Epigenetic Drug Targets Research Articles

Non-canonical nitric oxide signalling and DNA methylation: Inflammation induced epigenetic alterations and potential drug targets.

DNA methylation controls DNA accessibility to transcription factors and other regulatory proteins, thereby affecting gene expression and hence cellular identity and function. As epigenetic modifications control the transcriptome, epigenetic dysfunction is strongly associated with pathological conditions and ageing. The development of pharmacological agents that modulate the activity of major epigenetic proteins are in pre-clinical development and clinical use. However, recent publications have identified novel redox-based signalling pathways, and therefore novel drug targets, that may exert epigenetic effects. This review will discuss the recent developments in nitric oxide (NO) signalling on DNA methylation as well as potential epigenetic drug targets that have emerged from the intersection of inflammation/redox biology and epigenetic regulation.

EXTH-28. ISOCITRATE DEHYDROGENASE MUTATIONS PREDICT SENSITIVITY TO HISTONE DEACETYLASE INHIBITION AND ENHANCED HISTONE ACETYLATION DYNAMICS ONLY IN THE CONTEXT OF GLIOMA

Abstract Isocitrate dehydrogenase 1/2 mutations (IDHmut) define a subtype of glioma with profound epigenetic dysregulation through genomic hypermethylation. IDHmut gliomas are incurable and fatal, demonstrating new treatments are needed. These mutations are common in other solid tumors, including chondrosarcoma (CS) and intrahepatic cholangiocarcinoma (ICC). Histone deacetylase (HDAC) enzymes compose a class of epigenetic drug targets utilized clinically to treat cancer, but their potential against IDHmut gliomas has not been extensively studied. We previously showed that IDHmut glioma cultures exhibit pronounced sensitivity to the effects mediated by the FDA-approved HDAC inhibitor (HDACi) panobinostat. Here, we integrate proliferation and cytoxicity data to show that this effect is unique to gliomas. In the context of histone H3 acetylation (H3KAc), panobinostat increased H3KAc, on average, by 7.6-fold and 30.5-fold in IDHwt and IDHmut glioma cultures, respectively (p< 0.001). This difference in HDACi-mediated H3KAc based on IDH status was not significant for CS and ICC. IDHmut gliomas are more sensitive two other FDA-approved HDACis: belinostat and vorinostat. RNA-Seq analysis showed that panobinostat more greatly affected gene expression in IDHmut glioma cells, particularly in the context of gene downregulation. Similarly, H3KAc ChIP-Seq showed a decrease in promoter histone acetylation moreso in IDHmut glioma cultures treated with panobinostat: there is an average loss of 1,236 and 14,759 H3KAc peaks at gene promoters in IDHwt and IDHmut cultures, respectively. This suggests that IDHmut gliomas exhibit enhanced histone acetylation dynamics in response to HDACi. Furthermore, using a PDX glioma model, we show that only mice engrafted with IDHmut glioma exhibit a significant survival increase in response to the HDACi belinostat: median survival was extended by 14 days in IDHmut glioma-bearing mice (p= 0.01), whereas belinostat extended median survival by 4 days in IDHwt glioma-bearing mice (p= 0.11). These results suggest that HDACi may be an effective in treating IDHmut gliomas.

Abstract 2764: Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy

Abstract NanoBRET target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allowing the measurement of compound binding to a selected cellular target protein. Our NanoBret assay is scaled to 384-well plate format and Z’ analysis demonstrated the assay to be High Throughput Screening compatible. Protein kinases, RAS, and epigenetic proteins are very important drug targets. NanoBRET TE cellular assay has enabled the development of live-cell quantitative compound binding assays for >360 individual wild type and mutant protein kinases including serine/threonine-specific kinases, receptor tyrosine kinases, and all 20 cyclin-dependent kinases (CDKs). RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Our data demonstrate that NanoBRET TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), KRAS-G12A, KRAS-G12S, HRAS, HRAS-G12C, and HRAS-G12V fusion constructs co-transfected in live HEK293 cells. Our data also show NanoBRET cellular assay can be used to measure compound binding affinity to epigenetic drug target proteins such as BRD4, BRD4-BD1, BRD4-BD2, HDAC6, HDAC1, HDAC2, and HDAC3. Taken together, our results suggest NanoBRET TE cellular assay can serve as a great tool to facilitate new drug discovery against human cancers. Citation Format: Jianghong Wu, Yuan Wang, Shawn McGinley, Peter Gallagher, Li Liang, Alisha Di Lanni, Ashley Miles, Mohammad Haque, Yong Wan, Haiching Ma. Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2764.

Hydroxamic acid derivatives as selective HDAC3 inhibitors: computer-aided drug design strategies

Histone deacetylases (HDACs) are critical epigenetic drug targets that have gained significant attention in the scientific community for the treatment of cancer. The currently marketed HDAC inhibitors lack selectivity for the various HDAC isoenzymes. Here, we describe our protocol for the discovery of novel potential hydroxamic acid based HDAC3 inhibitors through pharmacophore modeling, virtual screening, docking, molecular dynamics (MD) simulation and toxicity studies. The ten pharmacophore hypotheses were established, and their reliability was validated by different ROC (receiving operator curve) analysis. Among them, the best model (Hypothesis 9 or RRRA) was employed for searching SCHEMBL, ZINC and MolPort database to screen out hit molecules as selective HDAC3 inhibitors, followed by different docking stages. MD simulation (50 ns) and MMGBSA study were performed to study the stability of ligand binding modes and with the help of trajectory analysis, to calculate the ligand-receptor complex RMSD (root-mean-square deviation), RMSF (root-mean-square fluctuation) and H-bond distance, etc. Finally, in-silico toxicity studies were performed on top screened molecules and compared with reference drug SAHA and established structure-activity relationship (SAR). The results indicated that compound 31, with high inhibitory potency and less toxicity (probability value 0.418), is suitable for further experimental analysis. Communicated by Ramaswamy H. Sarma

Design and Synthesis of Triazole-Containing HDAC Inhibitors That Induce Antitumor Effects and Immune Response.

Histone deacetylase (HDAC) is an epigenetic antitumor drug target, but most existing HDAC inhibitors show limited antitumor activity and their use is often accompanied by serious adverse effects. To overcome these problems, we designed and synthesized a series of triazole-containing compounds as novel HDAC inhibitors. Among them, compound 19h exhibited potent and selective inhibition of HDAC1, with good antiproliferative activity in vitro and an excellent pharmacokinetic profile. Compound 19h significantly inhibited the growth of human tumor xenografts in nude mice and murine tumor growth in immune-competent mice bearing MC38 colon cancer. In the MC38 model, 19h increased the ratio of splenic CD4+ T effector cells and promoted complete tumor regression in 5/6 animals when combined with the mPD-1 antibody. These results suggested that selective class I HDAC inhibitors exert direct tumor growth inhibition and indirect immune cell-mediated antitumor effects and are synergistic with immune checkpoint inhibitors.

EXTH-25. EXPLOITING EPIGENETIC DYSREGULATION IN ISOCITRATE DEHYDROGENASE MUTANT GLIOMA VIA THE USE OF HISTONE DEACETYLASE INHIBITORS

Abstract Isocitrate dehydrogenase 1/2 mutations (IDHmut) define a unique subtype of glioma that exhibits profound epigenetic dysregulation through DNA and histone hypermethylation. Though IDHmutgliomas are less aggressive than their IDH wildtype (IDHwt) counterparts, these tumors are still incurable and are invariably fatal. New treatments are therefore needed. Histone deacetylase (HDAC) enzymes compose a class of epigenetic drug targets already used in the clinic to treat other malignancies, but their potential against gliomas, specifically IDHmutgliomas, have not been extensively studied. We previously showed that IDHwt gliomas are more sensitive than IDHwt gliomas to the cytotoxic and antiproliferative effects mediated by the FDA-approved HDAC inhibitor panobinostat, and that this sensitivity was associated with a greater increase in histone acetylation in IDHmut glioma cultures. Follow-up RNA-Seq showed that panobinostat more greatly affected gene expression in IDHmut glioma cells, with 1999 significant differentially expressed genes showing a 10% or greater change in gene expression in IDHmut cells compared to 388 genes for IDHwt counterparts. This included the known HDAC target and cell-cycle inhibitor CDKN1A, with panobinostat causing a 1.9-fold greater upregulation of CDKN1A in IDHmut cells than IDHwt cells (p= 0.045). In immunocompromised mice intracranially engrafted with IDHmut or IDHwt glioma, 15 mg/kg intraperitoneally-administered panobinostat was detectable after 6 hours within the intracerebral tumors via mass spectrometry at a concentration of 52.4 ng/g (150.1 nM), and extended the survival of IDHmut glioma-engrafted mice (Median: 4 days; Mean: 9.4 days), but not IDHwt glioma-engrafted mice (IDHmut: logrank p= 0.044; IDHwt: logrank p= 0.18). These results suggest that HDAC inhibition via panobinostat may be an effective therapy to treat IDHmutglioma patients. Ongoing studies, including ChIP-Seq, will determine specific sites of histone acetylation and HDACs that are responsible for the greater sensitivity of IDHmut gliomas to HDAC inhibition.

Decoding the Role of Epigenetics in Breast Cancer Using Formal Modeling and Machine-Learning Methods

Breast carcinogenesis is known to be instigated by genetic and epigenetic modifications impacting multiple cellular signaling cascades, thus making its prevention and treatments a challenging endeavor. However, epigenetic modification, particularly DNA methylation-mediated silencing of key TSGs, is a hallmark of cancer progression. One such tumor suppressor gene (TSG) RUNX3 (Runt-related transcription factor 3) has been a new insight in breast cancer known to be suppressed due to local promoter hypermethylation mediated by DNA methyltransferase 1 (DNMT1). However, the precise mechanism of epigenetic-influenced silencing of the RUNX3 signaling resulting in cancer invasion and metastasis remains inadequately characterized. In this study, a biological regulatory network (BRN) has been designed to model the dynamics of the DNMT1–RUNX3 network augmented by other regulators such as p21, c-myc, and p53. For this purpose, the René Thomas qualitative modeling was applied to compute the unknown parameters and the subsequent trajectories signified important behaviors of the DNMT1–RUNX3 network (i.e., recovery cycle, homeostasis, and bifurcation state). As a result, the biological system was observed to invade cancer metastasis due to persistent activation of oncogene c-myc accompanied by consistent downregulation of TSG RUNX3. Conversely, homeostasis was achieved in the absence of c-myc and activated TSG RUNX3. Furthermore, DNMT1 was endorsed as a potential epigenetic drug target to be subjected to the implementation of machine-learning techniques for the classification of the active and inactive DNMT1 modulators. The best-performing ML model successfully classified the active and least-active DNMT1 inhibitors exhibiting 97% classification accuracy. Collectively, this study reveals the underlined epigenetic events responsible for RUNX3-implicated breast cancer metastasis along with the classification of DNMT1 modulators that can potentially drive the perception of epigenetic-based tumor therapy.

Sirtuins-Novel Regulators of Epigenetic Alterations in Airway Inflammation.

Histone modification is an important epigenetic alteration, and histone deacetylases are involved in the occurrence and development of various respiratory diseases. Sirtuins (SIRTs) have been demonstrated to play an important role in the formation and progression of chronic inflammatory diseases of the respiratory tract. SIRTs participate in the regulation of oxidative stress and inflammation and are related to cell structure and cellular localization. This paper summarizes the roles and mechanisms of SIRTs in airway inflammation and describes the latest research on SIRT modulators, aiming to provide a theoretical basis for the study of potential epigenetic alteration-inducing drug targets.

Ki67 and LSD1 Expression in Testicular Germ Cell Tumors Is Not Associated with Patient Outcome: Investigation Using a Digital Pathology Algorithm.

TGCTs represent a model of curable disease afflicting especially young men. Defining tumor biological characteristics is crucial to increase current knowledge and tailor the best clinical management. Ki67, a potential prognostic marker, still exhibits heterogenous associations with patient outcomes, thus bringing the need of corroboration with larger cohorts in clinical practice. LSD1, an epigenetic enzyme, represents a future target for epigenetic drugs that may lower treatment-associated morbidity. This study aimed to assess Ki67/LSD1 immunoexpression across all TGCT histological subtypes and correlate it with clinicopathological features. Results were compared with an in silico analysis of the TCGA database. Immunohistochemistry for Ki67 and LSD1 was carried out in a cohort of 157 TGCT tumor samples and assessed using a digital pathology algorithm. LSD1 protein expression was explored in TGCT cell lines, including ATRA-differentiated clones. There was a significant positive correlation between Ki67 and LSD1 H-scores (rs = 0.182, p = 0.037). Ki67 positivity percentage and H-score were significantly higher in non-seminomas (p = 0.0316 and 0.0113, respectively). Expression was not significantly different according to clinicopathological features, including stage, IGCCCG prognosis-based system, or relapse/progression-free survival, which was corroborated by in silico analysis. Our study, making use of digital image analysis, does not confirm the utility of these biomarkers in a daily practice cohort. Although not affecting patient outcome in our cohort, LSD1 is expressed overall in TGCTs, suggesting sensitivity to LSD1 inhibitors.

Chidamide Suppresses the Growth of Cholangiocarcinoma by Inhibiting HDAC3 and Promoting FOXO1 Acetylation.

Inhibitors for histone deacetylases (HDACs) have been identified as epigenetic drug targets to treat a variety of malignancies through several molecular mechanisms. The present study is aimed at investigating the mechanism underlying the possible antitumor effect of the HDAC inhibitor chidamide (CDM) on cholangiocarcinoma (CCA). Microarray-based gene expression profiling was conducted to predict the expression of HDACs in CCA, which was validated in clinical tissue samples from CCA patients. Next, the proliferation, migration, invasion, autophagy, and apoptosis of human CCA QBC939 and SNU308 cells were measured following treatment with CDM at different concentrations. The acetylation level of FOXO1 in the nucleus and cytoplasm of QBC939 and SNU308 cells was determined after overexpression and suppression of HDAC3. A QBC939-implanted xenograft nude mouse model was established for further exploration of CDM roles in vitro. HDAC3 was prominently expressed in CCA tissues and indicated a poor prognosis for patients with CCA. CDM significantly inhibited cell proliferation, migration, and invasion of QBC939 and SNU308 cells, while inducing their autophagy and apoptosis by reducing the expression of HDAC3. CDM promoted FOXO1 acetylation by inhibiting HDAC3, thereby inducing cell autophagy. Additionally, CDM inhibited tumor growth in vivo via HDAC3 downregulation and FOXO1 acetylation induction. Overall, this study reveals that CDM can exhibit antitumor effects against CCA by promoting HDAC3-mediated FOXO1 acetylation, thus identifying a new therapeutic avenue for the treatment of CCA.

Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site.

Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.

Design and synthesis of HDAC inhibitors to enhance the therapeutic effect of diffuse large B-cell lymphoma by improving metabolic stability and pharmacokinetic characteristics

Histone deacetylases (HDAC) are clinically validated and attractive epigenetic drug targets for human cancers. Several HDAC inhibitors have been approved for cancer treatment to date, however, clinical applications have been limited due to the poor pharmacokinetics, bioavailability, selectivity of the HDAC inhibitors and most of them need to be combined with other drugs to achieve better results. Here, we describe our efforts toward the discovery of a novel series of lactam-based derivatives as selective HDAC inhibitors. Intensive structural modifications lead to the identification of compound 24g as the most active Class I HDAC Inhibitor, along with satisfactory metabolic stability in vitro (t1/2, human = 797 min) and the desirable oral bioavailability (F = 92%). More importantly, compound 24g showed good antitumor efficacy in a TMD-8 xenograft model (TGI = 77%) without obvious toxicity. These results indicated that Class I HDAC Inhibitor could be potentially used to treat certain diffuse large B-cell lymphoma therapeutics.

Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development.

Simple SummaryMost epithelial ovarian cancer (EOC) patients, although initially responsive to standard treatment with platinum-based chemotherapy, develop platinum resistance over the clinical course and succumb due to drug-resistant metastases. It has long been hypothesized that resistance to platinum develops as a result of epigenetic changes within tumor cells evolving over time. In this study, we investigated epigenomic changes in EOC patient samples, as well as in cell lines, and showed that profound changes at enhancers result in a platinum-resistant phenotype. Through correlation of the epigenomic alterations with changes in the transcriptome, we could identify potential novel prognostic biomarkers for early patient stratification. Furthermore, we applied a combinatorial RNAi screening approach to identify suitable targets that prevent the enhancer remodeling process. Our results advance the molecular understanding of epigenetic mechanisms in EOC and therapy resistance, which will be essential for the further exploration of epigenetic drug targets and combinatorial treatment regimes.Epithelial ovarian cancer (EOC) is the most lethal disease of the female reproductive tract, and although most patients respond to the initial treatment with platinum (cPt)-based compounds, relapse is very common. We investigated the role of epigenetic changes in cPt-sensitive and -resistant EOC cell lines and found distinct differences in their enhancer landscape. Clinical data revealed that two genes (JAK1 and FGF10), which gained large enhancer clusters in resistant EOC cell lines, could provide novel biomarkers for early patient stratification with statistical independence for JAK1. To modulate the enhancer remodeling process and prevent the acquisition of cPt resistance in EOC cells, we performed a chromatin-focused RNAi screen in the presence of cPt. We identified subunits of the Nucleosome Remodeling and Deacetylase (NuRD) complex as critical factors sensitizing the EOC cell line A2780 to platinum treatment. Suppression of the Methyl-CpG Binding Domain Protein 3 (MBD3) sensitized cells and prevented the establishment of resistance under prolonged cPt exposure through alterations of H3K27ac at enhancer regions, which are differentially regulated in cPt-resistant cells, leading to a less aggressive phenotype. Our work establishes JAK1 as an independent prognostic marker and the NuRD complex as a potential target for combinational therapy.

Myofibroblast Deficiency of LSD1 Alleviates TAC-Induced Heart Failure.

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Mimicking H3 Substrate Arginine in the Design of G9a Lysine Methyltransferase Inhibitors for Cancer Therapy: A Computational Study for Structure-Based Drug Design.

G9a protein methyltransferase is a potential epigenetic drug target in different cancers and other disease conditions overexpressing the enzyme. G9a is responsible for the H3K9 dimethylation mark, which epigenetically regulates gene expression. Arg8 and Lys9 of the H3 substrate peptide are the two crucial residues for substrate-specific recognition and methylation. Several substrate competitive inhibitors are reported for the potent inhibition of G9a by incorporating lysine mimic groups in the inhibitor design. In this study, we explored the concept of arginine mimic strategy. The hydrophobic segment of the reported inhibitors BIX-01294 and UNC0638 was replaced by a guanidine moiety (side-chain moiety of arginine). The newly substituted guanidine moieties of the inhibitors were positioned similar to the Arg8 of the substrate peptide in molecular docking. Additionally, improved reactivity of the guanidine-substituted inhibitors was observed in density functional theory studies. Molecular dynamics, molecular mechanics Poisson–Boltzmann surface area binding free energy, linear interaction energy, and potential mean force calculated from steered molecular dynamics simulations of the newly designed analogues show enhanced conformational stability and improved H-bond potential and binding affinity toward the target G9a. Moreover, the presence of both lysine and arginine mimics together shows a drastic increase in the binding affinity of the inhibitor towards G9a. Hence, we propose incorporating a guanidine group to imitate the substrate arginine’s side chain in the inhibitor design to improve the potency of G9a inhibitors.

First-in-human Phase 1 open label study of the BET inhibitor ODM-207 in patients with selected solid tumours

BackgroundBromodomain and extra-terminal domain (BET) proteins are reported to be epigenetic anti-cancer drug targets. This first-in-human study evaluated the safety, pharmacokinetics and preliminary anti-tumour activity of the BET inhibitor ODM-207 in patients with selected solid tumours.MethodsThis was an open-label Phase 1 study comprised of a dose escalation part, and evaluation of the effect of food on pharmacokinetics. ODM-207 was administered orally once daily. The dose escalation part was initiated with a dose titration in the initial cohort, followed by a 3 + 3 design.ResultsThirty-five patients were treated with ODM-207, of whom 12 (34%) had castrate-resistant prostate cancer. One dose-limiting toxicity of intolerable fatigue was observed. The highest studied dose achieved was 2 mg/kg due to cumulative toxicity observed beyond the dose-limiting toxicity (DLT) treatment window. Common AEs included thrombocytopenia, asthenia, nausea, anorexia, diarrhoea, fatigue, and vomiting. Platelet count decreased proportionally to exposure with rapid recovery upon treatment discontinuation. No partial or complete responses were observed.ConclusionsODM-207 shows increasing exposure in dose escalation and was safe at doses up to 2 mg/kg but had a narrow therapeutic window.Clinical trial registrationThe clinical trial registration number is NCT03035591.

Abstract 1745: Application of BET/CBP/p300 multi-bromodomain inhibitors as a novel therapeutic strategy for MLL-rearranged acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood leukemia. While the prognosis of patients with ALL has improved significantly, ALL with chromosomal rearrangements involving mixed-lineage leukemia (MLL) gene remains an incurable disease. Thus, development of a safe and efficacious therapeutic agent based on the biology of MLL rearrangement is of high importance to address this unmet medical need. Bromodomains are protein motifs that bind to acetylated histones and are recognized as new epigenetic drug targets in recent years. Bromodomain and extra-terminal motif proteins (“BET proteins”) are epigenetic ‘reader' proteins that are characterized by containing two of such bromodomains. BRD4, a member of BET protein family, is a key component of multi-protein complex that induce oncogene transcription at Super-Enhancers. Currently, small-molecule BET inhibitors are being investigated in the clinic as potential therapeutics for hematological cancers. CBP and p300, two paralogous histone acetyltransferases, also contain bromodomains and are considered as drug targets for cancer. Because BET and CBP/p300 proteins are key components of the etiology of MLL-rearranged (MLL-r) leukemia, an agent that simultaneously target both classes of proteins may have therapeutic advantages. We previously demonstrated that a novel BET inhibitor, CG13250, suppressed multiple myeloma cell proliferation in vitro and in vivo (Biochem Biophys Res Commun, 484:262-268, 2017) and subsequently synthesized additional BET inhibitors (Bioorg Med Chem Lett, 29:1220-1226, 2019). In the present study, we demonstrate that some of our BET inhibitors can also inhibit bromodomains of CBP/p300 proteins. One such compound, CN470, potently suppresses BRD4, CBP, and p300 with BROMOscan KD values of 33, 32, and 20 nM, respectively, and suppressed mRNA and protein expression of c-MYC as expected. CN470 potently inhibited the growth of MLL-r ALL cell lines in a dose-dependent manner and flow cytometric analyses showed that CN470 caused apoptosis in MLL-r ALL cells following a cell cycle arrest at the G1 phase. Moreover, co-immunoprecipitation assay demonstrated that CN470 reduced the binding of BRD4 to acetylated H3 (H3K27), an effect absent with a reference BET inhibitor, OTX-015. Taken together, CN470 may represent an effective strategy against MLL-r ALL cells. As a confirmatory study, we investigated the in vivo effects of CN470 by using SEMLuc/GFP cells in an orthotopic mouse model of MLL-AF4 leukemia. Mice were orally administered with CN470 (10 mg/kg) once daily resulting in a prolonged survival compared to vehicle control group (mean 37 days vs. 28 days; p=0.0246). In conclusion, simultaneous inhibition of BET/CBP/p300 with a multi-bromodomain inhibitor represents a promising novel therapeutic approach against MLL-r ALL. Citation Format: Natsuki Imayoshi, Makoto Yoshioka, Shyh-Ming Yang, Koshi Akahane, Yuki Toda, Shigekuni Hosogi, Takeshi Inukai, Seiji Okada, David J. Maloney, Jefferey W. Strovel, Eishi Ashihara. Application of BET/CBP/p300 multi-bromodomain inhibitors as a novel therapeutic strategy for MLL-rearranged acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1745.

Molecular Structure, Binding Affinity, and Biological Activity in the Epigenome.

Development of valid structure–activity relationships (SARs) is a key to the elucidation of pathomechanisms of epigenetic diseases and the development of efficient, new drugs. The present review is based on selected methodologies and applications supplying molecular structure, binding affinity and biological activity data for the development of new SARs. An emphasis is placed on emerging trends and permanent challenges of new discoveries of SARs in the context of proteins as epigenetic drug targets. The review gives a brief overview and classification of the molecular background of epigenetic changes, and surveys both experimental and theoretical approaches in the field. Besides the results of sophisticated, cutting edge techniques such as cryo-electron microscopy, protein crystallography, and isothermal titration calorimetry, examples of frequently used assays and fast screening techniques are also selected. The review features how different experimental methods and theoretical approaches complement each other and result in valid SARs of the epigenome.

The antischistosomal potential of GSK-J4, an H3K27 demethylase inhibitor: insights from molecular modeling, transcriptomics and in vitro assays

BackgroundSchistosomiasis chemotherapy is largely based on praziquantel (PZQ). Although PZQ is very safe and tolerable, it does not prevent reinfection and emerging resistance is a primary concern. Recent studies have shown that the targeting of epigenetic machinery in Schistosoma mansoni may result in severe alterations in parasite development, leading to death. This new route for drug discovery in schistosomiasis has focused on classes of histone deacetylases (HDACs) and histone acetyltransferases (HATs) as epigenetic drug targets. Schistosoma histone demethylases also seem to be important in the transition of cercariae into schistosomula, as well as sexual differentiation in adult worms.MethodsThe Target-Pathogen database and molecular docking assays were used to prioritize the druggability of S. mansoni histone demethylases. The transcription profile of Smp_03400 was re-analyzed using available databases. The effect of GSK-J4 inhibitor in schistosomula and adult worms’ motility/viability/oviposition was assessed by in vitro assays. Ultrastructural analysis was performed on adult worms exposed to GSK-J4 by scanning electron microscopy, while internal structures and muscle fiber integrity was investigated by confocal microscopy after Langeronʼs carmine or phalloidin staining.ResultsThe present evaluation of the potential druggability of 14 annotated S. mansoni demethylase enzymes identified the S. mansoni ortholog of human KDM6A/UTX (Smp_034000) as the most suitable druggable target. In silico analysis and molecular modeling indicated the potential for cofactor displacement by the chemical probe GSK-J4. Our re-analysis of transcriptomic data revealed that Smp_034000 expression peaks at 24 h in newly transformed schistosomula and 5-week-old adult worms. Moreover, this gene was highly expressed in the testes of mature male worms compared to the rest of the parasite body. In in vitro schistosome cultures, treatment with GSK-J4 produced striking effects on schistosomula mortality and adult worm motility and mortality, as well as egg oviposition, in a dose- and time-dependent manner. Unexpectedly, western blot assays did not demonstrate overall modulation of H3K27me3 levels in response to GSK-J4. Confocal and scanning electron microscopy revealed the loss of original features in muscle fibers and alterations in cell-cell contact following GSK-J4 treatment.ConclusionsGSK-J4 presents promising potential for antischistosomal control; however, the underlying mechanisms warrant further investigation.

Improving TRAIL-induced apoptosis in cancers by interfering with histone modifications.

Epigenetic regulation refers to alterations to the chromatin template that collectively establish differential patterns of gene transcription. Post-translational modifications of the histones play a key role in epigenetic regulation of gene transcription. In this review, we provide an overview of recent studies on the role of histone modifications in carcinogenesis. Since tumour-selective ligands such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are well-considered as promising anti-tumour therapies, we summarise strategies for improving TRAIL sensitivity by inhibiting aberrant histone modifications in cancers. In this perspective we also discuss new epigenetic drug targets for enhancing TRAIL-mediated apoptosis.