, suggesting a role for DNA methylation in promoting genetic instability and the survival of the adverse clone [34].
The importance of investigating DNA methylation status in specific genomic regions or single CpG sites has been underscored by a significant number of studies showing the immense potential for reliable biomarkers which could be employed for patient stratification.By quantitative methylation analysis (MassARRAY), Claus and colleagues identified a single non-promoter CpG (CpG +223) as critical for ZAP70 expression which can serve as a reliable prognostic biomarker for OS and TFS [35].Importantly, ZAP70_CpG+233 methylation was a superior biomarker as opposed to IGHV mutation status, CD38, or ZAP70 expression levels (Table 1) [35].
In a subsequent study, Oakes et al. identified an epigenetic signature (methylation maturation score-MMS) of 18 loci, which confirmed the existence of three distinct CLL subtypes.The three subtypes-termed high-, intermediate-, lowprogrammed CLL (HP-, IP-, LP-CLL)-were suggested to have arisen from the malignant transformation of normal counterparts with diverse maturation levels, and thereby different marks per se, prior to clonal evolution [11•].For instance, the LP-CLL subtype was associated with poor disease outcome, less maturity, and a germinal-center independent clonal history.Furthermore, they reported CLL-specific DNA methylation patterns linked to (i) hypermethylation as a result of blockage in hypomethylation and highly enriched for EBF1, BATF/AP-1, RUNX3 binding sites (B celldevelopment TFs) and (ii) hypomethylation in regions associated with B cell-activation TFs, such as NFAT and EGR [11•].
By using MassARRAY for the 18 loci epigenetic signature, D'Avola and colleagues investigated associations between the MMS and sIgM expression/function in 57 cases [12].They reported an inverse correlation between sIgM levels and MMS, and showed that the reduced sIgM and the high MMS within mutated IGHV CLL cases indicated an indolent disease subtype [12].
A later study employed pyrosequencing for the 5 CpGepigenetic signature discussed above in 135 CLL cases in order to validate strong prognostic significance by classifying patients into novel CLL epigenetic subgroups.Furthermore, stereotyped subset #2 (IGHV3-21/IGLV3-21 expressing cases) were included in this study and they were classified by methylation score as i-CLL subgroups, whereas their clinical classification resembled the n-CLL group [13].
Georgiadis and colleagues provided evidence that CLLlike changes at DNA methylation and transcriptome profiles existed more than 10 years before CLL diagnosis [14].These findings suggest that the path from normal counterpart to malignant transformation and further clonal evolution is a long, multi-step process, in which DNA methylation can be viewed not only as a contributor to disease pathogenesis but also as a tool for understanding pre-malignant evolutionary trajectory.
Recent work by Tomasz K. Wojdacz and colleagues has further strengthened our knowledge on the clinical significance of DNA methylation in CLL [15].High-throughput analysis on UK clinical trials' CLL samples identified DNA methylation signatures which can be used to divide IGHVmutated CLL into clinically relevant subgroups.Last but not least, investigators reported that the memory-like DNA methylation subgroup (m-CLL) can work as an independent marker of prolonged survival for patients treated with chemoimmunotherapy [15].
Overall, independent groups have discovered DNA methylation signatures which have been confirmed in follow-up studies and have significantly enhanced our understanding of CLL heterogeneity, clonal history, and evolution.

DNA Methylation and Transcriptional Regulation in CLL
Recent studies have advanced our understanding of the interplay between hypomethylation and hypermethylation in CLL, and highlighted their role in altering gene regulation to support malignant transformation and/or disease progression.In some cases, DNA methylation-mediated altered gene expression seems to be linked directly to key CLL pathways, whereas in other cases, DNA methylation patterns were identified as potential biomarkers without clear pathogenetic implications (Table 1).
In a metanalysis of publically deposited DNA methylation data, Kushwaha et al. reported regions that were consistently differentially methylated (hypo-or hyper-C-DMRs) in CLL [16].Hypo-C-DMRs were found enriched in regions harboring TF binding sites related to key signaling pathways (BCR, p53), whereas hyper-C-DMRs were linked to homeobox-and TATA box-containing regions [16].
In another study, it was shown that although the core IL10 promoter was largely unmethylated in CLL cases, two gene body regions (VMRs) exhibited variable methylation in association with IGHV mutation status.IL10 transcript levels were inversely correlated with DNA methylation of VMRs, but not the core promoter.VMR2 was located within transcriptional activation-associated H3K27ac peak regions, and harbored a functional STAT3 motif.This work underscores the importance of DNA methylation for gene regulation in regions outside the promoters [17].Hypomethylation in a putative enhancer for PD-1 harboring NFAT and STAT motifs was reported to increase PD-1 expression in CLL-derived CD8+ T cells.These T cells also exhibited disease-specific DNA methylation patterns, and top differentially methylated regions were included in CCR6 and KLRG [18].
In the list of genes (Table 1) regulated by DNA methylation, one should add ET-1, MCPH1, CRY1, and PAX9.DNA methylation levels of an ET-1 intron inversely correlate with expression [19].The MCPH1 was shown to have a tumorsuppressive function in M-CLL cases via binding to ANGPT2 promoter, recruitment of DNMTs, and subsequent silencing of ANGPT2 [20].In an array-based study, CRY1 and PAX9 expression was significantly higher in U-CLL, whereas a single CpG island of PAX9 was linked to IGHV mutation status.Although validation in external cohorts with quantitative approaches is required, these data suggest that DNA methylation is important for CRY1 and PAX9 gene regulation, and may have a prognostic value in CLL [21].
In a different setting, Hanney and colleagues worked with a Dnmt3aΔ/Δ model.They comparatively assessed global DNA methylation and gene expression in induced CLL and peripheral T cell lymphoma (PTCL) tumors, including B-1a and CD8+ T cells as controls.Interestingly, their analysis revealed a significant overlap between human and mouse transcriptomes and DNA methylomes [22].
Filarsky et al. reported aberrant DNA methylation for KLF4 and LILRA4 and provided evidence for a tumorsuppressive function for KLF4, which can be exerted upon NOTCH signaling inhibition [23].Another link between Notch signaling and aberrant DNA methylation is suggested by Arruga and colleagues, who reported promoter hypermethylation of DUSP22 in NOTCH1-mutated CLL [24].Exploiting the CRISPR/Cas9 system in a CLL cell line, they showed that methylation of DUSP22 is regulated by NICD through a circuit including DNMT3a, RBPJk, and HDAC1 [24].Interesting work on a cohort of 112 patients revealed that the two promoters of LPL show heterogeneous DNA methylation levels across CLL cases correlated to TFS [25].Wolf and colleagues showed that aberrant NFATC1 promoter hypomethylation was inversely correlated with expression levels, and that inhibition of NFAT activity increased apoptotic cell rate [26].
Finally, in a recent study by Papakonstantinou and colleagues, TP63 was reported to be hypomethylated and overexpressed in stereotyped subset #8 CLL cases [27].By array-based genome-wide DNA methylation profiling in poor prognostic CLL cases, the researchers showed that stereotyped subset #8 exhibits distinct DNA methylation patterns as opposed to non-subset IGHV-unmutated CLL cases and subset #6 cases [27].
Overall, the list of genes that are differentially methylated at their promoter or non-promoter regulatory regions is constantly expanding.However, observations require further investigation with state-of-the art technologies, validation in independent cohorts, or proof-of-concept experimental evidence to establish a clear mechanistic view of each gene's role in CLL molecular pathogenesis (Table 2).

DNA Hydroxymethylation in CLL
DNA hydroxymethylation was recently discovered as another important mark with active role in epigenetic reprogramming.It is the first product from TET-related oxidation in the process of active demethylation as described in different biological processes including primordial germ cells and zygotic development [28,29].Interestingly, several studies have associated 5-hmC with transcriptional activation mediated by enhancers, both in embryonic and differentiated cells [28,30].Like DNA methylation, hydroxymethylation has also been observed to be deregulated in various human pathologies such as degenerative diseases like Parkinson's, Alhzeimer's, and different cancer types [31,36].In the first thorough study exploring 5 hydroxymethylcytosine (5-hmC) in CLL pathogenesis, Wernig-Zorc et al. reported global loss of 5-hmC and identified aberrant 5-hmC and 5-mC patterns in gene body regions [37].Interestingly, they reported TET enzyme-mediated 5-hmC enrichment in regulating the differential expression of three oncogenes: NSMCE1, TUBGCP3, and TUBGCP6 [37].

Chromatin Dynamics and Histone Modifications
Advances in technologies for epigenome-wide screening provide insights and competent knowledge to the research community related to the complex mechanisms of chromatin configuration and dynamic modification of histones.A small number of studies in that direction-which will now be discussed-provide the first chromatin accessibility maps, report links to transcription factor networks and affected gene expression profiles, and above all, underscore the prognostic, predictive, and therapeutic potential value of chromatin dynamics and histone marks in CLL.

Profiling the Regulatory Chromatin Landscape in CLL
The seed for the emerging interest on chromatin in CLL has been shown by a study reporting a role for CHD2, an important chromatin remodeler in CLL pathogenesis.CHD2 was recurrently mutated in M-CLL and integrative analysis on a genomic, transcriptomic, and epigenomic level suggested that CDH2 mutation affects its DNA-binding properties and localization, leading to altered chromatin states [38].In another early study, the identification of an SNP (rs539846) located within a super-enhancer (H3K27ac marks) of BCL2-modifying factor (BMF) linked it with disruption of a RELA binding site and decreased BMF expression [39].
The first effort towards large-scale analysis and mapping of chromatin accessibility in CLL was reported by Rendeiro et al., who profiled 88 CLL cases by ATAC-seq [40•].Interestingly, ATAC-seq profiles were distinct between U-CLL and M-CLL, whereas one or two intermediate subtypes were revealed, based on chromatin accessibility patterns.Furthermore, they performed an RNA-seq/ChIPmentation integrative analysis, including H3K4me1, H3K27ac, and H3K27me3 histone marks and developed a bioinformatics pipeline for linking integrated profiles to clinical and molecular sample annotations.Their analysis revealed a pan-CLL gene regulatory network which included SP1/2/3, CTCF, EGR, E2F, and PAX5 factors, as well as subtype-specific networks [40•].
A recent pioneer analysis of the reference CLL epigenome, together with extensive analysis of regulatory chromatin landscape, sheds light on important knowledge gaps in CLL epigenomics.Interestingly, it showed that chromatin dynamics mirrored normal B cell maturation and regulatory elements were de novo reprogrammed in CLL or associated with CLL subtypes.In general, U-CLL harbored more active and open chromatin than M-CLL.Among de novo-active regions in CLL, an enrichment for NFAT, FOX, and TCF/LEF binding sites was reported, highlighting their functional and therapeutic potential.Finally, a link between genetic alterations and chromatin configuration was observed for MYD88 mutations and trisomy 12 [41].
Another recent important study delved further into the CLL epigenome and provided novel insights for CLL enhancer landscape and transcription factor dependencies.Ott and colleagues reported CLL-specific enhancer-related core regulatory circuits (CRCs) [42•].Super enhancers were found to mediate CRCs, including "usual suspects"such as CXCR4, CD74, PAX5, CD5, KRAS, and BCL2.Further functional work showed that PAX5 is a core regulator of CLL super enhancers, which regulates cell survival, whereas BET inhibitors block the super enhancer CRCs and promote apoptosis in vitro and in vivo [42•].
A significant advance in our knowledge of CLL epigenomics has arisen from a comprehensive study which dissected the aberrant epigenetic circuitry in CLL [43•].Mallm and colleagues reported that CLL-specific chromatin features included (i) localized changes at regulatory enhancer and promoter elements and (ii) large-scale chromatin reorganization (> 1 Mb).In analogy to other cancers, partially methylated domains (PMDs) linked to repressive chromatin and gene silencing was a CLL trait demarcated by CTCF.Authors further reported a CLL-specific TF network based on aberrant chromatin features, which included key epigenetic molecules (i.e., SIN3, NuRD, SWI/SNF).H3K4me3 marks were found to be redistributed in CLL alongside nucleosome  gain at promoters and extensive changes of enhancer activity.Finally, a CLL-specific network centered around TFs targeting 17 central binding motifs was presented, describing gaining for NFAT, TCF4, and LEF1 motifs, and loss for EBF1 and AP-1 motifs in CLL.
A deeper understanding of the relationship between intraleukemic epigenetic and transcriptional diversity has been achieved via recent integrative analysis of CLL's epigenetic landscape.Pastore and colleagues reported corrupted coherence across different layers of the CLL genome and epigenome.An extensive chromatin rewiring at H3K27ac marks, mediated in particular by NFAT and TCF/LEF, was observed.These H3K27ac peaks showed hypomethylation at super enhancers regions, resulting preferentially in intermediate DNAme levels.H3K27me3-marked genes were incompletely silenced through the formation of PRC2 complex, reflecting to a set of dynamic changes on chromatin states tested.Interestingly, mutually exclusive (H3K27ac/ H3K27me3) histone marks were co-mapped.This work suggests intraleukemic diversity is linked to stochastic cell activation of alternate gene programs, and leads to an admixture of cells with diverging identities [44].
All in all, the last few years' high-impact research studies have provided valuable resources related to the CLL chromatin landscape, including the until recently understudied histone modifications.These studies have significantly strengthened our knowledge of links between CLL phenotype, clinical outcome, and aberrant features of the CLL epigenome beyond the DNA methylome.

Histone-Modifying Enzymes: Targets for Therapy
Since the dysfunction of histone-modifying enzymes (HME) has been linked to human cancer initiation and progression, the CLL research community has placed significant emphasis on investigating HME.While initial studies have focused on canonical HME functions (catalyzing histone modifications), a few recent studies have explored the nonenzymatic roles of HME in transcriptional regulation [45].
An early study reported that the global levels of histone deacetylase (HDAC) can be an independent prognostic marker for treatment-free (TFS) and overall survival (OS) [46].Later, Zhou and colleagues reported global histone H3/H4 hypoacetylation, H3K9 hypermethylation, and overexpression of SIRT1 and EZH2 in CLL patients [47], whereas Parker et al. discovered recurrent deletions and mutations (7 %) of the histone methyltransferase SETD2 [48].
In another study, the association between NOTCH1 mutations and low CD20 expression was suggested to be HDACmediated.Interestingly, HDAC inhibition by Valproic acid was shown to upregulate both MS4A1 and CD20 [49].Later work tested the in vivo efficancy of valproate in del13q/ NOTCH1wt CLL patients.Although in vivo treatment ↑: upregulation of gene expression /enhanced/increased levels of [..]; ↓: downregulation of gene expression levels/enhanced/increased levels of each condition described; BTKi, Bruton's tyrosine kinase inhibitor: CD20, B-lymphocyte antigen CD20; CLL, chronic lymphocytic leukemia; CRC, core regulatory circuit; Decitabine, 5-aza-2′-deoxycytidine; Epi-silenced, epigenetically silenced; EZH2, enhancer of zeste homolog 2; GSI, γ-Secretase inhibitor; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor or inhibition; H3K27me3, histone H3 trimethylation at lysine 27; H3K4me3, histone H3 trimethylation at lysine resulted in global H3K9ac, it did not induce CD20 expression.In contrast, in vitro treatment of E95 CLL cell line resulted in CD20 upregulation.This finding was attributed to simultaneous induction of H3K9ac and EZH2-mediated H3K27me3 by valproate at the CD20 promoter in vivo, highlighting the need for combinatorial therapies [50].Independent studies showed that expression of HDAC6 is upregulated in patient samples, cell lines, and euTCL1 transgenic mice.These findings, as well as functional in vivo HDAC6 inhibition, underlined the potential therapeutic value of HDAC6 inhibition for CLL treatment.Furthermore, it was shown that coadministration of an HDAC6 inhibitor (ACY738) with BTK inhibitor Ibrutinib displayed synergistic cell death and improved overall compared with either single agent in vivo [51].
EZH2 appears to be an HME of great interest.An early study showed that EZH2 is overexpressed in poor prognostic CLL cases and correlated with H3K27me3, whereas EZH2 inhibition in vitro promoted apoptosis [52].Recently, inhibition of EZH2 in combination with PI3K inhibition (Idelalisib) has been reported to effectively minimize both canonical and non-canonical EZH2 functions [53].Furthermore, the oncogenic potential of EZH2 via its antiapoptotic effect was shown to be regulated by microenvironmental signals [54].
Finally, a recent study by Filiu-Braga et al. explored the role of other HMEs, namely the KDM4 histone demethylase family.Interestingly authors linked KDM4B and KDM4C expression to poor prognostic CLL patient subgroups [55].
Although all aforementioned studies have indicated an important role of HME in CLL, there is a therapeutic potential in light of combinatorial therapies (i.e., BCR signaling inhibitors and HME inhibitors) which is not yet systematically explored.Given the significant number and diversity of HME, together with canonical and recently emerging non-canonical functions [45], it is now evident that in-depth understanding of HMEspecific functions is perquisite for designing a successful epigenetically relevant combinatorial therapy.

BCR Targeted Therapies and the Epigenome
B cell receptor signaling pathway inhibitors (BCRi) have shifted the treatment paradigm in CLL over the last 5 years [56].Dissection of the epigenomic and transcriptomic landscape of CLL in parallel with the advent of new therapies targeting key biological pathways has led to an opportunity for a more pharmaco-epigenomics based approach in CLL research, which has recently emerged as a field with great potential for understanding treatment resistance.
To that end, a pioneering study by Schmidl et al. shed light on the epigenetic remodeling profiles in association with BTK inhibition.The team employed a systematic and innovative approach from matched CLL patients pre-and post-Ibrutinib, to identify treatment-specific vulnerabilities for drug combinations to overcome Ibrutinib resistance.They scanned the epigenetic cell state and regulatory profiles by ATAC-seq and combined the data with single-cell chemosensivity analysis, which included 131 drugs.Interestingly, Ibrutinib induced a significant loss (92%) of accessible regions, while fewer cases (8%) gained chromatin accessibility upon treatment.At a pathway level, downregulation of NF-κΒ and BCR signaling as well as specific enrichment for proteasome regulation and autophagy was reported.The study reported an ibrutinib-induced gain of CLL cell selectivity for proteasome inhibitors, PLK1 inhibitors, and mTOR inhibitors, and sets the ground for novel combinatorial therapies [57•].
Another recent work by Holmes et al. elucidates the ibrutinib-induced changes in chromatin configuration of CLL.Authors report a remarkable loss of both H3K27ac and H3K27me3 marks together with EZH2 expression after 14 days treatment [58].The IR-induced lymphocytosis tended to be quicker in treatment-naïve (TN) and delayed in relapsedrefractory (RR) patients.Interestingly, this delay was linked to H3K4me3 maintenance at PRC2 targets only in RR patients arguing for a mechanism of partial reprogramming that takes place during the process of relapse.Overall, these findings suggest a mechanism that CLL cells deploy to adaptively respond to Ibrutinib by histone hypomethylation and therefore passage from a proliferative state into quiescence [58].

Non-coding RNAS
The emerging role of non-coding RNAs (ncRNAs) in gene regulation and pathogenesis of numerous malignancies has been underscored by previous key reviews [59][60][61].Studies exploring the molecular background and mechanisms mediated either through microRNAs (miRs) or long, non-coding RNAs (lncRNAs) showed that these molecules possess significant regulatory roles and influence to a critical point the epigenetic portrait of CLL.From a clinical standing, the clinical significance of microRNAs (miRs) in CLL has been well documented [62,63], whereas more recent studies shed light on other types of non-coding RNAs.
In 2016, Bottoni et al. identified a set of miRNAs which directly downregulate BTK expression in CLL [64].In a group of 83 patients, the HDAC complex was recruited to miR promoters to silence their expression.HDAC inhibition increased miR promoter H3K4me3 and decreased BTK protein levels.This work suggests that HDACi therapies may target Ibrutinib resistant CLL via miR-based epigenetic silencing of BTKassociated miRNAs [64].
The miR-34b/c (part of the TP53 network) has been shown to be epigenetically silenced in cancer.In CLL, miR-34b/c shared promoter (with BTG4) is aberrantly hypermethylated in almost half of CLL cases.Promoter DNA methylation levels inversely correlated with expression and transfection of microRNA-34b/c genes into HG3 CLL cells increased apoptosis underlying their tumor-suppressive role, which is epigenetically silenced in CLL [65].
Based on previous findings for aberrant promoter DNA methylation of miR-708 in CLL, Baer C et al. explored the effects and pathway of downstream targets related to its tumor-suppressor function [66].Remarkably, they found that miR-708 strongly interacted and inhibited NF-κΒ signaling, and therefore expression of target genes through endogenous repression of IKKβ.Overall, this tumor-suppressive function was found to be reversed in CLL and mostly in U-CLL subset as hypermethylation of a distinct enhancer located downstream of miR-708 promoter drove the CLL cell to an epigenetic-mediated silenced state.Hence, this effect led to lower miR-708 expression, and was associated with highrisk characteristics in CLL patients [66].
Another example for miR transcriptional inhibition is miR-3151.A tumor-suppressor role has been proposed for this miR.In CLL specifically, miR-3151 promoter was mostly hypermethylated and demethylation treatment resulted in miR-3151 expression, subsequent tumor suppression and enhanced apoptosis via downregulation of a MEK/ERK PI3K/AKT signaling [67].
In another study, miR-26A1 was epigenetically repressed in CLL via promoter hypermethylation.Higher miR-26A1 methylation was linked to poor prognosis; therefore, a tumorsuppressive role was suggested for miR-26A1.miR-26A1 expression inversely correlated with the oncogenic driver EZH2 in vitro, further supporting a tumor-suppressive function of this miR [68].Also, a later study reports an inverse correlation between EZH2 and miR-26A1 expression in CLL lymph node samples [69].
A large-scale study by Rochetti and colleagues investigated lncRNA expression in a cohort of 217 CLL patients and reported 24 lncRNAs as being deregulated in CLL [70].Concurrently, a significant percentage of CLL-specific differentially methylated genes were mapped to lncRNAs.Two n o v e l l n c R N A s ( h y p e r m e t h y l a t e d C R N D E a n d hypomethylated AC012065.7)were further validated and correlated with an inferior outcome [71].Finally, another study suggested a tumor-suppressive role for lncRNA BM742401 in CLL via enhanced apoptosis.Interestingly, BM742401 expression was regulated by promoter DNA methylation [72].

Conclusion
CLL epigenomics has witnessed a rapid growth, very much linked to recent technological advances.The transition from array-based methods to whole-genome sequencing and single-cell approaches has been followed by an exponential growth of high-throughput data generation and high-impact publication in the field.This growth has had a large impact on the understanding of epigenetic mechanisms and shed plenty of light onto CLL ontogeny and subset characterization, including novel prognostic considerations.
From the global DNA methylome studies to single CpG biomarker discoveries, and from histone modification mapping to non-coding RNA contribution to leukemogenesis, epigenetics has emerged beyond doubt as a key element of current CLL translational research.
In the near future, CLL epigenomics could involve more single-cell approaches, as those may help us to better tackle intraclonal epigenomic diversification and understand emerging treatment resistance.The dissection of the epigenome of non-leukemic cells is yet another important direction, along with the characterization of the epigenomes of the various cellular components of the CLL niche.The impact of epigenetic treatments on non-CLL cells and how these affect the cross-talk between CLL cells and the microenvironment is a question that also remains to be systematically explored by epigenomic analyses.

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Fig. 1
Fig.1Overview of CLL epigenetics.Microenvironmental signals shape to a critical point CLL B cell fate and B cell receptor (BCR) possess a central role into this translational process.A significant number of proteins have been reported to be regulated by DNA methylation in CLL.For instance, ZAP70, TP63, NFATc1, and others (dark grey boxes) have been found to be upregulated by DNA hypomethylation (promoter/gene body/cis-regulation) either in CLL as a whole or only in specific CLL subsets with diverse prognosis.Other molecules such as KLF4, DUSP22, and various miRs were downregulated and epigenetically silenced via DNA promoter hypermethylation or gene body/cis-regulatory element DNA methylation changes (light yellow boxes).Furthermore, molecules expressed either in the cytoplasm or in the nucleus such as tumor suppressive microRNAs (i.e., miR-708), long non-coding RNAs (i.e., CRNDE) and histone-modifying enzymes (HME) (i.e., EZH2) have been found to be epigenetically dysregulated in CLL.These molecules act as epigenetic regulators and are either downregulated or aberrantly overexpressed affecting the downstream signaling cascade, the epigenome and transcriptome.Interestingly, aberrant expression and function of various factors have been found to be actively mediated by HDACs (black boxes) or EZH2 enzymes (light grey boxes).Through a dynamic process, HMEs (HDAC, EZH2, SETD2, CHD2 and KDM4) can alter chromatin configuration and TF dependencies for regulatory areas (i.e., promoters, enhancers).These epigenetic changes are the catalytic switch for enabling or blocking gene transcription and not surprisingly, HME inhibition has shown to be the most promising strategy of epigenetic therapy in CLL.The figure was created with BioRender.com.AP-1, activator protein 1; BTK, Bruton's tyrosine kinase; BCR, B cell receptor; CHD2,

Table 2
An overview of treatment-related epigenetic studies in CLL