Inhibition of Jumonji demethylases reprograms severe dilated cardiomyopathy and prolongs survival.

Hypertrophic/dilated cardiomyopathy, often a prequel to heart failure, is accompanied by maladaptive transcriptional changes that contribute to arrythmias and contractile misfunction. Transgenic mice constitutively expressing high levels of calcineurin are known to develop extreme heart hypertrophy, which progresses to dilated cardiomyopathy, and to die several weeks after birth. Here, we characterized aberrant transcriptional and epigenetic pathways in this mouse model and established a pharmacological approach to treat established cardiomyopathy. We found that H3K4me3 (trimethyl histone 3 lysine 4) and H3K9me3 (trimethyl histone 3 lysine 9) Jumonji histone demethylases are markedly increased at the protein level and show enhanced enzymatic activity in diseased hearts. These epigenetic regulators continued to increase with time, further affecting cardiac gene expression. Our findings parallel the lower H3K4me3 and H3K9me3 levels seen in human patients. Inhibition of Jumonji demethylase activities in vivo results in lower histone demethylase enzymatic function in the heart and higher histone methylation levels and leads to partial reduction of heart size, reversal of maladaptive transcriptional programs, improved heart function, and prolonged survival. At the molecular level, target genes of transcription factor myocyte enhancer factor 2 are specifically regulated in response to pharmacological or genetic inhibition of Jumonji demethylases. Similar transcriptional reversal of disease-associated genes is seen in a second disease model based on cardiac mechanical overload. Our findings validate pharmacological inhibitors of Jumonji demethylases as potential therapeutics for the treatment of cardiomyopathies across disease models and provide evidence of the reversal of maladaptive transcriptional reprogramming leading to partial restoration of cardiac function. In addition, this study defines pathways of therapeutic resistance upregulated with disease progression.

JIB-04, a Pan-Inhibitor of Histone Demethylases, Targets Histone-Lysine-Demethylase-Dependent AKT Pathway, Leading to Cell Cycle Arrest and Inhibition of Cancer Stem-Like Cell Properties in Hepatocellular Carcinoma Cells.

JIB-04, a pan-histone lysine demethylase (KDM) inhibitor, targets drug-resistant cells, along with colorectal cancer stem cells (CSCs), which are crucial for cancer recurrence and metastasis. Despite the advances in CSC biology, the effect of JIB-04 on liver CSCs (LCSCs) and the malignancy of hepatocellular carcinoma (HCC) has not been elucidated yet. Here, we showed that JIB-04 targeted KDMs, leading to the growth inhibition and cell cycle arrest of HCC, and abolished the viability of LCSCs. JIB-04 significantly attenuated CSC tumorsphere formation, growth, relapse, migration, and invasion in vitro. Among KDMs, the deficiency of KDM4B, KDM4D, and KDM6B reduced the viability of the tumorspheres, suggesting their roles in the function of LCSCs. RNA sequencing revealed that JIB-04 affected various cancer-related pathways, especially the PI3K/AKT pathway, which is crucial for HCC malignancy and the maintenance of LCSCs. Our results revealed KDM6B-dependent AKT2 expression and the downregulation of E2F-regulated genes via JIB-04-induced inhibition of the AKT2/FOXO3a/p21/RB axis. A ChIP assay demonstrated JIB-04-induced reduction in H3K27me3 at the AKT2 promoter and the enrichment of KDM6B within this promoter. Overall, our results strongly suggest that the inhibitory effect of JIB-04 on HCC malignancy and the maintenance of LCSCs is mediated via targeting the KDM6B-AKT2 pathway, indicating the therapeutic potential of JIB-04.

Development of EphA2 siRNA-loaded lipid nanoparticles and combination with a small-molecule histone demethylase inhibitor in prostate cancer cells and tumor spheroids.

BACKGROUND: siRNAs hold a great potential for cancer therapy, however, poor stability in body fluids and low cellular uptake limit their use in the clinic. To enhance the bioavailability of siRNAs in tumors, novel, safe, and effective carriers are needed. RESULTS: Here, we developed cationic solid lipid nanoparticles (cSLNs) to carry siRNAs targeting EphA2 receptor tyrosine kinase (siEphA2), which is overexpressed in many solid tumors including prostate cancer. Using DDAB cationic lipid instead of DOTMA reduced nanoparticle size and enhanced both cellular uptake and gene silencing in prostate cancer cells. DDAB-cSLN showed better cellular uptake efficiency with similar silencing compared to commercial transfection reagent (Dharmafect 2). After verifying the efficacy of siEphA2-loaded nanoparticles, we further evaluated a potential combination with a histone lysine demethylase inhibitor, JIB-04. Silencing EphA2 by siEphA2-loaded DDAB-cSLN did not affect the viability (2D or 3D culture), migration, nor clonogenicity of PC-3 cells alone. However, upon co-administration with JIB-04, there was a decrease in cellular responses. Furthermore, JIB-04 decreased EphA2 expression, and thus, silencing by siEphA2-loaded nanoparticles was further increased with co-treatment. CONCLUSIONS: We have successfully developed a novel siRNA-loaded lipid nanoparticle for targeting EphA2. Moreover, preliminary results of the effects of JIB-04, alone and in combination with siEphA2, on prostate cancer cells and prostate cancer tumor spheroids were presented for the first time. Our delivery system provides high transfection efficiency and shows great promise for targeting other genes and cancer types in further in vitro and in vivo studies.

Targeting KDM4 family epigenetically triggers antitumour immunity via enhancing tumour-intrinsic innate sensing and immunogenicity.

Despite the remarkable clinical efficacy of cancer immunotherapy, considerable patients fail to benefit from it due to primary or acquired resistance. Tumours frequently hijack diverse epigenetic mechanisms to evade immune detection, thereby highlighting the potential for pharmacologically targeting epigenetic regulators to restore the impaired immunosurveillance and re-sensitise tumours to immunotherapy. Herein, we demonstrated that KDM4-targeting chemotherapeutic drug JIB-04, epigenetically triggered the tumour-intrinsic innate immune responses and immunogenic cell death (ICD), resulting in impressive antitumour effects. Specifically, JIB-04 induced H3K9 hypermethylation through specific inhibition of the KDM4 family (KDM4A-D), leading to impaired DNA repair signalling and subsequent DNA damage. As a result, JIB-04 not only activated the tumour-intrinsic cyclic GMP-AMP synthase (cGAS)-STING pathway via DNA-damage-induced cytosolic DNA accumulation, but also promoted ICD, releasing numerous damage-associated molecular patterns. Furthermore, JIB-04 induced adaptive resistance through the upregulation of programmed death-ligand 1 (PD-L1), which could be overcome with additional PD-L1 blockade. In human tumours, KDM4B expression was negatively correlated with clinical outcomes, type I interferon signatures, and responses to immunotherapy. In conclusion, our results demonstrate that targeting KDM4 family can activate tumour-intrinsic innate sensing and immunogenicity, and synergise with immunotherapy to improve antitumour outcomes.

Triple Combinations of Histone Lysine Demethylase Inhibitors with PARP1 Inhibitor-Olaparib and Cisplatin Lead to Enhanced Cytotoxic Effects in Head and Neck Cancer Cells.

PARP inhibitors are used to treat cancers with a deficient homologous recombination (HR) DNA repair pathway. Interestingly, recent studies revealed that HR repair could be pharmacologically impaired by the inhibition of histone lysine demethylases (KDM). Thus, we investigated whether KDM inhibitors could sensitize head and neck cancer cells, which are usually HR proficient, to PARP inhibition or cisplatin. Therefore, we explored the effects of double combinations of KDM4-6 inhibitors (ML324, CPI-455, GSK-J4, and JIB-04) with olaparib or cisplatin, or their triple combinations with both drugs, on the level of DNA damage and apoptosis. FaDu and SCC-040 cells were treated with individual compounds and their combinations, and cell viability, apoptosis, DNA damage, and gene expression were assessed using the resazurin assay, Annexin V staining, H2A.X activation, and qPCR, respectively. Combinations of KDM inhibitors with cisplatin enhanced cytotoxic effects, unlike combinations with olaparib. Triple combinations of KDM inhibitors with cisplatin and olaparib exhibited the best cytotoxic activity, which was associated with DNA damage accumulation and altered expression of genes associated with apoptosis induction and cell cycle arrest. In conclusion, triple combinations of KDM inhibitors (especially GSK-J4 and JIB-04) with cisplatin and olaparib represent a promising strategy for head and neck cancer treatment.

JIB-04 Has Broad-Spectrum Antiviral Activity and Inhibits SARS-CoV-2 Replication and Coronavirus Pathogenesis.

Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types, including primary human bronchial epithelial cells, against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens. IMPORTANCE The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, is an ongoing public health disaster worldwide. Although several vaccines are available as a preventive measure and the FDA approval of an orally bioavailable drug is on the horizon, there remains a need for developing antivirals against SARS-CoV-2 that could work on the early course of infection. By using infectious reporter viruses, we screened small-molecule inhibitors for antiviral activity against SARS-CoV-2. Among the top hits was JIB-04, a compound previously studied for its anticancer activity. Here, we showed that JIB-04 inhibits the replication of SARS-CoV-2 as well as different DNA and RNA viruses. Furthermore, JIB-04 conferred protection in a porcine model of coronavirus infection, although to a lesser extent when given as therapeutic rather than prophylactic doses. Our findings indicate a limited but still promising utility of JIB-04 as an antiviral agent in the combat against COVID-19 and potentially other viral diseases.

Jumonji Histone Demethylase Inhibitor JIB-04 as a Broad-Spectrum Antifungal Agent.

Invasive fungal infections are emerging as a global public health problem. The lack of effective antifungal drugs is the bottleneck of clinical antifungal treatment. To identify novel antifungal agents with new mechanisms of action, JIB-04, a Jumonji histone demethylase inhibitor, was identified to possess broad-spectrum antifungal activity by a cell-based screen. Particularly, JIB-04 effectively inhibited Jumonji demethylase activity and ergosterol biosynthesis of Cryptococcus neoformans cells, leading to in vitro and in vivo anti-Cryptococcus activity. It also significantly inhibited the virulence factors of C. neoformans including biofilm, melanin, capsule, and surface hydrophobicity. Thus, JIB-04 was validated as a potent antifungal agent for the treatment of cryptococcal meningitis and Jumonji histone demethylase was preliminarily identified as a potential target for the development of novel antifungal therapeutics.

JIB-04, a histone demethylase Jumonji C domain inhibitor, regulates phenotypic switching of vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotype switching is critical for neointima formation, which is the major cause of restenosis after stenting or coronary artery bypass grafting. However, the epigenetic mechanisms regulating phenotype switching of VSMCs, especially histone methylation, are not well understood. As a main component of histone lysine demethylases, Jumonji demethylases might be involved in VSMC phenotype switching and neointima formation. METHODS AND RESULTS: A mouse carotid injury model and VSMC proliferation model were constructed to investigate the relationship between histone methylation of H3K36 (downstream target molecule of Jumonji demethylase) and neointima formation. We found that the methylation levels of H3K36 negatively correlated with VSMC proliferation and neointima formation. Next, we revealed that JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) could increase the methylation levels of H3K36. Furthermore, we found that JIB-04 obviously inhibited HASMC proliferation, and a cell cycle assay showed that JIB-04 caused G2/M phase arrest in HASMCs by inhibiting the phosphorylation of RB and CDC2 and promoting the phosphorylation of CHK1. Moreover, JIB-04 inhibited the expression of MMP2 to suppress the migration of HASMCs and repressed the expression of contraction-related genes. RNA sequencing analysis showed that the biological processes associated with the cell cycle and autophagy were enriched by using Gene Ontology analysis after HASMCs were treated with JIB-04. Furthermore, we demonstrated that JIB-04 impairs autophagic flux by downregulating STX17 and RAB7 expression to inhibit the fusion of autophagosomes and lysosomes. CONCLUSION: JIB-04 suppresses the proliferation, migration, and contractile phenotype of HASMCs by inhibiting autophagic flux, which indicates that JIB-04 is a promising reagent for the treatment of neointima formation.

A UHPLC-MS/MS method for the quantification of JIB-04 in rat plasma: Development, validation and application to pharmacokinetics study.

Methylation of lysine by histone methyltransferases can be reversed by lysine demethylases (KDMs). Different KDMs have distinct oncogenic functions based on their cellular localization, stimulating cancer cell proliferation, reducing the expression of tumor suppressors, and/or promoting the development of drug resistance. JIB-04 is a small molecule that pan-selectively inhibits KDMs, showing maximal inhibitory activity against KDM5A, and as secondary targets, KDM4D/4B/4A/6B/4C. Recently, it was found that JIB-04 also potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines via the inhibition of a new target, serine hydroxymethyltransferase 2. Pharmacokinetic characterization and an analytical method for the quantification of JIB-04 are necessary for the further development of this small molecule. Herein, a sensitive, specific, fast and reliable UHPLC-MS/MS method for the quantification of JIB-04 in rat plasma samples was developed and fully validated using a SCIEX 6500+ triple QUAD LC-MS system equipped with an ExionLC UHPLC unit. The chromatographic separation was achieved on a reverse phase ACE Excel 2 Super C18 column with a flow rate of 0.5 mL/min under gradient elution. The calibration curves were linear (r2 > 0.999) over concentrations from 0.5 to 1000 ng/mL. The accuracy (RE%) was between -7.4% and 3.7%, and the precision (CV%) was 10.2% or less. The stability data showed that no significant degradation occurred under the experimental conditions. This method was successfully applied to the pharmacokinetic study of JIB-04 in rat plasma after intravenous and oral administration and the oral bioavailability of JIB-04 was found to be 44.4%.

Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases.

Little is known about the role of the three Jumonji C (JmjC) enzymes in Plasmodium falciparum (Pf). Here, we show that JIB-04 and other established inhibitors of mammalian JmjC histone demethylases kill asexual blood stage parasites and are even more potent at blocking gametocyte development and gamete formation. In late stage parasites, JIB-04 increased levels of trimethylated lysine residues on histones, suggesting the inhibition of P. falciparum Jumonji demethylase activity. These epigenetic defects coincide with deregulation of invasion, cell motor, and sexual development gene programs, including gene targets coregulated by the PfAP2-I transcription factor and chromatin-binding factor, PfBDP1. Mechanistically, we demonstrate that PfJmj3 converts 2-oxoglutarate to succinate in an iron-dependent manner consistent with mammalian Jumonji enzymes, and this catalytic activity is inhibited by JIB-04 and other Jumonji inhibitors. Our pharmacological studies of Jumonji activity in the malaria parasite provide evidence that inhibition of these enzymatic activities is detrimental to the parasite.

Targeting of Histone Demethylases KDM5A and KDM6B Inhibits the Proliferation of Temozolomide-Resistant Glioblastoma Cells.

Lysine histone demethylases (KDMs) are considered potential therapeutic targets in several tumors, including glioblastoma (GB). In particular, KDM5A is involved in the acquisition of temozolomide (TMZ) resistance in adult GB cells and UDX/KDM6B regulates H3K27 methylation, which is involved in the pediatric diffuse intrinsic pontine glioma (DIPG). Synthetic inhibitors of KDM5A (JIB 04 and CPI-455) efficiently block the proliferation of native and TMZ-resistant cells and the KDM6B inhibitor GSK J4 improves survival in a model of DIPG. The aim of our work was to determine if GSK J4 could be effective against GB cells that have acquired TMZ resistance and if it could synergize with TMZ or JIB 04 to increase the clinical utility of these molecules. Standard functional and pharmacological analytical procedures were utilized to determine the efficacy of the molecules under study when used alone or in combination against native GB cells and in a model of drug resistance. The results of this study indicated that although GSK J4 is active against native and TMZ-resistant cells, it does so at a lower efficacy than JIB 04. Drug combination studies revealed that GSK J4, differently from JIB 04, does not synergize with TMZ. Interestingly, GSK J4 and JIB 04 strongly synergize and are a potent combination against TMZ-resistant cells. Further studies in animal models will be necessary to determine if this combination of molecules might foster the development of novel therapeutic approaches for glioblastoma.

Jumonji Inhibitors Overcome Radioresistance in Cancer through Changes in H3K4 Methylation at Double-Strand Breaks.

We have uncovered a role for Jumonji inhibitors in overcoming radioresistance through KDM5B inhibition. Pharmacological blockade of Jumonji demethylases with JIB-04 leads to specific accumulation of H3K4me3 at sites marked by γH2AX and impaired recruitment of DNA repair factors, preventing resolution of damage and resulting in robust sensitization to radiation therapy. In DNA-repair-proficient cancer cells, knockdown of the H3K4me3 demethylase KDM5B, but not other Jumonji enzymes, mimics pharmacological inhibition, and KDM5B overexpression rescues this phenotype and increases radioresistance. The H3K4me3 demethylase inhibitor PBIT also sensitizes cancer cells to radiation, while an H3K27me3 demethylase inhibitor does not. In vivo co-administration of radiation with JIB-04 significantly prolongs the survival of mice with tumors even long after cessation of treatment. In human patients, lung squamous cell carcinomas highly expressing KDM5B respond poorly to radiation. Thus, we propose the use of Jumonji KDM inhibitors as potent radiosensitizers.

Taxane-Platin-Resistant Lung Cancers Co-develop Hypersensitivity to JumonjiC Demethylase Inhibitors.

Although non-small cell lung cancer (NSCLC) patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.