Review published: 16 October 2019 doi: 10.3389/fgene.2019.00986 Recent Advances in the Targeting of Epigenetic Regulators in B-Cell Non- Hodgkin Lymphoma Marcelo L. Ribeiro 1,2*, Diana Reyes-Garau 1, Marc Armengol 1, Miranda Fernández-Serrano 1 and Gaël Roué 1* 1Laboratory of Experimental Hematology, Department of Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, Autonomous University of Barcelona, Barcelona, Spain, 2 Laboratory of Immunopharmacology and Molecular Biology, Sao Francisco University Medical School, Braganca Paulista, São Paulo, Brazil In the last 10 years, major advances have been made in the diagnosis and development of selective therapies for several blood cancers, including B-cell non-Hodgkin lymphoma (B-NHL), a heterogeneous group of malignancies arising from the mature B lymphocyte compartment. However, most of these entities remain incurable and current treatments Edited by: are associated with variable efficacy, several adverse events, and frequent relapses. Jiucun Wang, Thus, new diagnostic paradigms and novel therapeutic options are required to improve Fudan University, China the prognosis of patients with B-NHL. With the recent deciphering of the mutational Reviewed by: Naoko Hattori, landscapes of B-cell disorders by high-throughput sequencing, it came out that different National Cancer Center Research epigenetic deregulations might drive and/or promote B lymphomagenesis. Consistently, Institute (Japan), over the last decade, numerous epigenetic drugs (or epidrugs) have emerged in the Japan Maurizio D'Esposito, clinical management of B-NHL patients. In this review, we will present an overview of the Italian National Research Council most relevant epidrugs tested and/or used so far for the treatment of different subtypes (CNR), Italy of B-NHL, from first-generation epigenetic therapies like histone acetyl transferases *Correspondence: Marcelo L. Ribeiro (HDACs) or DNA-methyl transferases (DNMTs) inhibitors to new agents showing selectivity mlribeiro@vhio.net for proteins that are mutated, translocated, and/or overexpressed in these diseases, Gaël Roué including EZH2, BET, and PRMT. We will dissect the mechanisms of action of these groue@vhebron.net epigenetic inhibitors, as well as the molecular processes underlying their lack of efficacy Specialty section: in refractory patients. This review will also provide a summary of the latest strategies being This article was submitted to employed in preclinical and clinical settings, and will point out the most promising lines of Epigenomics and Epigenetics, a section of the journal investigation in the field. Frontiers in Genetics Keywords: B-cell lymphoma, DNMT, EZH2, HDAC, PRMT inhibitor, BET bromodomain inhibitor (BETi), Received: 11 June 2019 combination therapy Accepted: 17 September 2019 Published: 16 October 2019 Citation: INTRODUCTION Ribeiro ML, Reyes-Garau D, Armengol M, Fernández-Serrano M Characteristics of B-Cell Non-Hodgkin Lymphoma (B-NHL) and Roué G (2019) Recent Advances At the origin of 4% of all cancers and more than 90% of the cases of lymphoma, B-NHLs comprise in the Targeting of Epigenetic Regulators in B-Cell Non-Hodgkin a heterogeneous group of lymphoid neoplasms. According to the last World Health Organization Lymphoma. hematopoietic and lymphoid tumor classification, more than 40 distinct entities are categorized, Front. Genet. 10:986. according to a combination of morphological, immunophenotypic, genetic, and clinical doi: 10.3389/fgene.2019.00986 features, having each entity its own clinical course and requiring specific treatments (Table 1) Frontiers in Genetics | www.frontiersin.org 1 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL TABLE 1 | Classification of B-cell non-Hodgkin lymphoma. Name Cell of origin Genetic aberration Involved genes Frequency (%) References B-ALL Hematopoietic stem cell or Hyperdiploidy – 31* (Yeoh et al., 2002) B-cell progenitor t(12;21)(p13.2;q22.1) ETV6/RUNX1 28* t(1;19)(q23;p13.3) TCF3/PBX1 9* t(9;22)(q34.1;q11.2) BCR/ABL1 5* CLL/SLL Naïve (unmutated IGHV del(13q14.3) – 54 (Haferlach et al., 2007) subset) or memory (mutated – SF3B1 21 (Landau et al., 2015) IGHV subset) B cell Trisomy of 12 – 14 del(11q) ATM 12 LPL Post-follicular B cell – MYD88 90 (Hunter et al., 2014) del(6q) – 43 – CXCR4 27 ARID1A 17 NMZL Post-germinal center Gains of 3p – 24 (Rinaldi et al., 2011) marginal zone B cell Gains of 18q – 24 del(6q23) TNFAIP3 16 EMZL-MALT Post-germinal center marginal Trisomy of 3 – 31 (Streubel et al., 2004) zone B cell del(6q23) TNFAIP3 30 (Rinaldi et al., 2011) t(11;18)(q21;q21) BIRC3/MALT1 13 Trisomy of 18 – 11 SMZL Marginal zone B cell with or del(7q) – 26 (Rinaldi et al., 2011) without antigen exposure Gains of 3q – 20 (Parry et al., 2015) del(17p13) or mutation TP53 16 – KLF2 12 HCL Late activated memory B cell – BRAF 100 (Tiacci et al., 2011) – MAP2K1 48 (Waterfall et al., 2013) FL Germinal center B cell t(14;18)(q32;q21) IGH/BCL2 89 (Horsman et al., 1995) – KMT2D 82 (Okosun et al., 2014) – CREBBP 64 – TNFRSF14 35 MCL Peripheral B cell of the inner t(11;14)(q13;q32) IGH/CCND1 57 (Beà et al., 1999) mantle zone Gain of 3q26 – 49 (Beà et al., 2013) del(11q) or mutation ATM 41 del(17p13) or mutation CCND1 34 TP53 27 DLBCL-GCB Peripheral mature B cell of – KMT2D 46 (Dubois et al., 2016) germinal center origin del(1p36) or mutation TNFRSF14 38 (Scholtysik et al., Gains of 2p16 REL 30 2015)(Roulland et al., t(18q21) BCL2 28 2018) DLBCL-ABC Peripheral mature B cell of del(9p21) CDKN2A 47 (Scholtysik et al., germinal center exit or post- del(6q21) or mutation PRDM1 41 2015)(Dubois et al., germinal center – KMT2D 41 2016) – MYD88 28 BL Germinal center B cell t(8;14)(q24;q32) MYC/IGH 77 (Toujani et al., 2009) –Other t(8q24) ID3 58 (Schmitz et al., 2012) TCF3 29 MYC 8–15 *Frequency in pediatric cases. B-ALL, B-cell acute lymphocytic leukemia; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; LPL, lymphoplasmacytic lymphoma; NMZL, nodal marginal zone lymphoma; EMZL-MALT, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue; SMZL, splenic marginal zone lymphoma; HCL, hairy cell leukemia; FL, follicular lymphoma; MCL, mantle cell lymphoma; DLBCL-GCB, diffuse large B-cell lymphoma of germinal center B-cell subtype; DLBCL-ABC, diffuse large B-cell lymphoma of activated B-cell subtype; BL, Burkitt lymphoma. (Campo et al., 2011; Scott and Gascoyne, 2014; Swerdlow these processes, the VDJ heavy chain is formed, followed by et al., 2016). Originated from either mature or immature VJ light-chain gene rearrangement, which allows the pre-B B cells, B-NHLs are characterized by the proliferation of cells to express intracytoplasmic μ-heavy chains. Subsequently, lymphocytes, mainly in lymphoid organs and in extranodal immature immunoglobulin (Ig)-positive B cells are formed. tissues. Their diversity can often be traced to a particular stage Within the lymph node, and in contact with a determined of differentiation, from the bone marrow where the normal antigen, naïve B cells can mature into IgM-secreting plasma precursor B cell is originated to secondary lymphoid tissues cells or may proliferate into primary follicles to form germinal where B cells undergo multiple rounds of selection before their center (GC) centroblasts. Upon maturation, they further differentiation into plasma cells or memory B cells. During differentiate into centrocytes, which give place to memory B Frontiers in Genetics | www.frontiersin.org 2 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL cells or plasma cells. Within the GC, somatic hypermutation in Altered Chromatin-Modifying Enzymes the Ig heavy or light chain variable region (IGHV or IGHL) genes in B-NHL leads to increased antigen affinity. Contrary to the general belief that only accumulations of Although tightly regulated, the B-cell differentiation DNA mutations might lead directly to the development of process and especially the antibody diversification phase tumorigenic processes, it has been progressively reported can be accompanied by inherited events that may favor a growing subset of epigenetic alterations lying at the basis lymphomagenesis, such as chromosomal translocations, of many malignancies, including those occurring in lymph oncogene activation, and/or inactivating mutations in tumor nodes. Interestingly, in B-cell lymphomas, certain somatic suppressor genes. Infection by determined viruses, such as the mutations in chromatin-modifying enzymes account for Epstein–Barr virus, has also been involved in lymphomagenesis. several epigenetic alterations, suggesting that an aberrant The malignant counterparts of the early B-cell differentiation epigenetic landscape in B-NHL may be a consequence of steps account for B lymphoblastic lymphomas, which harbor genetic alterations associated with a particular lymphoma high similarity with B progenitor cells. On the other hand, subtype. For instance, deleterious and/or loss of function mantle cell lymphomas (MCLs) and a subset of chronic mutations in the histone acetyltransferase CREB binding lymphocytic leukemia (CLL) with unmutated IGHV are protein (CREBBP) or the E1A binding protein 300 (EP330) thought to derive from naive B cells and pre-GC mature B have been reported in about 40% of DLBCL and FL patients cells expressing the CD5 surface marker. Other GC-originated as well as in other lymphoma subtypes (Morin et al., 2011; lymphomas, including follicular lymphoma (FL), Burkitt’s Pasqualucci et al., 2011b). Recurrent point mutations in the lymphoma (BL), a subset of diffuse large B-cell lymphoma histone acetyl transferase (HAT) recruiting gene myocyte (DLBCL), and Hodgkin’s lymphoma (HL), present mutations enhancer binding factor 2B (MEF2B) have been also described in IGHV gene. Additional entities, including marginal zone in 15% of FL and 13% of DLBCL patients with germinal center lymphoma (MZL), lymphoplasmacytic lymphoma, CLL with B cell (DLBCL-GCB) subtype (Morin et al., 2011). Although somatic IGHV mutation, another subset of DLBCL, and no mutations have been reported in the genes coding for multiple myeloma (MM) correspond to post-GC cells. Each histone deacetylases (HDACs), several members of this lymphoma subtype retains key features of their cell of origin family like HDAC1, 2, and 6 can be overexpressed in DLBCL, as judged by the similarity of immunophenotype, histological in association with a decrease in the DNA accessibility to the appearance, and gene expression profiles (Seifert et al., 2013) transcription machinery (Marquard et al., 2009). (Table 1). The putative normal B-cell counterpart of each In addition to mutations in chromatin‐regulatory proteins, B-cell lymphoma is summarized in Figure 1. epigenetic modifications at chromatin level are also commonly In the last decade, loads of evidences have suggested an observed in B-NHL as a result of profound changes in DNA association between the frequent alterations in chromatin state methylation patterns. Indeed, while hypo- and hyper-DNA and epigenetic regulators observed in B-NHL patients, and methylation status have been linked to the pathogenesis of disease formation and progression. several cancer subtypes, somatic mutations in epigenetic genes codifying for DNA methylation regulators have been particularly well associated to a repressed chromatin state and to malignant processes in B-NHL (Esteller et al., 2001; Hassler et al., 2013). Among the main reported alterations, activating mutations in enhancer of zeste homolog 2 (EZH2), a histone methyltransferase (HMT) gene, were found in 22% of DLBCL- GCB patients and 7% of FL patients (Morin et al., 2010). Further loss‐of‐function mutations were observed in the histone-Lysine N-Methyltransferase 2D (MLL2/KMT2D) gene in about 90% of FL and 30% of DLBCL patients (Morin et al., 2011; Pasqualucci et al., 2011b; Lohr et al., 2012). Concretely, MLL2 presents a defective SET domain when mutated by either truncation or frameshift mutations, leading to a reduced H3K4 methylation activity (Shilatifard, 2008; Morin et al., 2011; Pasqualucci et al., 2011b; Lohr et al., 2012). Hence, B-NHL occurrence as a result of disruption in FIGURE 1 | Major B-cell non-Hodgkin lymphoma subtypes arise from epigenetic mechanisms has generated a strong rationale to different cell of origin within the lymph node. Mantle cell lymphomas target epigenetic and chromatin regulators for drug discovery (MCL) arise from naive B cells or germinal center (GC) B cells found within attempts. To address these alterations, several Food and Drug the mantle zone. Marginal zone lymphomas initiate from naive B cells or GCB that have entered the marginal zone. GCB are the origin of follicular Administration (FDA)–approved epigenetic-modulating lymphomas (FL), Burkitt lymphoma (BL), and diffuse large B-cell lymphomas agents, whose clinical use has been mainly restrained so far (DLBCL) when still in the germinal center. This last DLBCL appears to also to other hematological malignancies (Popovic et al., 2013), form GCB within the marginal zone or from fully developed memory B cells. are now being made available for their evaluation in B-NHL. Frontiers in Genetics | www.frontiersin.org 3 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL These agents include the HDAC inhibitors romidepsin (FK228, Shaknovich and collaborators demonstrated the relevance of depsipeptide), vorinostat (suberanilohydroxamic acid, SAHA), DNA methylation in defining the molecular DLBCL subtypes panobinostat (LBH589), and belinostat (PXD101); the DNA (Shaknovich et al., 2010). It was further proposed that DNMT1 methyltransferase (DNMT) inhibitors (hypomethylating and DNMT 3B overexpression may play a role in malignant agents, HMAs) azacitidine (5-azacytidine) and decitabine progression of these tumors (Amara et al., 2010) and also in BL (5-aza-2′-deoxycytidine); and the isocitrate dehydrogenase neoplasm (Robaina et al., 2015). In line with this, the disruption (IDH) inhibitors enasidenib (AG-221) and ivosidenib (AG- of DNA methylation pattern is correlated with disease severity 120) (Table 2). and patient survival in DLBCL and FL (De et al., 2013). Considering that the majority of cancers, including B-NHL, harbor an altered DNA methylation pattern, and also taking TARGETING WRITER EPIGENETIC into account the reversibility of this alteration, the idea to ENZYMES modulate the methylation machinery to restore a “normal” DNA methylation state has attracted great attention in cancer DNMT Inhibitors treatment (Azad et al., 2013). The first two DNA methylation DNA methylation is responsible for the control of gene expression epigenetic compounds (DNMTi) ratified by the FDA and the and for maintaining genomic stability during embryogenesis and European Medicines Agency for cancer treatment, azacitidine tissue differentiation (Meissner, 2010). This process is clonally and decitabine (Jones et al., 2016), were initially described as inherited and preserved in daughter cells, and occurs through promising chemotherapeutic agents against myelodysplastic the inclusion of a methyl group at cytosine residues in CpG syndrome (MDS) and acute myeloid leukemia (AML), although dinucleotides (Figure 2). It is carried out by the DNMTs, namely with moderate efficacy and high toxicity (Li et al., 1970; Vogler DNMT1 which primarily mediates maintenance methylation et al., 1976). In further trials, low-dose decitabine and azacitidine during cell division, and DNMT3A and 3B that regulate de novo demonstrated to be effective in these patients, improving both DNA methylation (Belinsky et al., 2003; Hermann et al., 2004). the response and the overall survival (OS), leading to their DNA methylation is thought to have a significant role in the further approval (Table 2 and Figure 3) (Silverman et al., 2002; regulation of lymphoid compartment, as it has been demonstrated Fenaux et al., 2009; Lübbert et al., 2016). In B-NHL patients, that differential recruitment of DNMT1, DNMT3A, and two phase I studies using decitabine have been completed so far, DNMT3B and consequent specific DNA methylation patterns but the response to therapy and the effect on DNA methylation are determined at early stages during lymphopoiesis and B-cell were moderate (Stewart et al., 2009; Blum et al., 2010). Currently, activation (Shaknovich et al., 2011; Lai et al., 2013). azacitidine and decitabine are being evaluated alone or in While on the one hand, DNA methylation is essential for combination in approximately 10 active clinical trials involving cell homeostasis, on the other hand, disturbance in methylation relapsed/refractory R/R B-NHL patients (Table 3). Considering pattern have been widely described in cancer. Changes in CpG the preliminary data of these trials, it seems premature to methylation are indeed commonly associated with malignant conclude that DNMTis can be used as monotherapy in B-NHL. transformation and tumor progression (Berdasco and Esteller, Although the mechanism of action of DNMTi is not well 2010). In addition, accumulating evidences suggest that understood, the activity of decitabine and azacitidine is known aberrant epigenetic regulation, including DNA methylation, to involve their incorporation into the DNA of proliferating cells, exerts an important role in regulating each cancer’s hallmarks followed by irreversible inhibition of DNMT1 enzymatic activity (Flavahan et al., 2017). Illustrating this relationship in B-NHL, and the addressing of this latest to proteasomal degradation TABLE 2 | FDA-approved epigenetic drugs for hematological malignancies. Agent Target Indication Year of approval Current development Azacitidine DNMT MDS 2004 CMML AML Decitabine DNMT MDS 2006 Atherosclerosis AML Vorinostat HDAC CTCL 2006 Romidepsin HDAC CTCL 2009 HIVAutism PTCL 2011 Belinostat HDAC PTCL 2014 Ovarian cancer CTCL Panobinostat HDAC MM 2015 CML MDS Breast cancer Prostate cancer DNMT, DNA methyltransferase; HDAC, histone deacetylase; MDS, myelodysplastic syndrome; CMML, chronic myelomonocytic leukemia; AML, acute myeloid leukemia; CTCL, cutaneous T-cell lymphoma; PTCL, peripheral T-cell lymphoma; MM, multiple myeloma. Frontiers in Genetics | www.frontiersin.org 4 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL FIGURE 2 | Mechanisms of action of common epigenetic enzymes. Histone methylation is regulated by histone methyltransferases (HMTs), such as the EZH2 subunit of polycomb repressive complex 2 (PRC2), and histone demethylases (HDMTs). DNA methylation is established by DNA methyltransferases (DNMTs) and reversed by several enzymes like TET hydroxymethylases. Demethylation of both histones and DNA is inhibited by 2-hydroxyglutarate (2-HG), produced from α-ketoglutarate (α-KG) by mutant forms of IDH1/2 enzymes (mIDH). Histone acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Bromodomain-containing proteins, such as CREB-binding protein (CBP), BET, or the BRD9 subunit of the SWI/SNF complex, bind to acetylated residues of histones. and in vivo (Li et al., 2002; Clozel et al., 2013), although in other studies a less drastic and transient effect was observed (Karpf, 2004; McGarvey et al., 2006; Egger et al., 2007). Furthermore, DNMT inhibition is also linked to the demethylation of gene bodies, leading to oncogene downregulation (Wong et al., 2013; Yang et al., 2014). Several new DNMTis have been developed in the last decade with potential activity in hematological malignancies. Among them, thioguanine (2-amino-1,7-dihydro-6H-purine-6-thione (6-tG)) has been approved by FDA to treat AML patients (Munshi et al., 2014). Its mechanism of action involves its incorporation into DNA, decrease in DNMT activity and DNA methylation, blockade of DNA and RNA synthesis, and ultimately cell death (Hogarth et al., 2008; Yuan et al., 2011; Flesner et al., 2014). Recently described as an experimental DNMTi, 5-fluoro-2′- FIGURE 3 | Timeline of FDA approvals of epigenetic-modulating therapies in hematological cancers. Source: https://www.accessdata.fda.gov/scripts/cder/daf/ deoxycytidine (FdCyd) is currently undergoing a phase I/II clinical trial in combination with other drugs (Kinders et al., 2011; Newman et al., 2015). Its mechanism of action involves the (Ghoshal et al., 2005; Juttermann et al., 2006). Accordingly, ability to block DNMT-dependent DNA methylation (Jones and two main molecular effects have been described for DNMTi Taylor, 1980; Beumer et al., 2008). 5,6-Dihydro-5-azacytidine is inhibitors: (1) a global demethylation of gene promoters (mainly a reduced, hydrolytically stable form of 5-azacytidine nucleoside tumor suppressor genes) and (2) the activation of immune (Beisler et al., 1979). The mechanism of action is very similar system and the triggering of an anti-tumor immune response to that described for azacytidine, with the advantage of a lower (Groudine et al., 1981; Almstedt et al., 2010; Goodyear et al., 2010; toxicity (Avramis et al., 1989). However, its evaluation in clinical Chiappinelli et al., 2015;). As an illustration, in DLBCL it has settings revealed a reduced response rate and the rise of significant been described that decitabine can reverse DNA methylation and adverse effects (Samuels et al., 1998). Zebularine is another restore expression of important cancer-related pathways in vitro DNMTi, which has been previously described as tumor-selective Frontiers in Genetics | www.frontiersin.org 5 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL TABLE 3 | Selected examples of epigenetic drugs under clinical evaluation in B-NHL patients as single agents. Epigenetic drug class Drug Diseases Sponsor Trial identifier HDAC inhibitor Vorinostat FL, indolent B-NHL, MCL Merck Sharp & Dohme Corp. NCT00875056 Chidamide (Epidaza) R/R B-NHL Sun Yat-sen University NCT03245905 Abexinostat R/R FL Xynomic Pharmaceuticals, Inc. NCT03934567 Mocetinostat (MGCD0103) DLBCL, FL Memorial Sloan Kettering Cancer Center and NCT02282358 MethylGene Inc. Ricolinostat(ACY-1215) R/R NHL Acetylon Pharmaceuticals Inc. and Columbia NCT02091063 University Panobinostat CLL Peter MacCallum Cancer Centre, Australia NCT01658241 CDX101 Advanced lymphomas Cancer Research UK and Oxford NCT01977638 University Hospitals IDH1 inhibitor Ivosidenib(AG-120) Advanced hematologic malignancies Agios Pharmaceuticals, Inc. NCT02074839 with an IDH1 mutation BET bromodomaininhibitor CPI-0610 Progressive lymphomas Constellation Pharmaceuticals NCT01949883 BMS-986158 Lymphomas Dana-Farber Cancer Institute NCT03936465 Molibresib (GSK525762) NHL GlaxoSmithKline NCT01943851 EZH1/2 inhibitor DS-3201b R/R B-NHL Daiichi Sankyo Co., Ltd. NCT02732275 CPI-1205 Progressive B-cell lymphomas Constellation Pharmaceuticals NCT02395601 Tazemetostat R/R B-NHL Eisai Co., Ltd. NCT03009344 Tazemetostat R/R NHL with EZH2 gene mutations National Cancer Institute (NCI) NCT03213665 PRMT inhibitor JNJ-64619178 R/R B-NHL Janssen Research & Development, LLC NCT03573310 GSK3326595 MDS, AML GlaxoSmithKline NCT03614728 GSK3368715 DLBCL GlaxoSmithKline NCT03666988 DNMT inhibitor Decitabine R/R DLBCL Mingzhi Zhang, Zhengzhou University NCT03579082 EED inhibitor MAK683 Advanced DLBCL Novartis Pharmaceuticals NCT02900651 EED, embryonic ectoderm development. Source: https://clinicaltrials.gov/. inhibitor of DNMTs (Cheng et al., 2004). Although there are a lot of heterochromatic regions and downregulation of the nearby of evidences, both in vitro and in vivo, indicating the potential of genes (Bracken and Helin, 2009; Ferrari et al., 2014) (Figure 2). zebularine as a demethylating agent in a wide range of tumors In B lymphocytes, EZH2 becomes expressed and inhibited in a (Agrawal et al., 2018), its poor bioavailability has prevented its cyclic manner. First, in pre-B lymphocytes, induction of EZH2 introduction into clinical trials (Ben-Kasus et al., 2005). More expression is required for an optimal V(D)J recombination. Later recently, 4′-thio-2′-deoxycytidine (TdCyd) and its 5-aza analog, on, during the migration to lymphoid tissues, it is downregulated 5-aza-TdCyd, have been reported to downregulate DNMT1 and until the GC reaction occurs, after which it becomes re-expressed to exhibit anti-tumor activity in vitro and in human leukemia to allow the silencing of the anti-proliferative genes cyclin- and lung cancer xenografts (Thottassery et al., 2014). Among dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent these last molecules, TdCyd has already entered into phase I kinase inhibitor 1A (CDKN1A1) and the pro-differentiation genes clinical evaluation (NCT02423057 and NCT03366116). Further interferon regulatory factor 4 (IRF4) and PR domain zinc finger molecules were developed with superior anti-tumoral efficacy protein 1 (PRDM1/BLIMP1) during the somatic hypermutation and included guadecitabine (SGI-110), a second-generation and isotype switch processes. Finally, EZH2 becomes repressed DNMTi that harbors an improved DNA methylation inhibition when mature B cells leave the GC (Velichutina et al., 2010; in solid tumors both in vitro and in vivo (Chuang et al., 2010; Béguelin et al., 2013). Gain-of-function mutations in EZH2 Srivastava et al., 2015). A phase I clinical trial has provided have been reported in several solid tumors and hematological promising results in patients with MDS and AML (Issa et al., cancers. The consequence of those mutations in GC lymphocytes 2015). Fluorocyclopentenylcytosine (RX-3117) is a cytidine is the irreversible silencing of certain cell cycle checkpoint and analog that presents an anti-tumor activity in a large set of tumor plasma cell differentiation genes (Béguelin et al., 2013). The main cells and in vivo. Its mechanism of action is associated with an gain-of-function mutation identified in DLBCL and FL patients inhibition of DNMT1 (Choi et al., 2012). This agent is being includes a tyrosine deletion (Y641) at the EZH2 SET domain that evaluated in a phase II study with R/R pancreatic or advanced increases the levels of H3K27me3, promoting a repressed state of bladder cancer (NCT02030067). cell differentiation and the repression of tumor suppressor genes (Morin et al., 2010; McCabe et al., 2012a). Similar effects have been described as a consequence of the A677G mutation in EZH2, EZH2 Inhibitors which has been characterized in multiple human lymphoma EZH2 constitutes the catalytic subunit of the polycomb cell lines. A change in the substrate preferences accounts for repressive complex 2 (PRC2). Its structure is composed by a the aberrant H3K27me3 levels observed in cells bearing EZH2 SET domain, typical in chromatin-associated regulators of gene mutant forms. Indeed, wt EZH2 displays preference for less expression (Xiao et al., 2003). It catalyzes histone H3 lysine 27 methylated substrates whereas Y641 and A667G mutants prefer tri-methylation (H3K27me3) and the subsequent formation either substrates with higher methylation levels or show equal Frontiers in Genetics | www.frontiersin.org 6 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL affinity for all three substrates (H3K73me0, me1, and me2) and the mutant EZH2 forms, is being evaluated in clinical studies (McCabe et al., 2012a). Interestingly, these gain-of-function to treat R/R B-NHL and MM patients (NCT03456726) (Knutson EZH2 variants expressed in GC B-cell lymphomas seem to et al., 2014; Gulati et al., 2018), reaching an overall response rate synergize with BCL2 deregulation, favoring the progression of of 38% in a phase I clinical trial (Italiano et al., 2018). these malignancies (Béguelin et al., 2013). On the other hand, Despite first promising results, single-agent treatment with overexpression of wt EZH2 has been also reported in B-NHL EZH2 inhibitors is in general slightly effective in aggressive (Van Kemenade et al., 2001; Visser and Gunster, 2001), with a lymphomas. Among the possible mechanism(s) of resistance, positive correlation being observed between EZH2 transcript overactivation of the phosphatidylinositol 3-kinase (PI3K) and levels, tumor aggressiveness, and disease prognosis (Abd Al mitogen-activated protein kinase (MAPK) pathways has been Kader et al., 2013). Taking into account these considerations, it identified in GSK126-resistant DLBCL cells (Bisserier and looks reasonable that inhibiting EZH2 activity could result in a Wajapeyee, 2018). Thus, it looks reasonable to prioritize the potential therapeutic strategy to treat B-NHL. discovery of new drug combination associating EZH2 inhibitors In this context, many efforts directed to develop highly with other compounds targeting key signaling pathways in order selective EZH2 inhibitors have been made in the last decade. to prevent and/or overcome the occurrence of EZH2i resistance EZH2 activity was initially targeted by means of the carbocyclic in lymphoid neoplasm with mutated EZH2. adenosine analog 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylhomocysteine hydrolase. DZNep promotes a global increase in the levels of 5-adensylhomocystein and a further PRMT Inhibitors inhibition in the activity of many methyltransferases, including A conserved biological mechanism within all eukaryotic EZH2. Nevertheless, due to its mechanism of action, it resulted to organisms, from yeast to higher mammals, is arginine be too unspecific as many other methyltransferases were similarly methylation (Migliori et al., 2010). This post-translational affected. In 2012, a small chemical compound named El1 with a modification is mediated by N-arginine methyltransferases good capacity to inhibit the Y641 mutant and wt EZH2 form (PRMTs), which catalyze the transfer of a methyl group, from was evaluated for the treatment of DLBCL. This compound was SAM to the omega nitrogens found in terminus guanidine designed as a competitive inhibitor of the EZH2 methyl group group of an arginine residue of the side chain. This transfer donor S-adenosyl--methionine (SAM). Unlike DZNep, El1 showed may occur in one or both nitrogens (Bedford and Clarke, a 10,000-fold selectivity for EZH2 over other HMTs and a 90-fold 2009). Among the nine different members of the PRMT family selectivity over EZH1 methyltransferase. This compound promoted (Schubert et al., 2003), PRMT1 is the major enzyme responsible a global decrease in methyl donor availability, leading to a lower for arginine methylation followed by PRMT5, according to global levels of H3K27me3 (Qi et al., 2012). Other subsequent the observation that PRMT1 and PRMT5 knockout mice die compounds directed specifically against EZH2 are the dual at an early stage during development whereas mice lacking EZH2/1 inhibitors UNC1999, with a potent capacity to suppress any of the other seven PRMTs are fully viable (Hadjikyriacou H3K27me3 and H3K27me2 levels and to inhibit proliferation of et al., 2015). Protein modifications performed by PRMTs mixed lineage leukemia (MLL)-rearranged cells, and the OR-S1 are traditionally related to important genetic processes such and OR-S2 inhibitors, which were assessed for the treatment of as DNA repair and gene transcription, among others. More DLBCL, AML, and MM (Konze et al., 2013; Honma et al., 2017). recently, PRMT functions have been linked to carcinogenesis Later on, EPZ0005687 and GSK126, two selective and SAM- and metastasis, giving to these enzymes the status of potent competitive EZH2 inhibitors with a higher inhibitory capacity therapeutic targets in a variety of cancers where they are for the mutant EZH2 form, were developed and tested in DLBCL overexpressed, including colon, breast, prostate, and lung and FL (McCabe et al., 2012b; Knutson et al., 2014). In 2014, cancers, neuroblastomas, leukemias, and B-cell lymphoma GSK126 entered into phase I clinical trials with B-NHL and MM (Yoshimatsu et al., 2011). patients (NCT02082977) (Zeng et al., 2016; Yap et al., 2018), but Within this family, upregulation of PMRT1 and PRMT5 unfortunately that study had to be discontinued as a consequence has been widely associated with hematological malignancies of insufficient therapeutic activity, evidencing the need to keep (Greenblatt et al., 2016; Smith et al., 2018). In particular, the working in the improvement of those inhibitors. Also in 2014, CPI- expression and function of PMRT5 have been extensively 360 and its more potent and stable analog, CPI-169, were reported examined during lymphomagenesis, as this enzyme is highly to be effective EZH2 inhibitors for the treatment of several B-NHL expressed in primary samples and cell lines from different subtypes (Vaswani et al., 2016). An improved version of these latest, leukemia and lymphoma subtypes, where it promotes the CPI-1205, showed a higher oral bioavailability and was first tested repression of tumor suppressors such as the retinoblastoma in preclinical studies with xenograft mouse models generated from proteins. In these models, experimental studies have human B-NHL cell lines and further challenged in phase I trials for suggested that PRMT5 upregulation may be caused by the treatment of DLBCL (NCT02395601). overexpression of MYC and NOTCH oncogenes (Wang et al., Valemetostat (DS-3201) is another potent wild-type (wt) 2008). In transformed DLBCL, the S-methyl-5’-thioadenosine and mutant EZH1/2 inhibitor that demonstrated a strong anti- phosphorylase (MTAP) gene encoding for a critical methionine proliferative effect against NHL, DLBCL, and T-cell lymphoma metabolism enzyme is deleted due to its proximity to the tumor (Maruyama et al., 2017). Currently, tazemetostat (EPZ‐6438), suppressor gene CDKN2A (Dreyling et al., 1998), and this another SAM competitive inhibitor with a high affinity for the wt phenomenon sensitizes cancer cells to PRMT5 inactivation Frontiers in Genetics | www.frontiersin.org 7 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL (Marjon et al., 2016). A remarkable interplay has also been and the cyclic peptide trapoxin (Bradner et al., 2010; Di Costanzo described between PRMT5 and the B cell lymphoma 6 (BCL6) et al., 2014). Taking into account that HDACs can also modulate oncogene during the lymphomagenesis in the GC (Lu et al., the function of several non-histone proteins regulating a number 2018), suggesting that pharmacological inhibition of arginine of physiological processes (Lane and Chabner, 2009), and that methylation could be of special interest in BCL6-driven HDACs can simultaneously exert pro- and anti-leukemic lymphoma. Regarding PRMT1, an interesting interaction activities (Heideman et al., 2013; Santoro et al., 2013), blocking exists between this enzyme and EZH2 in DLBCL-GCB individual HDACs with isotype-selective inhibitors specific tumors. Indeed, recent works have reported an increase in for one or two classes of HDACs might represent a strategy of PRMT1-related histone arginine methylation in DLBCL-GCB choice for the treatment of lymphoid tumors. In line with this, cells resistant to EZH2 inhibition, in association with BCL-2 the isotype-selective HDACis include the benzamides entinostat overexpression and modulation of the B-cell receptor (BCR) (MS-275, SNDX-275) and mocetinostat (MGCD0103) (Fournel downstream signaling, supporting the rational association et al., 2008; Vannini et al., 2004), the hydroxamic acid derivative of EZH2 and PRMT1 inhibitors in DLBCL patient samples rocilinostat (ACY-1215) (Santo et al., 2012), and the cyclic (Goverdhan et al., 2017). peptide romidepsin, which show preference for HDAC1-6-8, Among the multiple functional inhibitors that have been HDAC6, and HDAC1-2, respectively (Lemoine and Younes, developed to target the different members of the family, 2010). Several HDACis like vorinostat, mocetinostat, and PRMT1 and PRMT5 small molecule inhibitors have already entinostat can be administered orally; conversely, other agents shown great potential against B-NHL, either alone or upon like romidepsin are given intravenously (Batlevi et al., 2016; their combination with other agents. As an illustration, Mann et al., 2007; Younes et al., 2011; Holkova et al., 2017). By promising results have been obtained with the specific inhibiting the catalytic activity of their target HDAC(s), these PRMT5 inhibitor EPZ015666 (GSK3235025) when used as compounds impair the formation of HDAC–substrate complexes, single agent in in vitro and in vivo models of MCL (Chan- thus altering the transcriptomic pattern of the malignant cells as Penebre et al., 2015). well as the activity of non‐histone proteins, ultimately leading to growth arrest, differentiation, and induction of apoptosis (Qiu et al., 2000). Of importance, when compared to their malignant TARGETING ERASER EPIGENETIC counterparts, healthy tissues are generally unaffected by HDACis ENZYMES: HDAC INHIBITORS (Mai et al., 2005). A number of preclinical studies have highlighted a role By favoring an open chromatin state, histone acetylation allows for HDACi therapy in a range of B-cell lymphoma, including numerous transcription factors to bind DNA and to activate DLBCL, HL, and BL, either alone or in combination with gene expression. At the same time, acetylated histones increase other epidrugs such as HMAs, with small molecule agents or DNA accessibility to transcriptional activators and counteract with standard chemotherapeutics (Buglio et al., 2008; Kretzner the function of transcriptional repressors (McClure et al., 2018). et al., 2011; Kewitz et al., 2012; Ageberg et al., 2013; Klein et al., Acetylation of histones and non-histone proteins is regulated 2013; Rozati et al., 2016; Garrido Castro et al., 2018). Among through a correct balance between HAT and HDAC activities. these studies, the weak HDACi valproic acid was shown to Among these enzymes, the most advanced subfamily is human overcome DLBCL cell resistance to the standard R-CHOP HDACs, which have been classified into four classes according to (rituximab, cyclophosphamide, doxorubicin, vincristine, their sequence homology, activity, and subcellular localization. prednisone) chemotherapeutic regimen (Ageberg et al., 2013). In HDACs 1, 2, 3, and 8 constitute class I. HDAC 4, 5, 6, 7, 9, and preclinical models of DLBCL and MCL, panobinostat, belinostat, 10 belong to class II. Class III includes sirtuin 1 (SIRT1) and depsipeptide, and vorinostat were shown to evoke tumor growth sirtuin 7 (SIRT7), two NAD-dependent structurally unrelated arrest, differentiation, and/or apoptosis in vitro and/or in vivo, protein deacetylases (Minucci and Pelicci, 2006). Finally, class mediated by the accumulation of DNA damage upon PARP IV is represented by HDAC11. In contrast to class II HDACs trapping (Valdez et al., 2018), G1 cell cycle arrest consequent which show a heterogeneous expression pattern, class I HDACs to an increase in the expression of the cyclin-dependent kinase are found at particularly high levels in lymphoid cell lines and inhibitor p21, acetylation of histone H3 (Xue et al., 2016), or primary tumors, suggesting a predominant role of these latest in transcriptional activation of the BCL-2 family proapoptotic lymphomagenesis. Accordingly, the design of HDAC inhibitors members BIM, BMF, and NOXA (Kalac et al., 2011; Xargay- (HDACis) in lymphoid malignancies has been mainly centered Torrent et al., 2011). on this latest group of enzymes (Gloghini et al., 2009). Based on these preclinical studies, several HDACis have Several structurally distinct classes of HDACis have been entered clinical trials under different modalities (monotherapies developed. These molecules can be divided into five chemical or in combination). Many of these trials have been conducted groups: hydroxamic acids, cyclic peptides, electrophilic ketones, in DLBCLs, FLs, and HLs using HDACis, either alone or in short-chain fatty acids, and benzamides. Pan-HDACis have combinatorial therapies (Watanabe et al., 2010; Stathis et al., the capacity to inhibit almost all HDACs with the exception of 2011; Younes et al., 2012; Oki et al., 2013; Ogura et al., 2014; class III HDACs and include the hydroxamic acid derivatives Chen et al., 2015; Morschhauser et al., 2015) (Table 2 and vorinostat, givinostat (ITF2357), abexinostat, panobinostat, Figure 3). As monotherapy, HDACis have shown a wide range belinostat, and trichostatin A, the carboxylate sodium butyrate, of response in lymphoma patients, varying from complete Frontiers in Genetics | www.frontiersin.org 8 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL remissions (CRs) to no response. In the absence of biomarkers et al., 2016). In a second phase IB trial, the drug has been tested for prediction of clinical outcome, the molecular mechanisms of in patients with R/R DLBCL and showed a response rate of resistance are poorly understood. Vorinostat was the first proved 37%, with a higher effect in MYC-altered versus MYC-unaltered in relapsed B-NHL patients, including FL, MZL, and MCL. In patients (Oki et al., 2017). As a result of these encouraging a phase II study including relapsed FL, non-FL indolent NHL initial data, this agent is currently being evaluated in a phase and MCL patients, oral vorinostat showed low levels as a single II study including DLBCL patients, and also in a phase I trial agent, with the exception of FL, in which an overall response rate involving pediatric patients with lymphomas (NCT02674750 (ORR) of 47–49% (referring to the proportion of patients with and NCT02909777). tumor size reduction of a predefined amount and for a minimum time period) and a CR rate of 23% was observed (Kirschbaum et al., 2011; Ogura et al., 2014). This agent was also tolerated, but TARGETING READER EPIGENETIC displayed limited activity in another phase II trial against R/R ENZYMES DLBCL, with only 1/18 patients presenting complete response (Crump et al., 2008). BET Inhibitors With the pan-HDACis abexinostat and quisinostat, or the Among the post-translational modifiers with ability to orchestrate class-specific mocetinostat and entinostat, the response rates chromatin organization, bromodomain (BD)-containing proteins were quite variable (from 12% to 56%), and mostly dependent are readers of Ac-K residues at the N-terminal histone tails. They on the drug and on the lymphoma subtype. The most robust act as scaffolds that enable histone attachment to the chromatin responses were obtained with abexinostat in FL patients (56% and form active multi-protein transcription complexes, thereby ORR). This latest drug showed a unique pharmacokinetic profile modulating chromatin dynamics and ultimately diversifying and an optimized oral dosing schedule that allowed for a superior gene expression (Filippakopoulos et al., 2012; Chaidos et al., anti-tumoral activity. In a recent phase II study with patients 2015; Smith and Zhou, 2016). This family of proteins contains 46 with R/R B-NHL or CLL, among the evaluable patients the ORR members, comprising nuclear proteins with HAT or HMT activity, was 28%, with highest responses observed in FL patients (ORR chromatin remodelers, helicases, transcription co-activators, and 56%) and DLBCL (ORR 31%) (Ribrag et al., 2017). A phase II mediators or scaffold proteins. They are subdivided into eight clinical trial with mocetinostat in patients with R/R DLBCL and subfamilies (I to VIII), based on their structure and sequence FL showed promising results (Batlevi et al., 2017), whereas for similarities. Subfamily II is the most studied one and includes entinostat only one B-NHL patient has been included in phase II the bromodomain-containing proteins mBRDT, BRD2, BRD3, trial; therefore, no conclusion can be made on its efficacy in this and BRD4 (Padmanabhan et al., 2016). Besides the presence subgroup of patients (Kummar et al., 2007). of two bromodomains (BD1 and BD2) that allow acetylated Similar to DNMTis, the effectiveness of the first-generation chromatin recognition, these proteins harbor an extra-terminal HDACis carries significant toxicity and is limited to domain, which is responsible for protein–protein interactions. hematopoietic malignancies, which makes them challenging This bromodomain and extra-terminal (BET) subfamily has thus to combine (Suraweera et al., 2018). It is believed that part of the capacity to act as protein adaptors facilitating the recruitment this toxicity may be related to the capacity of HDACis to alter of chromatin remodelers and transcription factors for further directly the function of many non-histone proteins. Toxicity initiation and elongation of transcription (Delmore et al., 2011; may also be due to widespread activity across HDAC isoforms; Chaidos et al., 2015; Padmanabhan et al., 2016). Several reports therefore, the focus of second-generation HDACi discovery have highlighted the importance of the BET proteins action was to enhance the discrimination over HDAC family members over DNA enhancers for the regulation of certain oncogenes (Galli et al., 2010; Knipstein and Gore, 2011; Younes et al., expression (Lovén et al., 2013). Altogether, these studies make 2011; Santo et al., 2012; Evens et al., 2016). In this context, BET proteins attractive therapeutic targets in cancer. targeting HDAC6 was associated to the upregulation of CD20 As interfering with this family of proteins may serve as a and consequent enhanced efficacy of anti-CD20 monoclonal strategy to address transcription irrespective of the presence antibody therapy (Bobrowicz et al., 2017). Also, tucidinostat of epigenetic mutations, BET proteins inhibitors have been a (CS055/chidamide), the first oral subtype-selective HDACi, was significant area of focus in the last decade, in cancer but also in approved for the treatment of refractory/relapsed PTCL by the inflammation, fibrosis, and heart diseases (Vakoc, 2015). Drug China Food and Drug Administration. This compound inhibits developmental studies have paid special attention to the Ac-K HDAC1, HDAC2, HDAC3, and HDAC10, and has entered a binding sites in the bromodomains, as these deep hydrophobic phase II clinical trial as single-agent treatment for patients with pockets with conserved asparagine and/or aspartate residues R/R B-NHL (NCT03245905) based on preliminary evidences of make BET proteins highly druggable (Cox et al., 2016). Indeed, clinical activity in DLBCL (Yang et al., 2018). the most common drug targeting approach in this family has Another approach to maximize efficacy with manageable been the development of small molecules that could block the toxicity consists in developing dual inhibitors. In this field, lysine-binding pocket and disrupt the interactions between BDs CUDC-907, a novel first-in-class oral small molecule inhibitor and the Ac-K on chromatins (Smith and Zhou, 2016). of both HDAC (class I and II) and PI3K (class Iα, β, and δ), In 2005, a first bromodomain inhibitor developed by the Zhou has demonstrated excellent levels of activity (55% ORR) and laboratory, namely NP1, has the ability to target the BD of the tolerability in DLBCL patients in a phase IA clinical trial (Younes P300/CBP-associated factor transcriptional coactivator (Zeng Frontiers in Genetics | www.frontiersin.org 9 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL et al., 2005). This step was followed by the discovery in 2006 of and IL4R. Accordingly, in a mouse model of CLL, exposure MS7972, a weakly binding fragment specific for CREBBP-BD, to the BET inhibitor PLX51107 promoted an expression hindering its binding to acetylated p53 (Sachchidanand et al., downmodulation of several tumor-associated genes, followed 2006). Among BET proteins, the first target considered to be by consistent reduction in tumor burden (Ozer et al., 2018). druggable was BRD4, as a pioneering RNAi base unveiled its According to these promising results, in the last years critical role in the maintenance of AML. In this study, authors a number of clinical leads have entered into trials for the found that BRD4-dependent transcriptional activity could be treatment of hematological patients. Nevertheless, several side efficiently targeted by the pan-BET thieno-triazolo-1,4-diazepine effects have been reported including some bone marrow and (+)-JQ1 (Filippakopoulos et al., 2010; Zuber et al., 2011). This gastrointestinal toxicity that has forced to dose discontinuation class of diazepine-based small molecule inhibitors, which also or reduction. Nowadays, 18 BET inhibitors are being assessed includes the benzodiazepine I-BET151 (GSK1210151A) (Dawson in clinical trials either as single agents or in combination with et al., 2011) and I-BET762 (GSK525762) (Mirguet et al., 2013) other compounds (Table 4). While the data from various solid (NCT01943851), utilizes the methyltriazolo-diazepine ring tumor trials look mitigated, several BETis including birabresib system as the acetyl-mimetic. Further studies demonstrated that (OTX015, MK-8628), molibresib (GSK525762), RO6870810/ inhibition of BRD4 by (+)-JQ1 unveiled the MYC downregulation TEN-010, and mivebresib (ABBV-075) have demonstrated and, consequently, a genome-wide inhibition of its target genes remarkable clinical efficacy in myeloproliferative disorders, (Filippakopoulos et al., 2010; Delmore et al., 2011). These results while other small molecule inhibitors such as PFI-1, BI-894999, underlined significant preclinical activity of this inhibitor in FT-1101, INCB-54329, and CPI0610, a pharmacological MYC-driven B-NHL, including the aggressive, so-called “double derivative of CPI203, are currently undergoing human clinical hit” lymphoma (DHL), characterized by simultaneous oncogenic trials in these patients (Table 3). Among these different activation of MYC and/or BCL2/BCL6 (Johnson-Farley et al., molecules, molibresib has demonstrated an 18.5% ORR in 2015). Accordingly, (+)-JQ1 could increase survival of mice various subtypes of NHLs including a CR in a DLBCL case xenografted with MYC-driven lymphoma, including those (Dickinson et al., 2018). CPI0610 has also been evaluated in ones bearing either TP53 deletions or intrinsic resistant to the a phase I clinical trial (NCT01949883) in 64 R/R FL, DLBCL, topoisomerase II inhibitor etoposide (Hogg et al., 2016). or HL patients, showing leading to a complete remission in These promising results from (+)-JQ1 encouraged the one FL case and in four DLBCL patients (Blum et al., 2018). In development of BET inhibitors with similar chemical structure, addition, the compound INCB057643 is currently being tested including the BRD4 inhibitor CPI203 characterized by a in a third phase I trial involving lymphoma patients, including higher bioavailability profile in mice (Normant et al., 2012; some FL and DLBCL cases. In this evaluation trial, a CR has King et al., 2013). This agent displayed remarkable efficacy in been achieved in one FL case whereas in two other patients, different preclinical models of B-NHL, either as single agent the disease has been stabilized (Forero-Torres et al., 2017). In or in combination with the BCL-2 antagonist venetoclax in the dose-escalation, open-label, phase I study with OTX015, DHLs (Esteve-Arenys et al., 2018), in DLBCL-ABC (Ceribelli et al., 2014) and in both ABC and GCB subtypes of DLBCL in combination with blockade of the CXCR4 chemokine receptor TABLE 4 | Drug combinations with non-approved epigenetic agents in B-NHL. (Recasens-zorzo et al., 2018). In these studies, BRD4i activity Epigenetic drug Drug Agent used in Trial identifier was mainly related to the blockade of MYC transcriptional class combination program. This is of special interest, as despite its central role in multiple hematological malignancies, including various HDAC inhibitor CUDC-907 Rituximab, venetoclax, NCT01742988 subtypes of B-NHL, direct targeting of MYC was considered and bendamustine Entinostat Isotretinoin NCT00098891 impossible until the demonstration that BET inhibition Molibresib NCT03925428 could regulate MYC activity in varied contexts, thanks to Mocetinostat Azacitidine NCT00543582 alleviation of BRD4 occupancy on MYC super-enhancers. EZH2 inhibitor Tazemetostat Fluconazole, NCT03028103 Importantly, beside MYC, different anti-apoptotic proteins like omeprazole, and BCL-2 and MCL-1 are also downregulated, either by direct repaglinide Atezolizumab and NCT02220842 transcription repression or as a downstream consequence of obinutuzumab BRD4 antagonism (Vakoc, 2015). Unlike the expected general Prednisolone NCT01897571 effects of BET inhibition in the elongation of transcription of PF 06821497 SOC NCT03460977 several genes, changes in the expression of only a small subset BET inhibitor Molibresib Entinostat NCT03925428 RO6870810 Venetoclax and NCT03255096 of genes was observed in cultures and/or animals receiving rituximab this therapy, suggesting that bromodomain inhibitors might INCB057643 Gemcitabine, NCT02711137 be suitable modulators of certain disease-associated genes. As paclitaxel, rucaparib, an illustration, high levels of BRD4 co-localize in CLL cells abiraterone, ruxolitinib, with super-enhancer sites of genes and microRNAs belonging and azacitidine FT-1101 Azacitidine NCT02543879 to the BCR-mediated signaling pathway with possible tumor- initiating activity, including miR-21, miR-15, TCL1, IL21R, Source: https://clinicaltrials.gov/. Frontiers in Genetics | www.frontiersin.org 10 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL FIGURE 4 | Epigenetic-targeted effects on immuno-oncology mechanisms. In lymphoma B cells, DNMT, HDAC, and BET inhibitors (DNMTi, HDACi, BETi) regulate the expression of MHC class I and PD-1 ligands (PD-L1 and PD-L2). In effector T cells, DNMTi also upregulates the expression of PD-1 and CTLA-4, which leads to T-cell exhaustion. The effects of HDACi on FoxP3 decrease the infiltration of regulatory T cells into the tumor. Lastly, EZH2i decreases the regulatory and increases effector T-cell population in the microenvironment. a 47% complete remission was reported in 17 DLBCL cases; (which targets bromodomain-containing protein II) (Sheppard however, objective responses were seen in only three DLBCL et al., 2018), the bivalent BET inhibitors AZD5153 and MT1 (a patients and clinical activity in other six B-NHL patients JQ1-derived BETi) (Rhyasen et al., 2016; Tanaka et al., 2016), (NCT01713582) (Amorim et al., 2016). More recently, the BETis and the so-called BET-PROTACs (QCA570, dBET6, BETd-260, molibresib, CC-90010, and INCB054329 are being challenged and ARV-771) that drive BET proteins to their degradation by in clinical trials including various hematological malignancies proteolysis-targeted chimera (Raina et al., 2016; Winter et al., (NCT02431260, NCT01943851, and NCT03220347), but no 2017; Qin et al., 2018 ). These molecules have shown both to data have been released so far. promote apoptosis in MCL-derived cells resistant to the first- Although at the moment most of the tested compounds aimed in-class Bruton’s kinase (BTK) inhibitor ibrutinib as well as to at inhibiting BET bromodomains are pan-BET inhibitors, many increase survival compared to OTX015-treated MCL xenografts efforts are being focused in targeting BET proteins in a more (Sun et al., 2018). Although promising results have been reported specific and novel way. These new approaches include ABBV-744 for this new generation of BET-targeting agents in preclinical Frontiers in Genetics | www.frontiersin.org 11 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL FIGURE 5 | Different strategies for combining drugs targeting epigenetic regulators in B-cell lymphoma. (A) Epigenetic drugs can be used to overcome (left axis) or to circumvent (right axis) malignant B-cell resistance to targeted agents or to standard chemotherapeutic regimens. (B) Pre-exposure of tumor cells to epigenetic drugs induces profound modifications of cell transcriptional profile, thus priming them to the cytotoxic effect of chemotherapeutic and targeted agents. Frontiers in Genetics | www.frontiersin.org 12 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL studies, their therapeutic window when moving to clinical trials or the other constitute the core component of the BAF complex. has still to be evaluated. They contain BDs within their structure that recognize and contact acetyl groups present in histone proteins (Wang et al., 1996). Although they share similarities in their domain composition, Non-BET Bromodomain-Containing they interact with different families of transcription factors what Proteins: the Histone Acetyltransferase confers to them specific functions in the BAF complex (Kadam and CREB-Binding Protein (CBP) Emerson, 2003). As previously mentioned, chromatin modifications can regulate BRG1 has been reported to be the most frequently mutated protein several important features of cell function. Among these of the BAF complex in cancer. Classically, it has been described as a modifications, histone lysine acetylation is generally associated tumor suppressor gene as inactivating mutations of its protein have with activation of gene expression (Shahbazian and Grunstein, been found in numerous solid tumors like breast, lung, gastric, 2007). HAT enzymes can deposit acetyl marks on histones and bladder, colon, ovarian cancers, and melanomas (Atlas et al., 2012; modify chromatin structure. Such marks are also recognized by Hodis et al., 2012; Jelinic et al., 2014), but also in determined B-NHL bromodomains, thus adding a second level of regulation of the subtype like DLBCL and MCL (Cuadros et al., 2017). Concretely, transcription process (Kouzarides, 2007). The transcriptional these loss-of-function mutations lead to the upregulation of the co-activators CBP/p300 are highly homologous, multifunctional pro-survival gene BCL2L1 in MCL, conferring to this malignancy proteins that encode a single bromodomain each and possess primary resistance to treatment or eventually relapse after dual HAT activity (Chen and Li, 2011; Delvecchio et al., 2013). CBP/ exposure to ibutrinib and venetoclax (Agarwal et al., 2019). Other p300 act as transcriptional co-factors, involved in the regulation studies described BRG1 as a potent oncogene, since its function was of several biological processes (Dancy and Cole, 2015). Animal required for AML progression in mice, through its binding to MYC studies have shown that CBP and p300 are required for the enhancer region and consequent aberrant expression of this second generation and activity of normal hematopoietic stem cells oncogene (Shi et al., 2013; Buscarlet et al., 2014). as well as for adult hematopoietic stem cell maintenance and Beside BRG1, several BRG-/BRM-associated factors (BAF function (Chan et al., 2011; Rebel et al., 2002). Consequently, subunits) participate in tumoral progression. Two of these subunits, CBP ablation has a direct impact on the quiescence, apoptosis, namely the AT-Rich Interaction Domain 1A (ARID1A/BAF250A) and self-renewal of adult hematopoietic stem cells (Chan et al., and its homologous ARID1B/BAF250B, contain domains capable 2011) and CBP/p300 have a tumor suppressor role in mice of recognizing and binding to AT-enriched genomic regions and models (Kung et al., 2000; Kang-Decker et al., 2004; Chan et al., C terminus region, stimulating the activation of transcription in a 2011). This role of CBP and p300 as tumor suppressors has glucocorticoid receptor-dependent manner. The presence of each been also observed in B-NHL, where its inactivating mutation of them in the complex is mutually exclusive, suggesting specific is a common event in FL and DLBCL, providing a rationale for roles at concrete genomic regions (Wang et al., 2004). employing drugs with the capacity to modulate acetylation and Mutations that truncate the ARID1A sequence and promote deacetylation processes in these tumors (Cerchietti et al., 2010; its degradation have been widely characterized in endometrial Mullighan et al., 2011; Pasqualucci et al., 2011a). carcinoma (Kandoth et al., 2013), colon cancer (Atlas et al., 2012), stomach cancer (Wang et al., 2011), bladder cancer (Gui et al., 2011), neuroblastoma (Sausen et al., 2013), and pancreatic CHROMATIN REMODELERS: SWI/SNF or hepatocellular carcinoma (Biankin et al., 2012; Fujimoto AND BRG1 AND ARID1 et al., 2012), evidencing the role of this protein in preventing tumoral progression. Similar to the mutations reported for The SWItch/Sucrose Non-Fermentable (SWI/SNF) complex was ARID1A, truncating mutations have also been identified for initially discovered in yeast. It is composed by polypeptides associated ARID1B although in a lesser frequency and most of them with a subset of proteins codified by the SWI1, SWI2, SNF2, associated with neurodevelopmental disorders (Santen et al., 2012) SWI3, SWI5, and SWI6 genes (Pazin and Kadonaga, 1997). This or neuroblastomas (Lee et al., 2017). ARID1B knockdown has complex regulates gene transcription by altering DNA–nucleosome been reported to destabilize the SWI/SNF complex and inhibit interactions at expenses of ATP consumption, thus facilitating or cell proliferation in both ARID1A-mutant cancer cell lines and impeding the accession of the transcription machinery at concrete primary tumor cells, suggesting that this protein could constitute genomic regions (Workman and Kingston, 2002). Several studies an interesting therapeutic target for the treatment of ARID1A- have reported its capacity to repair nucleotide excisions and DNA mutant tumors (Helming et al., 2014). double-strand breaks by homologous recombination (Chai et al., 2005). The mammalian analog of the SWI/SNF complex (mSWI/ SNF) is the BRG1-Associated Factors (BAF) complex. It comprised INDIRECT INHIBITION OF EPIGENETIC approximately 11 subunits encoded by 19 distinct genes assembled DYSREGULATION BY IDH INHIBITORS in different combinations according to its specific molecular mechanism of action, and in a concrete genomic region. Two of the The enzyme isocitrate dehydrogenase (IDH) catalyzes the BAF components are the human Brahma (hBRM, also SMARCA2) conversion of isocitrate into α-ketoglutarate (α-KG) by oxidative and the Brahma-related gene 1 (BRG1, also SMARCA4). These decarboxylation using NADP+ as a cofactor. The IDH1 isomer proteins are ATPase subunits (Khavari et al., 1993) and either one is located in the cytosol and the peroxisomes, whereas IDH2 is Frontiers in Genetics | www.frontiersin.org 13 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL found in the mitochondria. IDH enzymes play an important role be some room to evaluate, alone or in combination with standard in the tricarboxylic (TCA) or Krebs’ cycle, but are also related with chemotherapy, some of the molecules exhibiting clinical activity other cellular functions such as the regulation of redox balance in non-lymphoid patients. (Dang et al., 2016; Dang and Su, 2017). Mutations in IDH genes are most commonly found in the R132 codon of IDH1 and the R172 and R140 codons of IDH2, which correspond to evolutionarily COMBINATION INVOLVING EPIGENETIC- conserved residues in the enzyme active site which is critical TARGETING APPROACHES for substrate binding. Mutant forms of IDH have much lower catalytic activity and are associated with metabolic alterations. Concomitant Targeting of Different More importantly, mutant IDH enzymes gain neomorphic Epigenetic Modulators activity as they convert α-KG into 2-hydroxyglutarate (2-HG). In recent years, thanks to the many works directed to characterize Under homeostatic conditions, 2-HG is only produced by errors and get a better understanding of the human epigenome, it in catalysis and it is maintained at low levels due to the action of came out that more than 50% of the human cancers account for 2-HG-hydroxigenases (2-HGHD). Unlike in bacteria and plants, aberrant changes in chromatin organization at certain genomic 2-HG has no known physiological function in mammals (Dang regions, as a consequence of mutations in enzymes involved in and Su, 2017). 2-HG is structurally similar to α-KG and acts as a the regulation of chromatin structure (You and Jones, 2012; The competitive inhibitor, blocking the activity of α-KG-dependent Cancer Genome Atlas Research Network, 2013). Changes in the dioxygenases. This group of enzymes includes the TET family activity of these chromatin modifiers can lead not only to the of hydroxylases, which participate in DNA demethylation, and initiation of a tumor formation process but also to its progression, the JMJ domain-containing histone demethylases (Dang and Su, metastasis, development of drug resistances, and further relapse 2017). The consequent aberrant hypermethylation of both DNA and/or escape from immune surveillance (Jones et al., 2016). and histones has been associated to a blockade in differentiation Therapeutic modulation of such alterations can be achieved with in hematopoietic cells (Figueroa et al., 2010; Losman et al., chemical compounds that broadly affect the structure of the 2013), hepatocytes (Saha et al., 2014), and mesenchymal stem DNA such as DNMTis, histone HDACis, or BETis (Figure 4). cells (Jin et al., 2015), among other cell types. While single-agent clinical trials with these compounds have Homozygous missense mutations in both IDH1 or IDH2 been conducted with some success in MDS or R/R AML have been described in several cancer types, including glioma, patients receiving azacitidine (Scott, 2016; Schuh et al., 2017) cholangiocarcinoma, and hematological tumors, such as or in R/R FL, MZL, and MCL patients treated with vorinostat AML and MDS (Dang et al., 2016). Although infrequent, (Kirschbaum et al., 2011; Ogura et al., 2014), the association of mutations have also been found in lymphoid malignancies like these agents with other compounds has also been tested. As an angioimmunoblastic T-cell lymphomas (Cairns et al., 2012) example, the combinatorial treatment with vorinostat and the and acute lymphocytic leukemia, both in pediatric (Andersson sirtuin inhibitor niacinamide was evaluated in R/R NHL and HL et al., 2011; Tang et al., 2012) and adult cases (Kang et al., cases (NCT00691210) (Amengual et al., 2013), but it achieved a 2009; Abbas et al., 2010; Zhang et al., 2012). Dysregulation of modest efficiency with an ORR below 50% (Olsen et al., 2007). the IDH pathway has also been reported in CLL, as leukemic Other examples include the combination of panobinostat with B cells from these patients show overexpression of IDH1 and decitabine which displayed synergistic caspase-dependent cell lower levels of IDH2 when compared to healthy B cells (Van death in DLBCL cells (Kalac et al., 2011) or the combination of Damme et al., 2016). romidepsin with the antimetabolite pralatrexate for the treatment Two IDH inhibitors have been recently approved by the of relapsed PTCL (Amengual et al., 2018). FDA for the treatment of R/R AML in adults. Enasidenib (AG- A different therapeutic approach consists in targeting 221) targets IDH2 with R172S, R172K, and R140Q mutations, specifically certain chromatin regulatory proteins to achieve a whereas ivosidenib (AG-120) targets IDH1 with susceptible more restricted effect in the transcription of a concrete subset of mutations, such as R132H and R132C (Han et al., 2019). Other genes. Promising examples are the inhibition of the DOT1-like non-approved IDH inhibitors are currently in clinical trials (DOT1L) histone H3K79 methyltransferase with pinometostat involving patients with advanced hematological cancers. Among (EPZ-5676) in adults with MLL/KMT2A-driven leukemia these molecules, AG-881 is a pan-inhibitor of both IDH1 and (NCT02141828) (Stein et al., 2018) or inhibition of histone IDH2 that can penetrate the blood–brain barrier, while IDH305, H3K4 and K9 demethylation by the lysine-specific demethylase 1 FT-2102, and BAY-1436032 are IDH1-specific inhibitors (Dang (LSD1) inhibitor seclidemstat, currently being assessed in clinical et al., 2016; Montalban-Bravo and DiNardo, 2018). At the trials to treat refractory Ewing sarcomas (NCT03600649). preclinical level, the pharmacological IDH2 inhibitor AGI- Combinations with chemical compounds that broadly 6780 displayed synergistic cytotoxicity in MCL and BL cell affect an epigenetic mark and a specific inhibitor of a lines in combination with the proteasome inhibitor carfilzomib, chromatin-modifying enzyme, such as the EZH2 inhibitor mediated by the blockade of tricarboxylic acid cycle and the GSK126 and romidepsin, have also been assessed in decrease in ATP levels, as a consequence of enhanced IDH2 preclinical studies with DLBCL-GCB cell lines, leading enzymatic inhibition (Bergaggio et al., 2019). Thus, although to synergistic tumor growth inhibition effects in mice activating mutations of IDH genes are rare in B-NHL, there may (Lue et al., 2019). Another example of the strategies currently Frontiers in Genetics | www.frontiersin.org 14 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL evaluated in clinical studies is the concomitant treatment 2015). Ageberg and collaborators also showed that valproic of drug-resistant MM with panobinostat and bortezomib acid sensitizes to CHOP and enhances the CHOP ability to (NCT01083602) (Richardson et al., 2016). induce apoptosis in DLBCL cell lines (Ageberg et al., 2013). Finally, and in concordance with the concept that acquired Subsequently, it has been shown in a small set of DLBCL resistance to chemotherapy is tightly linked to changes in patients that the administration of valproate before R-CHOP chromatin structure, many efforts have been made in identifying treatment upregulated the CD20 levels and increased the combinational strategies associating different types of cytotoxic efficacy of anti-CD20-based therapy (Damm et al., 2015). drugs to small molecule regulators of chromatin modifiers. As an Recently, the VALFRID phase I trial (NCT01622439) showed example, the dinitroazetidine derivative RRx-001 administered in that valproate when added to standard R-CHOP therapy combination with radiation, chemotherapy, or immunotherapies is secure, tolerable, and increases OS in DLBCL patients promotes the generation of reactive oxygen and nitrogen species, (Drott et al., 2018). The efficacy of vorinostat combined with leading to the oxidation of the cysteines present at the catalytic cyclophosphamide, etoposide, and prednisone (R-CVEP) was sites of DNMTs and HDACs. This phenomenon entrains evaluated in aged patients with R/R DLBCL (NCT00667615); the inhibition in DNMT and HDAC enzymatic activities, however, the R-CVEP association did not reach the criteria with subsequent alterations in the chromatin structure. The for cohort expansion (Straus et al., 2015). Similarly, the therapeutic benefits of this compound have been assessed in combination of vorinostat with R-CHOP was evaluated in the phase II clinical trials both as a radio- and chemo-sensitizer, as SWOG S0806 phase I/II trial (NCT00972478) without success well as a way to prone tumor response to conventional therapies in DLBCL patients (Persky et al., 2018). Panobinostat was (NCT02215512, NCT02452970, NCT02096341, NCT02871843) tested in combination with conventional therapy and although (Oronsky et al., 2017; Zhao et al., 2017). the data from the clinical trial NCT01238692 suggested that as a single agent this drug induces a durable response in a subset of R/R DLBCL patients, its combination with rituximab did not Combination of Epigenetic Drugs With improve the response rate (Assouline et al., 2016). Similarly, Other Classes of Anti-Tumoral Drugs Barnes and collaborators observed that this combination was The use of epigenetic agents combined with other anti-tumoral effective in a minority of DLBCL patients heavily pretreated drugs may represent the future of epigenetic-targeted therapies (NTC01282476) (Barnes et al., 2018). The combination with (Figure 5). The rationale of such combinations would be, on the immunomodulatory drug (IMiD) lenalidomide was assessed in one hand, to benefit from the transcriptional effects of targeting a phase I/II clinical trial in patients with R/R HL (NCT01460940); epigenome. Indeed, growing evidences are showing that however, the combination was not advantageous over single- epigenetic therapy, using DNMTi or HDACi, in combination agent treatment and raised relevant concerns regarding the with conventional therapy or immunotherapy, might be an toxicity (Maly et al., 2017). Finally, preclinical data have shown up-and-coming step toward the development of new and efficient that belinostat exhibits synergistic cytotoxic activity in DLBCL cancer treatment strategies (Brahmer et al., 2012; Sharma and cell lines when associated to the microtubule-interfering drug Allison 2015; Topalian et al., 2015; Issa et al., 2017). Accordingly, vincristine, mediated by the prevention of cell polyploidy the acquired capacity of tumors to resist chemotherapy is related (Havas et al., 2016). with changes in the cancer cell’s epigenome, which might affect Regarding EZH2 inhibitors, combinatorial treatments with directly the cell cycle and/or some key apoptosis regulators tazemetostat and the anti-programmed death-ligand 1 (PDL1) (Fodale et al., 2011). antibody atezolizumab (NCT02220842), prednisone alone, or In a phase I study, Clozel and collaborators proposed a new combined with other components of CHOP regimen are currently approach to beaten chemotherapy resistance in DLBCL patients. being evaluated in patients with refractory DLBCL (NCT02889523) The authors demonstrated a high rate of complete remission (Gulati et al., 2018). Moreover, combinations with EZH2 inhibitors when a 5-day exposure to azacitidine followed by treatment with and inhibitors of the BCR signaling cascade such as ibrutinib, the R-CHOP was employed. Mechanistically, the treatment leads spleen tyrosine kinase (SYK) inhibitor tamatinib, the mammalian to the demethylation of the chemoresistance-associated gene target of rapamycin (mTOR) inhibitor everolimus, or MAPK SMAD1 and subsequent chemosensitization (Clozel et al., 2013). inhibitor have also been challenged in pre-clinical models of Based on these results, an ongoing phase I study using azacitidine DLBCL (Brach et al., 2017; Lue et al., 2017). Other therapeutic combined with R-CHOP in therapy-naive DLBCL, grade 3B FL, strategies currently assessed in pre-clinical studies for the or transformed FL patients is showing promising preliminary treatment of MM consist in combining the inhibitor tazemetostat results (NCT02343536). Finally, the safety and tolerability of with IMiDs such as lenalidomide or pomalidomide (Dang et al., adding oral azacitidine to R-ICE therapy is being evaluated in 2016), glucocorticoid receptor agonists (dexamethasone or R/R DLBCL patients (NCT03450343). prednisolone), proteasome inhibitors (bortezomib or ixazomib) Regarding HDACi, in vitro studies have demonstrated that (Drew et al., 2017), or HDACis (Issa et al., 2017). this class of agents can synergize with chemotherapy. Globally, Finally, in combination with the CDK4/6 inhibitor palbocilib, these trials have had mixed heterogeneous results. Among the the BETi JQ1 has shown synergistic activity in MCL in vitro and in potently successful studies, in indolent B-NHL the vorinostat/ vivo (Sun et al., 2015). Another member of the CDK family, CDK9, rituximab combination exhibited a nice activity with an is a core component of the assembly of the positive transcription acceptable safety profile and durable responses (Chen et al., elongation factor complex (P-TEFb), which is recruiting by BRD4. Frontiers in Genetics | www.frontiersin.org 15 October 2019 | Volume 10 | Article 986 Ribeiro et al. Epigenetic Drugs in B-NHL In relation with this, the BETi BI-894999 shows profound synergy with standard chemotherapy, allowing to decrease the chemotherapy with CDK9 inhibitors alvocidib and LDC000067 in both in vitro doses and to limit toxicities and adverse effects, co-administration of and in vivo models of hematological malignancies (Doroshow et al., two epigenetic modulators like DNA hypomethylating agents and 2017). Among other promising combinations, CPI203 combined HDAC inhibitors, for example, can also show synergistic molecular with the proteasome inhibitor bortezomib or with lenalidomide effects, resulting in increased antitumor activity. was particularly efficient in aggressive bortezomib-resistant MCL In the light of the large number of drugs currently in clinical tumors (Moros et al., 2014), and GS-5829 synergistically interacted development in B-NHL patients, selection of the most relevant with venetoclax or with BCR-interfering agents in preclinical models targeted therapies will be extremely important to move the field of DLBCL, MCL, and/or CLL (Bates et al., 2016; Kim et al., 2017). ahead. Epigenetic drugs with more specific targets, such as EZH2 inhibitors or BRD4 inhibitors, but also the newer epigenetic agents like PRMT5 and IDH inhibitors, are also of great interest, CONCLUSIONS as demonstrated by a particularly rapid translation from bench to bedside within the past 5 years. Besides the well-known genomic changes, several epigenetic Despite these considerable advances in epigenetic drug therapy modifications that result in an altered chromatin state and in B-cell lymphoma, there is still some way to go before reaching alterations in the DNA methylation status have been described in a complete overview of the complex landscape of the epigenetic lymphoma cells. In general, these alterations favor the malignant modifications occurring during the lymphomagenesis, and much transformation and/or tumor progression. Among the mechanisms work is still to be done to improve the rationale use of epigenetic that may apply to several lymphoma entities, epigenetic activation drugs in lymphoma patients. According to the promising reports of suppressors of lineage fidelity leads to downregulation of lineage- from several trials involving the newest agents and the most specific genes, while additional silencing of essential transcription innovative drug combinations in B-NHL patients with relapse factors through H3K27 trimethylation avoids the restoration of disease, it seems that we are entering a very exciting era for the the cell type characteristic expression program. Therefore, there is field of epigenetics in lymphoma. undoubtedly an important clinical role for epigenetic drugs across the spectrum of lymphoid malignancies, including B-NHL. In the last decade, the progresses in the awareness of epigenetic AUTHOR CONTRIBUTIONS changes in lymphoma cells have paved the way for targeted therapy alternatives employing epigenetic drugs. Treatment approaches such MR, DR, MA, MF and GR made a substantial contribution to all as HDAC inhibition or DNMT blockade have shown remarkable aspects of the preparation of this manuscript. activity in specific subsets of lymphoma patients who remained unresponsive to or relapsed after standard therapy. These drugs have FUNDING already been added into routine use for patients with a particular lymphoma/leukemia subtype and are the most broadly studied now. The authors received financial support from Fondo de Investigación However, the identification of biomarkers of clinical sensitivity/ Sanitaria PI15/00102 and PI18/01383, European Regional resistance to these agents is still needed in order to better identify Development Fund (ERDF) “Una manera de hacer Europa” (to GR). those lymphoma patients suitable for treatment with these drugs, The authors received fundings from TG Therapeutics and Celgene and for the design of rationally based targeted combination therapies. 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