Available online at www.sciencedirect.com ScienceDirect Towards a more precise therapy in cancer: Exploring epigenetic complexity Fernando P. Cossío1,2, Manel Esteller3,4,5,6 and María Berdasco7,8 Abstract For a complete overview see the Issue and the Editorial A plethora of preclinical evidences suggests that pharmaco- Available online 29 May 2020 logical targeting of epigenetic dysregulation is a potent strategy https://doi.org/10.1016/j.cbpa.2020.04.008 to combat human diseases. Nevertheless, the implementation 1367-5931/© 2020 The Author(s). Published by Elsevier Ltd. This is an of epidrugs in clinical practice is very scarce and mainly limited open access article under the CC BY license (http://creativecommons. to haematological malignancies. In this review, we discuss org/licenses/by/4.0/). cutting-edge strategies to foster the chemical design, the bio- logical rationale and the clinical trial development of epidrugs. Keywords Specifically, we focus on the development of dual hybrids to Epidrugs, DNA methylation, Histone modifications, Dual inhibitors, exploit multitargeting of key epigenetic molecules deregulated Multitargeting, Synthetic lethality, Chemoresistance, Immunotherapy. in cancer; the study of epigenetic-synthetic lethality in- Abbreviations teractions as a mechanism to address loss-of-function muta- BET, bromo- and extra-terminal domain proteins; DNMT, DNA methyl- transferase; HAT, histone acetyltransferase; HDAC, histone deacety- tions, and the combination of epidrugs with other therapies lase; HDMT, histone demethylase; HMT, histone methyltransferase; such as immunotherapy to avoid acquired chemoresistance MBD, methyl-binding domains proteins; ROS, reactive oxygen species. and increase therapy sensitivity. By exploring these chal- lenges, among others, the field of epigenetic chemical biology will increase its potential for clinical benefit, and more effective Introduction strategies targeting the aberrant epigenome in cancer are For the last two decades, epigenetic dysregulation has likely to be developed both in haematological and solid been recognized as a key factor contributing to human tumours. disorders. This is boosting an increasing number of Addresses studies into the field of epigenetic drug discovery [1,2]. 1 Kimika Fakultatea, Kimika Organikoa I Saila, Universidad del País Epidrugs, defined as small-molecule inhibitors that Vasco – Euskal Herriko Unibertsitaea, and Donostia International target either the epigenome or enzymes with epigenetic Physics Center (DIPC), San Sebastián-Donostia, Spain activity, have been developed for the three categories of 2 Centro de Innovación en Química Avanzada (ORFEO –CINQA), epigenetic regulators (writers, readers and erasers). Spain 3 Cancer Epigenetics Group, Cancer and Leukemia Epigenetics and Although writers are responsible for adding chemical Biology Program (PEBCL), Josep Carreras Leukaemia Research groups to histones or DNA (e.g., histone acetyl- Institute (IJC), Badalona, Barcelona, Catalonia, Spain transferases (HATs), histone methyltransferases 4 Centro de Investigación Biomédica en Red Cáncer (CIBERONC), (HMTs) or DNA methyltransferases (DNMTs); erasers Madrid, Spain remove them (e.g., histone deacetylases (HDACs) or 5 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barce- lona, Catalonia, Spain histone demethylases (HDMTs). In addition, epige- 6 Physiological Sciences Department, School of Medicine and Health netic modifications are recognized by a set of reader Sciences, University of Barcelona, Barcelona, Catalonia, Spain domains that are recruited to specific epigenetic marks 7 Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Cata- and act as effector proteins (e.g., methyl-binding do- lonia, Spain 8 mains proteins or bromo- and extra-terminal (BETs) Epigenetic Therapies Group, Experimental and Clinical Hematology Program (PHEC), Josep Carreras Leukaemia Research Institute (IJC), domain proteins). At present, the FDA-approved Badalona, Barcelona, Catalonia, Spain epidrugs include therapies with the following HDAC inhibitors: Vorinostat and Romidepsin for refractory Corresponding author: Berdasco, María (mberdasco@carrer- cutaneous T cell lymphoma, belinostat for peripheral T asresearch.org) cell lymphoma or panobinostat for multiple myeloma. Approvals also included the DNMT inhibitor Decita- Current Opinion in Chemical Biology 2020, 57:41–49 bine which is administrated in patients with haemato- This review comes from a themed issue on Chemical Genetics and logical malignancies, such as myelodysplastic Epigenetics syndromes, acute myeloid leukaemia, and chronic Edited by Akane Kawamura and Arasu Ganesan myelomonocytic leukaemia. After the first- and second- generations of epidrugs, possibilities for epidrug www.sciencedirect.com Current Opinion in Chemical Biology 2020, 57:41–49 42 Chemical genetics and epigenetics development are now being explored in erasers, and for optimizing the clinical translation of epigenetic BETand methyl-binding domains proteins inhibitors are therapy. undergoing clinical evaluation for efficacy in different cancer settings [2]. From the magic bullet paradigm to multitarget epigenetic inhibitors Despite their promise, there many challenges to be Multitarget therapeutic strategies can involve separate resolved for efficient use of epidrugs in the treatment of molecules that give rise to well-known combined thera- human cancer, including the lack of specificity of pies. An alternative strategy consists of incorporating two epidrugs, disappointing success in solid tumours and the biologically active units directed to their respective acquisition of drug chemoresistance leading to higher therapeutic targets. These moieties are connected by a risk of tumour relapse. Herein, we review the cutting- spacer component bound to both units by covalent bonds. edge approaches in the field of chemical biology and This strategy has yielded dual inhibitors, designed as molecular biology that are currently being taken to ‘inhibitor(1)dspacerdinhibitor(2)’ compounds, which improve the translation of epidrug therapy into clinical in turn act against epigenetic and nonepigenetic enzymes practice. Although still in its infancy, the interesting [3]. In the last three years, several additional examples concept of epidrug multitargeting, the potential of the have been reported (Figures 1 and 2). epigenetic-based synthetic lethality strategies and the use of epidrugs in combination with other therapies As far as the design, chemical synthesis and biological (such as immunotherapy) are introduced as alternatives validation of novel epigenetic/nonepigenetic dual Figure 1 Dual inhibitors involving epigenetic and nonepigenetic targets (2017–2019). DBD, DNA-binding domain; DNMT1, DNA methyltransferase 1; HAT, histone acetyltransferase; HDAC1, histone deacetylase 1; IDO1, indoleamine 2,3-dioxygenase 1; NAMPT, nicotinamide phosphoribosyltransferase; PD1, programmed cell death protein 1; VEGFR, vascular endothelial growth factor receptor. Current Opinion in Chemical Biology 2020, 57:41–49 www.sciencedirect.com Epidrug development in advanced precision oncology Cossío et al. 43 Figure 2 Dual inhibitors involving solely epigenetic targets (2017–2019). DNMT1, DNA methyltransferase 1; EZH1, enhancer zeste homologue 1; EZH2, enhancer zeste homologue 2; HDAC1, histone deacetylase 1; LSD1, lysine specific demethylase 1; G9a (EHMT2), histone–lysine N-methyltransferase 2. therapeutic agents are concerned (Figure 1), an inter- Other recent developments in dual epigenetic/nonepi- esting novel approach consists of incorporating into the genetic inhibitors are gathered in Figure 1. Inhibitor 2 is same chemical entity several bioactive components not quite different to other dual covalent bioactive mole- connected by covalent bonds. Thus, Ruan et al. [4] cules because it combines a DNA-binding synthesized a reactive oxygen species (ROS)eresponsive domain consisting of a poly(1H-pyrrole) oligomer and a polymer by condensation of polyvinyl alcohol with a HAT inhibitor [7]. This dual molecule results in a boronic diacid that creates a N1-(4-boronobenzyl)-N3- sequence-specific inhibitor that shows promising inhib- (4-boronophenyl)-N1,N1,N3,N3-tetramethylpropane- itory properties and antiproliferative effects in the 1,3-diaminium (TSPBA) cross-linker, which can be upregulation of p53 genes thus initiating p53-dependent decomposed in vivo in the presence of ROS. This apoptosis. Many recently reported molecules include hydrogel was combined with pH-sensitive HDACs as epigenetic targets. Both hydroxamic acids and CaCO3 nanoparticles (NPs) to generate a combined amides derived from ortho-phenylenediamines, well- ROS/Hþ biosensitive depot that can encapsulate bioac- known chelating groups for Zn(II)-dependent HDACs tive species to be released into tumour microenviron- [2], have been reported. Dual inhibitor 3 includes two ments. These latter molecules were Zebularine [5], a groups that simultaneously inhibit nicotinamide known demethylating reagent, and an antibody against phosphoribosyltransferase and HDAC1, thus combining programmed death-1 (PD1) receptor [6], which is highly relevant metabolic and epigenetic targets [8]. expressed in immune cells, including CD8þ Tcells. The The former inhibitory moiety consists of a substituted combined Zebularine-polyvinyl alcohol-TSBPA-aPD1- thiourea and the latter includes the orthophenylene- CaCO3-NP chemical entity 1 demonstrated its efficacy amine Zn(II)-chelating group. Both units are covalently in the inhibition of tumour growth and in prolonging the connected by a very short para-phenylene unit. This survival time of B16F10 melanoma bearing tissues [6]. molecule showed excellent activity and efficiently This approach establishes a link between combined and induced cell apoptosis and autophagy. In addition, it dual therapies in cancer, as well as between epigenetic showed promising in vivo antitumour activity in the and immunotherapies (later discussed in this review). HCT116 xenograft model [8]. In the same vein, www.sciencedirect.com Current Opinion in Chemical Biology 2020, 57:41–49 44 Chemical genetics and epigenetics compound 4 shows a design based on the hydroxamic Other structural analogues [18] of 9 showed also group as the chelating group of HDAC1. This dual in- promising dual G9a/DNMT1 inhibitory activity. Other hibitor appears to be more potent than 3, and also dual inhibitors shown in Figure 2 involve HDACs as one showed promising results with the HCT116 xenograft of the classes of epigenetic targets. Compounds 10 [19], model [9]. Compound 5 also contains the amide 11 [20] and 12 [21] incorporate hydroxamic acid units as HDAC1-inhibitory group, whereas the second bioactive HDAC-chelating groups, whereas 13 [22] possesses the moiety consists of an heterocyclic oxime that binds alternative ortho-phenylenediamine unit as the indoleamine 2,3-dioxygenase 1 [10]. This latter haem- preferred chelating group to HDAC1.Compound 10 also containing dioxygenase catalyses the transformation of inhibits lysine specific demethylase 1 (LSD1), an L-Trp into N-formylkynurenines, which results in tryp- enzyme that is present in the CoREST corepressor tophan depletion and subsequent inhibition of the pro- complex and demethylates mono- and di-methylated liferation of T lymphocytes [10]. Therefore, dual H3K4. The trans-aminocyclopropyl moiety of 10 is inhibitor 5 also connects epigenetic and immunothera- responsible for the inhibition of LSD1, with a potency peutic targets. This compound inhibited both enzymes within the low micromolecular range [19]. Compound in the nanomolar range (Figure 1) and showed excellent 11 combines HDAC1,6 nM inhibition with micromolar in vivo antitumour activity in the murine LLC tumour binding to DNMT1 [20]. In contrast, compound 12 model [10]. Finally, compounds 6 and 7 demonstrated inhibits both HDACs and G9a, thus combining deace- their inhibitory potency of class I HDACs and vascular tylases and methyl transferases as dual targets [21]. endothelial growth factor (VEGFR) [11]. Inhibition of Finally, compound 13 (Corin) inhibits HDAC1 and the first epigenetic target was accomplished in 6 by a LSD1, thus targeting the CoREST complex by encom- moiety analogous to the known HDAC inhibitor MS-275 passing the chief features of Entinostat and Tranylcy- [12], whereas the second target is incorporated in 6 and 7 promine [22], and showed very promising therapeutic by means of a combined 2-methyl-2H-imidazole/pyrim- potential in slowing tumour growth in a murine mela- idine polyheterocycle similar to that which is present in noma xenograft. approved VEGFR inhibitor Pazopanib [13]. Both com- pounds exhibited HDACi and VEGFRi activities com- parable with those found for MS-275 and pazopanib, Targeting loss-of-function by synthetic aside additional activities not present in the separate lethality epigenetic approaches units. In addition, compound 5 showed good pharmaco- Several examples of epigenetic therapy take advantage kinetic profiles and oral bioavailability (72%), as well as a of the mutations found in all major classes of epigenetic promising antitumour efficacy in the HT-29 xenograft proteins and have been explored as targets for therapy model. [23]. From a mechanistic consideration, mutations can be divided into two categories: loss- or gain-of function Among dual epigenetic inhibitors, recent examples of the epigenetic enzyme. Although it is possible to include a dual inhibitor of the HMTs enhancer zeste inhibit gain-of-function of epigenetic enzymes (as homologues 2 and 1 (EHZ2 and EHZ1) triggering example overexpression of the HMTs EZH2 or MLL), trimethylation of the Lys-27 residue of histone 3 loss-of-function mutations are more difficult to target. (H3K27). Overexpression of EZH2 and subsequent An innovative approach based on the concept of syn- hypermethylation of H3K27 is present in the progres- thetic lethality has been implemented in recent years sion of PRC2-dependent tumours. Bioavailable com- opening up new strategies in drug development. pound 8 (also known as UNC1999) [14] inhibits both EZH2 and EZH1 (Figure 2). Most interestingly, it Synthetic lethality refers to a genetic interaction be- induced antimyeloma in vitro activity in combination tween two genes in which the loss of one of them has with proteasome inhibitor Bortezomib [15]. A poten- little effect on cellular viability, whereas loss of both tially interesting combination of epigenetic targets as- genes leads to cellular lethality. This interaction pro- sociates methylation processes in histones and DNA. In vides a therapeutic opportunity by inhibiting the second particular, the inhibition of HMT G9a (also known as partner (e.g., pharmacological inhibition) in those tu- EHMT2) methylates H3K9, which is overexpressed in mours with genetic mutations affecting the other part- many tumours, decreases cancer cell proliferation and ner. A normal cell without genetic mutations would hampers the development of metastasis [16,17]. 4- tolerate the pharmacological inhibition, while it would Aminoquinoline 9 (CM-272) is a promising dual G9a/ be lethal for the tumour cell without the two functional DNMT1 inhibitor, with potencies in the nanomolar genes (Figure 3a). After the first FDA-approved syn- range for both targets [16,17]. In addition, significantly thetic lethal drug therapy pairing BRCA1/2 mutations prolonged survival of acute myeloid leukaemia, Acute with PARP inhibitor olaparib treatment in ovarian cancer lymphoblastic leukemia (ALL) and Diffuse large B-cell [24], studies of synthetic lethal pairs involving lymphoma (DLBCL) xenogenic models was described. epigenetic-related synthetic lethal genes have been Current Opinion in Chemical Biology 2020, 57:41–49 www.sciencedirect.com Epidrug development in advanced precision oncology Cossío et al. 45 Figure 3 a Synthe c lethality Normal Cell Tumor Cell with muta ons GENE A GENE B MutGENE A GENE B GENE A MutGENE B MutGENE A MutGENE B X X X X Cell Viability Cell Viability Cell Viability Cell Lethality b Epigene c –related synthe c lethality approaches Tumor Cell with Epigene c muta on + epigene c inhibitor Normal Cell • MLL-fusion( (HMT) + DOT1L inhibitor (pimenostat) selected muta ons • ARID1A (HMT) + EZH2 inhibitor (GSK126) • SMARCB1 (CRF) + EZH2 inhibitor (EPZ-6438) • CREBBP (HAT) + EP300/CREBBEP inhibitor (CU329) Epigene c muta on + non-epigene c inhibitor • KMT2C (HMT) + PARP inhibitor (olaparib) • SETD2 (HMT) + PI3Kβ-AKT inhibitor (TGX221) GENE A GENE B MutGENE A GENE B Non-Epigene c muta on + epigene c inhibitor • GATA3 + G9a-GLP inhibitor (BIX101294) X X X • TP53 + EZH2 inhibitor(GSK126) Cell Viability Cell Lethality Current Opinion in Chemical Biology Epigenetic synthetic lethality. (a) Synthetic lethality refers to a genetic interaction between two genes (e.g., gene A and gene B) in which the loss of gene A or gene B has little effect on cellular viability, whereas loss of both genes leads to cellular lethality. (b) Epigenetic-related synthetic approaches as therapeutic opportunity in tumours with specific mutations. The pharmacological inhibition of one partner of the pair (e.g., pharmacological inhibition of gene B in tumours harbouring gene A mutations) could result in cellular lethality. A normal cell without genetic mutations would tolerate the pharma- cological inhibition of gene B. CRF, chromatin remodelling factor; HAT, histone acetyltransferase; HMT, histone methyltransferase. conducted, including synthetic lethality between Additional examples of synthetic lethality between epigenetic mutations and epigenetic inhibitors, epige- epigenetic alterations and epigenetic inhibitors exist, netic mutations and nonepigenetic inhibitors and specifically involving mutations in chromatin remodel- oncogene mutations and epigenetic inhibitors [25] ling proteins. Members of the SWI/SNF complex are (Figure 3b). frequently mutated in cancer, including mutations in the tumour suppressors ARID1A or SMARCB1 [23]. PRC2 is One pioneer example of epigenetic-related synthetic another crucial chromatin complex that includes the lethality entering in clinical trials was the use of in- HMT EZH2, and its deregulation is associated with hibitors of the HMT DOT1L, such as pinometostat, in multiple cancers [28]. To note that there is an epigenetic the treatment of MLL-fusion leukaemia [26]. The antagonism between SWI/SNF and PRC2 complexes, MLL-fusion results in DOT1L recruitment and which has been the focus of synthetic legal strategies. In epigenetic-mediated activation of well-known drivers of this way, pharmacological inhibition of EZH2 is a syn- tumorigenesis (e.g., HOXA9, MEIS1) together with thetic lethal strategy in tumours harbouring SWI/SNF proteins involved in protection of MLL fusion proteins mutations [28,29]. Treatment with GSK126, a specific from autophagic degradation (e.g., LAMP5) [27]. The inhibitor of EZH2, decrease tumorigenesis is preclinical pinometostat inhibition effect on DOT1L selectively models of ovarian tumours with ARID1A mutations [29]. killed MLL-fusion leukaemia cells but not normal cells Recently, an elegant work performed by Meyer et al. without the MLL genetic aberrations throughout [30] described relevant applications of synthetic downregulating LAMP5 and enhancing the selective lethality approaches involving HAT activities in lym- autophagic degradation of MLL oncoproteins [27]. phomas. Inactivating mutations of the CREBBP and www.sciencedirect.com Current Opinion in Chemical Biology 2020, 57:41–49 46 Chemical genetics and epigenetics EP300 HATs are mutually exclusive genetic alterations in potential in TP53-overexpressing prostate tumours and diffuse large B cell lymphoma and follicular lymphoma. represents new therapeutic opportunities for treatment However, treatments with small molecule inhibitors that of advanced solid cancers [34]. are selective for CREBBP and EP300 (i.e., the bromo- domain inhibitor CCS1477 and the preclinical HAT Epigenetic therapy in combination with domain inhibitor CU329) abolished the EP300- other drugs to boost immune response or dependency in CREBBP mutants and resulted in drug sensitivity diminished tumour proliferation in murine models [30]. In the recent years, results from preclinical and phase I/ II clinical trials support that, beyond their potential as Nonepigenetic inhibitors have been also explored in monotherapies, epigenetic drugs could have important synthetic lethality therapies involving tumours with roles in combination with other anticancer therapies. epigenetic alterations. PARP inhibitors have synthetic Epidrugs, especially HDACi and DNMTi, have been lethal effects with epigenetic enzymes in cancer. The tested in combination with: chemotherapy (e.g., vori- HMT KMT2C (also known as MLL3) gene has a high nostat plus capecitabine and Cisplatin in unresectable frequency in bladder cancer. The reduction of KMT2C gastric cancer [35]), radiotherapy (e.g., vorinostat plus activity results in decreased H3K27ac-dependent pelvic radiotherapy in gastric cancer [36]), hormonal expression of genes from the DNA repair pathways therapy (e.g., panobinostat plus bicalutamide in (particularly in the homologous recombination pathway) castration-resistant prostate cancer [37]) or targeted associated with higher endogenous DNA damage and therapies (e.g., Abexinostat plus the tyrosine kinase genomic instability in the tumour cells, and conse- inhibitor pazopanib in advanced renal cell carcinoma quentially increased sensitivity to the PARP inhibitor [38]). A synergistic effect to favour sensitization of the Olaparib in epithelial carcinomas [31]. Similarly, a mo- cancer cell to the giving therapy and for overcoming lecular interaction between the HMT SETD2, which acquired chemoresistance has been observed. catalyses the methylation at H3K36 enriched at pro- moters with active transcription, and PI3Kb kinase has Among the combinatorial possibilities, enhancing the been explored in preclinical models of renal cell carci- anticancer efficacy of immunotherapy through combi- noma [32]. Treatment with inhibitors of PI3Kb/AKT nation with epigenetic drugs is receiving the most pathway causes synthetic lethality with SET2D loss-of- attention [39]. Positive results for the immunogenicity of function and increased tumour inhibition in renal cancer tumour cells are described when epidrugs are adminis- cells [32]. trated together with immune checkpoint blockade therapy (anti-PD1/PDL1 therapy in combination with Finally, a synthetic lethal interaction between an onco- HDACi in nonesmall-cell lung cancer [40]) and adoptive gene mutation (nonepigenetic) and the epigenetic drug cellular immunotherapy (e.g., HDACi plus CD19-CAR has been described for the transcription factor GATA3 CTL therapy in non-Hodgkin’s lymphoma [41]). A and the HMTs G9A and GLP (also known as EHMT2 summary of the most recent examples of clinical trials is and EHMT1, respectively) in breast cancer [33]. provided in Table 1. Although the mechanisms of action Interestingly, not only loss-of-function but also gain-of- by which the epidrug therapies modulate the immune function of oncogenes could be exploited in epigenetic- response still need further investigation, the reactivation related synthetic lethality approaches. Treatment with of tumour-surface antigens, endogenous retroviruses and EZH2 inhibitors results in decreased metastatic proteins for the major complex of histocompatibility Table 1 Examples of current clinical trials involving epidrugs in combination with immunotherapy agents. BETi, bromodomain inhibitor; CRC, colorectal cancer; DNMTi, DNA methyltransferase inhibitor; HDACi, histone deacetylase inhibitor; HDMTi, histone demethylase inhib- itor; HNSCC, head and neck squamous cell carcinoma; HMTi, histone methyltransferase inhibitor; SGC, salivary gland cancer; NSCLC, non–small-cell lung cancer; RCC, renal cell carcinoma; UB, urinary bladder cancer. Clinical trial Clinical Epigenetic drug Immunotherapy agent Cancer type Status identifier trial phase NCT01928576 II Azacytidine (DNMTi), entinostat Nivolumab (anti-PD1) NSCLC Recruiting (HDACi) NCT02638090 I/II Vorinostat (HDACi) Pembrolizumab (anti-PD1) NSCLC Recruiting NCT02635061 I ACY 241 (HDACi) Nivolumab (anti-PD1) NSCLC Recruiting NCT03179930 II Entinostat (HDACi) Pembrolizumab (anti-PD1) Lymphoma Recruiting NCT02619253 I Vorinostat (HDACi) Pembrolizumab (anti-PD1) RCC, UB Recruiting NCT02453620 I Entinostat (HDACi) Ipilimumab (anti-CTLA-4), Breast cancer Recruiting nivolumab (anti-PD1) Current Opinion in Chemical Biology 2020, 57:41–49 www.sciencedirect.com Epidrug development in advanced precision oncology Cossío et al. 47 Table 1 (continued ) Clinical trial Clinical Epigenetic drug Immunotherapy agent Cancer type Status identifier trial phase NCT03552380 II Entinostat (HDACi) Ipilimumab (anti-CTLA-4), RCC Recruiting nivolumab (anti-PD1) NCT03250273 II Entinostat (HDACi) Nivolumab (anti-PD1) Cholangiocarcinoma, Recruiting pancreatic cancer NCT03278782 II Romidepsin (HDACi) Pembrolizumab (anti-PD1) Lymphoma Recruiting NCT03150329 I Vorinostat (HDACi) Pembrolizumab (anti-PD1) Lymphoma Recruiting NCT03220477 I Guadecitabine (DNMTi) plus Pembrolizumab (anti-PD1) NSCLC Recruiting mocetinostat (HDACi) NCT03903458 I Tinostamustine (HDACi) Nivolumab (anti-PD1) Cutaneous melanoma Recruiting NCT02546986 II CC-486 (DNMTi) or placebo Pembrolizumab (anti-PD1) NSCLC Active, not recruiting NCT02437136 I/II Entinostat (HDACi) Pembrolizumab (anti-PD1) NSCLC, cutaneous Active, not recruiting melanoma and CRC NCT02538510 I/II Vorinostat (HDACi) Pembrolizumab (anti-PD1) HNSCC, SGC Active, not recruiting NCT02032810 I Panobinostat (HDACi) Ipilimumab (anti-CTLA-4) Cutaneous melanoma Active, not recruiting NCT02909452 I Entinostat (HDACi) Pembrolizumab (anti-PD1) Advanced solid tumours Active, not recruiting NCT02395627 II Vorinostat (HDACi) plus Pembrolizumab (anti-PD1) Breast cancer Active, not recruiting tamoxifen (anti-oestrogen) NCT02512172 I Romidepsin (HDACi), Pembrolizumab (anti-PD1) CRC Active, not recruiting azacytidine (DNMTi) NCT02697630 II Entinostat (HDACi) Pembrolizumab (anti-PD1) Ocular melanoma Active, not recruiting NCT02915523 I/II Entinostat (HDACi) Avelumab (anti-PDL1) Epithelial ovarian Active, not recruiting cancer, peritoneal cancer NCT02708680 II Entinostat (HDACi) Atezolizumab (anti-PDL1) Breast cancer Active, not recruiting NCT02220842 I Tazemetostat Atezolizumab (anti-PDL1), Lymphoma Active, not recruiting obinutuzumab NCT03525795 I/II CPI-1205 (HMTi) Ipilimumab (anti-CTLA-4) Advanced solid tumours Active, not recruiting NCT02250326 II CC-486 (DNMTi) or paclitaxel Durvalumab (anti-PDL1) NSCLC Active, not recruiting (targeted therapy) NCT02959437 I/II Azacitidine (DNMTi) or Pembrolizumab (anti-PD1) plus Advanced solid tumours Active, not recruiting INCB057643 (BETi) or Epacadostat INCB059872 (HDMTi) HDACi, histone deacetylase inhibitor; DNMTi, DNA methyltransferase inhibitor; NSCLC, non–small-cell lung cancer; CRC, colorectal cancer; PD1, programmed cell death protein 1. could be mediators of the increased tumour visibility to Inhibition of a single epigenetic alteration can have the host immune system [42]. In spite of its preliminary global and local effects on chromatin conformation success, some frequent limitations of epidrugs also need affecting multiple biological processes (e.g., not only to be solved in combinational therapy, including the gene regulation but also DNA repair or DNA recombi- reduction of the toxicity and the secondary effects. Im- nation could be affected after treatments) and multiple provements in the immunotherapy schemes will also biological pathways (e.g., the HAT CBP/EP300 acety- benefit from systems biology approaches. In this regard, a lates lysine residues of histones H3 and H4, as well as recent mathematical model to predict synergies between the oncogene p53 [44]). Furthermore, the effect of BET inhibitors and anti-CTLA4 immunotherapy has targeting a specific mark could result in further changes been proposed for the optimization of combinatory in different modifications (e.g., treatments with HDAC therapy in breast cancer [43]. inhibitors could increase acetylation of histones but also affect histone methylation [45]). Therefore, research in Conclusions the field of systems biology applied to epigenetic com- A simplified vision of epigenetic regulation and the use plexes would allow us to exploit the benefits of targeting of inappropriate cohorts in clinical trials are undoubtedly complexity, such as the aforementioned strategies for limiting factors to the success of epidrugs as therapeutic development of dual inhibitors. agents. At present, we are envisioning an approach which moves “from the simplest to the most complex”. We are Undoubtedly, it is not only a question of chemical drug accepting that epigenetic modifications are not stand design. Basic research to unravel the mechanisms of alone processes because several epigenetic proteins action of tumour cell progression and response to ther- (and also not epigenetic) contribute to regulate chro- apy (e.g., molecular pathways associated with tumoral matin accessibility, as do several nonepigenetic proteins. evasion from the host immune system), the broader www.sciencedirect.com Current Opinion in Chemical Biology 2020, 57:41–49 48 Chemical genetics and epigenetics characterisation of the genetic and epigenetic mutations 8. Dong G, Chen W, Wang X, Yang X, Xu T, Wang P, Zhang W, Rao Y, Miao C, Sheng C: Small molecule inhibitors simulta- acting as drivers in a tumour cell and a better stratifi- neously targeting cancer metabolism and epigenetics: dis- cation of the patients entering clinical trials (i.e., covery of novel nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylase (HDAC) dual inhibitors. development of predictive biomarkers of response) J Med Chem 2017, 60:7965–7983. would contribute to the development of more adequate 9. Chen W, Dong G, Wu Y, Zhang W, Miao C, Sheng C: Dual clinical trials involving epigenetic drugs. In summary, NAMPT/HDAC inhibitors as a new strategy for multi- the studies reviewed here suggest promising opportu- targeting antitumor drug discovery. ACS Med Chem Lett 2018, 9:34–38. nities for targeting epigenetic alterations to make further advances in precision oncology. 10. Fang K, Dong G, Li Y, He S, Wu Y, Wu S, Wang W, Sheng C: * Discovery of novel indoleamine 2,3-dioxygenase 1 (IDO1) and histone deacetylase (HDAC) dual inhibitors. ACS Med Chem Lett 2018, 9:312–317. Author contributions An interesting dual inhibitor that connects epigenetic therapy with All authors contributed to all aspects of the manuscript. immunotherapy (see also ref. [4]). 11. Zang J, Liang X, Huang Y, Jia Y, Li X, Xu W, Chou CJ, Zhang Y: Discovery of novel pazopanib-based HDAC and VEGFR dual Funding inhibitors targeting cancer epigenetics and angiogenesis The authors thank CERCA Programme/Generalitat de simultaneously. J Med Chem 2018, 61:5304–5322. Catalunya for institutional support. Research at M.B. lab 12. Saito A, Yamashita T, Mariko Y, Nosaka Y, Tsuchiya K, Ando T, is supported by Instituto de Salud Carlos III cofunded Suzuki T, Tsuruo T, Nakanishi O: A synthetic inhibitor of his- tone deacetylase, MS-27-275, with marked in vivo antitumor by European Regional Development Funds (ERDF/ activity against human tumors. Proc Natl Acad Sci U S A 1999, FEDER) a way to build Europe (PI15/00638 and PI18/ 96:4592–4597. 00910). Research at F.P.C lab is supported by Spanish 13. Harris PA, Boloor A, Cheung M, Kumar R, Crosby RM, Davis- Ministerio de Ciencia e Innovación and FEDER Ward RG, Epperly AH, Hinkle KW, Hunter RN, Johnson JH, et al.: Discovery of 5-[[4-[(2,3-dimethyl-2 H-indazol-6-yl)methyl- (CTQ2016-80375-P and CTQ2014-51912-REDC), by amino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide Gobierno Vasco/Eusko Jaurlaritza (IT-324-07) and by (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor. J Med Chem 2008, 51:4632–4640. 2020 Framework Programme of the European Union (Euro-Cholangio-Net CA18122). 14. Konze KD, Ma A, Li F, Barsyte-Lovejoy D, Parton T, MacNevin CJ, Liu F, Gao C, Huang X-P, Kuznetsova E, et al.: An orally bioavailable chemical probe of the lysine methyl- Conflict of interest statement transferases EZH2 and EZH1. ACS Chem Biol 2013, 8: 1324–1334. M.B. discloses no conflicts of interest. F.P.C. and M.E. are consultants of Quimatryx. 15. Rizq O, Mimura N, Oshima M, Saraya A, Koide S, Kato Y, Aoyama K, Nakajima-Takagi Y, Wang C, Chiba T, et al.: Dual inhibition of EZH2 and EZH1 sensitizes PRC2-dependent References tumors to proteasome inhibition. Clin Cancer Res 2017, 23: Papers of particular interest, published within the period of review, 4817–4830. have been highlighted as: 16. José-Enériz ES, Rabal O, Agirre X, Oyarzabal J, Prosper F: Dual * of special interest epigenetic modifiers for cancer therapy. Mol Cell Oncol 2017, * * of outstanding interest 4. e1342748. 17. San José-Enériz E, Agirre X, Rabal O, Vilas-Zornoza A, Sanchez- 1. Berdasco M, Esteller M: Clinical epigenetics: seizing opportu- * Arias JA, Miranda E, Ugarte A, Roa S, Paiva B, Estella-Hermoso nities for translation. Nat Rev Genet 2018, https://doi.org/ de Mendoza A, et al.: Discovery of first-in-class reversible dual 10.1038/s41576-018-0074-2. small molecule inhibitors against G9a and DNMTs in hema- tological malignancies. Nat Commun 2017, 8:15424. 2. Ganesan A, Arimondo PB, Rots MG, Jeronimo C, Berdasco M: A very interesting approach to combined G9a and DNMTs epigenetic The timeline of epigenetic drug discovery: from reality to therapy that opens novel opportunities in cancer therapy. dreams. Clin Epigenetics 2019, 11:174. 18. López-López E, Prieto-Martínez F, Medina-Franco J: Activity 3. de Lera AR, Ganesan A: Epigenetic polypharmacology: from landscape and molecular modeling to explore the SAR of combination therapy to multitargeted drugs. Clin Epigenetics dual epigenetic inhibitors: a focus on G9a and DNMT1. Mol- [date unknown], 8:105. ecules 2018, 23:3282. 4. Ruan H, Hu Q, Wen D, Chen Q, Chen G, Lu Y, Wang J, Cheng H, 19. Duan Y-C, Ma Y-C, Qin W-P, Ding L-N, Zheng Y-C, Zhu Y-L, * Lu W, Gu Z: A dual bioresponsive drug delivery depot for Zhai X-Y, Yang J, Ma C-Y, Guan Y-Y: Design and synthesis of combination of epigenetic modulation and immune check- tranylcypromine derivatives as novel LSD1/HDACs dual in- point blockade. Adv Mater 2019, 31:1806957. hibitors for cancer treatment. Eur J Med Chem 2017, 140: A very interesting approach in the interface between combined and 392–402. dual therapies. 20. Ren Y, Sun Q, Yuan Z, Jiang Y: Combined inhibition of HDAC 5. Yoo CB, Cheng JC, Jones PA: Zebularine: a new drug for and DNMT1 induces p85a/MEK-mediated cell cycle arrest by epigenetic therapy. Biochem Soc Trans 2004, 32: dual target inhibitor 208 in U937 cells. Chinese Chem Lett 910 – 912. 2019, 30:1233–1236. 6. Topalian SL, Drake CG, Pardoll DM: Targeting the PD-1/B7- 21. Zang L, Kondengaden SM, Zhang Q, Li X, Sigalapalli DK, H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Kondengadan SM, Huang K, Li KK, Li S, Xiao Z, et al.: Structure Immunol 2012, 24:207–212. based design, synthesis and activity studies of small hybrid molecules as HDAC and G9a dual inhibitors. Oncotarget 2017, 7. Yu Z, Taniguchi J, Wei Y, Pandian GN, Hashiya K, Bando T, 8. Sugiyama H: Antiproliferative and apoptotic activities of sequence-specific histone acetyltransferase inhibitors. Eur J 22. Kalin JH, Wu M, Gomez AV, Song Y, Das J, Hayward D, Med Chem 2017, 138:320–327. * Adejola N, Wu M, Panova I, Chung HJ, et al.: Targeting the Current Opinion in Chemical Biology 2020, 57:41–49 www.sciencedirect.com Epidrug development in advanced precision oncology Cossío et al. 49 CoREST complex with dual histone deacetylase and deme- 35. Yoo C, Ryu M-H, Na Y-S, Ryoo B-Y, Lee C-W, Kang Y-K: Vori- thylase inhibitors. Nat Commun 2018, 9:53. nostat in combination with capecitabine plus cisplatin as a An innovative approach to dual inhibition of deacetylation and deme- first-line chemotherapy for patients with metastatic or unre- thylation of histones that opens novel possibilities in the field. sectable gastric cancer: phase II study and biomarker anal- ysis. Br J Cancer 2016, 114:1185–1190. 23. Pfister SX, Ashworth A: Marked for death: targeting epigenetic changes in cancer. Nat Rev Drug Discov 2017, 16:241–263. 36. Ree AH, Dueland S, Folkvord S, Hole KH, Seierstad T, Johansen M, Abrahamsen TW, Flatmark K: Vorinostat, a his- 24. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, tone deacetylase inhibitor, combined with pelvic palliative Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T: Specific killing radiotherapy for gastrointestinal carcinoma: the Pelvic Ra- of BRCA2-deficient tumours with inhibitors of poly(ADP- diation and Vorinostat (PRAVO) phase 1 study. Lancet Oncol ribose) polymerase. Nature 2005, 434:913–917. 2010, 11:459–464. 25. Yang H, Cui W, Wang L: Epigenetic synthetic lethality ap- 37. Ferrari AC, Alumkal JJ, Stein MN, Taplin M-E, Babb J, Barnett ES, proaches in cancer therapy. Clin Epigenetics 2019, 11:136. Gomez-Pinillos A, Liu X, Moore D, DiPaola R, et al.: Epigenetic therapy with panobinostat combined with bicalutamide 26. Daigle SR, Olhava EJ, Therkelsen CA, Basavapathruni A, Jin L, rechallenge in castration-resistant prostate cancer. Clin Boriack-Sjodin PA, Allain CJ, Klaus CR, Raimondi A, Scott MP, Cancer Res 2019, 25:52–63. et al.: Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood 2013, 122:1017–1025. 38. Aggarwal R, Thomas S, Pawlowska N, Bartelink I, Grabowsky J, * * Jahan T, Cripps A, Harb A, Leng J, Reinert A, et al.: Inhibiting 27. Wang W-T, Han C, Sun Y-M, Chen Z-H, Fang K, Huang W, histone deacetylase as a means to reverse resistance to Sun L-Y, Zeng Z-C, Luo X-Q, Chen Y-Q: Activation of the angiogenesis inhibitors: phase I study of Abexinostat plus lysosome-associated membrane protein LAMP5 by DOT1L Pazopanib in advanced solid tumor malignancies. J Clin serves as a bodyguard for MLL fusion oncoproteins to evade Oncol 2017, 35:1231–1239. degradation in leukemia. Clin Cancer Res 2019, 25:2795–2808. An excellent research of the benefits of combining histone desacety- 28. Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, lase inhibitors to improve sensibility to targeted therapy in solid Raimondi A, Porter Scott M, Chesworth R, Moyer MP, Copeland RA, cancers. et al.: Durable tumor regression in genetically altered malignant 39. Jones PA, Ohtani H, Chakravarthy A, De Carvalho DD: Epige- rhabdoid tumors by inhibition of methyltransferase EZH2. Proc netic therapy in immune-oncology. Nat Rev Cancer 2019, 19: Natl Acad Sci U S A 2013, 110:7922–7927. 151–161. 29. Bitler BG, Aird KM, Garipov A, Li H, Amatangelo M, 40. Levy BP, Giaccone G, Besse B, Felip E, Garassino MC, Domine Kossenkov AV, Schultz DC, Liu Q, Shih I-M, Conejo-Garcia JR, * * Gomez M, Garrido P, Piperdi B, Ponce-Aix S, Menezes D, et al.: et al.: Synthetic lethality by targeting EZH2 methyltransferase Randomised phase 2 study of pembrolizumab plus CC-486 activity in ARID1A-mutated cancers. Nat Med 2015, 21: versus pembrolizumab plus placebo in patients with previ- 231–238. ously treated advanced non-small cell lung cancer. Eur J 30. Meyer SN, Scuoppo C, Vlasevska S, Bal E, Holmes AB, Cancer 2019, 108:120–128. * * Holloman M, Garcia-Ibanez L, Nataraj S, Duval R, Vantrimpont T, An excellent research of the benefits of combining DNA methyl- et al.: Unique and shared epigenetic programs of the transferase inhibitors with immunotherapy in lung cancer patients. CREBBP and EP300 acetyltransferases in germinal center B 41. Torres-Collado A, Jazirehi A: Overcoming resistance of human cells reveal targetable dependencies in lymphoma. Immunity non-Hodgkin’s lymphoma to CD19-CAR CTL therapy by 2019, 51:535–547. e9. celecoxib and histone deacetylase inhibitors. Cancers (Basel) An innovative approach to synthetic lethality between epigenetic mu- 2018, 10:200. tations and epigenetic inhibitors. 42. Jones PA, Issa J-PJ, Baylin S: Targeting the cancer epigenome 31. Rampias T, Karagiannis D, Avgeris M, Polyzos A, Kokkalis A, for therapy. Nat Rev Genet 2016, 17:630–641. Kanaki Z, Kousidou E, Tzetis M, Kanavakis E, Stravodimos K, et al.: The lysine-specific methyltransferase KMT2C/MLL3 regulates 43. Lai X, Stiff A, Duggan M, Wesolowski R, Carson WE, Friedman A: DNA repair components in cancer. EMBO Rep 2019, 20. * * Modeling combination therapy for breast cancer with BET and immune checkpoint inhibitors. Proc Natl Acad Sci U S A 32. Terzo EA, Lim AR, Chytil A, Chiang YC, Farmer L, Gessner KH, * 2018, 115:5534–5539. Walker CL, Jansen VM, Rathmell WK: SETD2 loss sensitizes An example of how mathematical models are incorporated in basic cells to PI3Kb and AKT inhibition. Oncotarget 2019, 10. research for the optimization of combinatory therapy involving epige- An interesting example of synthetic lethality between epigenetic mu- netic mechanisms. tations and non-epigenetic inhibitors. 44. Wapenaar H, Dekker FJ: Histone acetyltransferases: chal- 33. Mair B, Konopka T, Kerzendorfer C, Sleiman K, Salic S, Serra V, lenges in targeting bi-substrate enzymes. Clin Epigenetics Muellner MK, Theodorou V, Nijman SMB: Gain- and loss-of- 2016, 8:59. function mutations in the breast cancer gene GATA3 result in differential drug sensitivity. PLoS Genet 2016, 12. e1006279. 45. Huang P-H, Plass C, Chen C-S: Effects of histone deacetylase inhibitors on modulating H3K4 methylation marks – a novel 34. Zhao Y, Ding L, Wang D, Ye Z, He Y, Ma L, Zhu R, Pan Y, Wu Q, cross-talk mechanism between histone-modifying enzymes. Pang K, et al.: EZH2 cooperates with gain-of-function p53 Mol Cell Pharmacol 2011, 3:39–43. mutants to promote cancer growth and metastasis. EMBO J 2019, 38. www.sciencedirect.com Current Opinion in Chemical Biology 2020, 57:41–49