Targeting epithelial-mesenchymal transition and apoptosis: novel histone methyltransferase inhibitors for colon cancer suppression.

Bushen Zhuyun Recipe improves endometrial receptivity through kruppel-like factor 4-mediated recruitment of mixed-lineage leukemia 1 to activate homeobox A10 transcription.

Gene expression and alternative splicing are observed to shift in different ways after binding of naringenin chalcone and HRH2 in 293T cells.

Abstract Aberrant epigenetic regulation contributes to tumor progression and immune evasion. Euchromatic histone-lysine N-methyl transferase 2 (EHMT2; G9a), a key mediator of H3K9 methylation–dependent transcriptional repressor, is frequently overexpressed in multiple cancers and has emerged as a promising epigenetic target. However, the pharmacological properties of EHMT2 inhibitors remain insufficiently characterized. Here, we compared the cellular exposure, pharmacokinetics, and antitumor and immunomodulatory efficacies of two widely used EHMT2 inhibitors, BIX-01294 (BIX) and UNC0642 (UNC). Although UNC exhibited stronger EHMT2 inhibition in enzymatic assays, it was less effective in reducing the viability of colorectal and pancreatic cancer cell lines. Western blot analysis confirmed that BIX induced H3K9me2 reduction, p62 degradation, and LC3-II accumulation more effectively than UNC in MIA PaCa-2 cells. Moreover, BIX exhibited consistently higher intracellular levels than UNC, with about 3–4-fold differences observed across concentrations (1 or 4 μM) and time points (2, 6, or 16 h). These results indicate that enhanced cellular uptake or retention contributes to the superior cellular efficacy of BIX, despite its lower EHMT2 inhibitory effect. In vivo pharmacokinetic studies in ICR mice demonstrated that BIX achieved approximately two-fold higher systemic exposure than UNC following intravenous administration. Both compounds exhibited poor oral bioavailability, consistent with their low Caco-2 permeability. In contrast, intraperitoneal (i.p.) administration resulted in the highest systemic exposure, with BIX achieving an area under the concentration-time curve extrapolated to infinity of 115,662.0 ± 7,872.7 ng·h/mL, compared with 58,106.3 ± 8,018.9 ng·h/mL for UNC. Accordingly, the i.p. route was selected for efficacy studies. In the MIA PaCa-2 xenograft model, BIX was administered at 20 and 40 mg/kg, and UNC was administered at 5 and 8 mg/kg. For UNC, further dose escalation was limited by its toxicity. Under these conditions, BIX showed greater tumor growth inhibition than did UNC. Therefore, BIX was used in an immuno-oncology combination study at 30 mg/kg to mitigate weight loss observed at 40 mg/kg. In the MC38 syngeneic model, combination treatment with BIX (30 mg/kg) and α–PD-L1 antibody (10 mg/kg) produced significantly greater tumor growth inhibition than either monotherapy. Consistently, immunofluorescence analysis of tumor tissues demonstrated increased infiltration of CD8α+ cytotoxic T cells and NK1.1+ natural killer cells in the combination treatment group. Collectively, these findings demonstrate that cellular exposure and systemic pharmacokinetics are key determinants of EHMT2 inhibitor–mediated antitumor and immune-enhancing effects. BIX showed superior in vitro and in vivo efficacy and functioned as a promising partner for immune checkpoint blockade. Nevertheless, dose-limiting toxicity and suboptimal pharmacokinetics highlight the need for additional preclinical evaluations and optimization of its pharmacokinetic properties. Citation Format: Sang Eun Park, Ji-Yoon Lee, Unju Lee, Seoyeong Kim, Soo Jin Oh, Jung-Jin Hwang. Impact of pharmacokinetic profiles on the in vivo antitumor efficacy of EHMT2 inhibitors combined with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-A006.

Disruptor of telomeric silencing 1-like (DOT1L), as the sole histone methyltransferase (HMT) for lysine 79 on histone H3 (H3K79), has become a promising therapeutic target for various diseases. DOT1L plays a critical role in the self-renewal of hematopoietic stem cells, B cell differentiation, and neural development. In acute leukemia (AML) carrying MLL rearrangements (MLL-r), this enzyme is aberrantly recruited to oncogene promoters, leading to excessive methylation of H3K79 and persistent activation of pro-proliferative transcription programs such as HOXA9. In addition, abnormal expression, mutations, protein interactions, and nonenzymatic functions of DOT1L play important roles in various diseases. Modulators targeting DOT1L have been developed for more than a decade, and compounds such as competitive inhibitors, protein-protein interaction (PPI) inhibitors, and degraders block DOT1L's pathogenic functions through various pharmacological mechanisms. Challenges and opportunities are discussed in this perspective, aiming to provide valuable insights for the future design of DOT1L modulators.

Histone methyltransferase EZH2 drives podocyte injury and senescence in diabetic nephropathy through STAT3 activation.

MORF4L1 regulation and its role in chromatin remodeling, DNA damage, cellular senescence, and cardiometabolic disease.

Neuroblastoma (NB) is a highly malignant pediatric extracranial tumor with genetic and epigenetic factors influencing its pathogenesis. Key genetic contributors include germline mutations in ALK and PHOX2B, as well as somatic amplification of MYCN. The epigenetic regulator EZH2, a histone methyltransferase of the PRC2 complex, is overexpressed in NB and linked to poor prognosis in advanced cases. Targeting EZH2 offers a promising therapeutic approach due to its role in tumor proliferation, survival, and maintenance of undifferentiated states. This study explores the use of acetylated human serum albumin nanoparticles (HSANPs) loaded with 4O4HPR for epigenetic therapy in NB, tested in vitro and in vivo using nude mice xenograft models. The nanoparticles enter cells via clathrin-mediated endocytosis and induce G2-M cell cycle arrest, mitochondrial depolarization, and the production of reactive oxygen species, which activate Caspase 3 and trigger apoptosis. Mechanistic studies show increased p53 acetylation, stabilizing p53 and inhibiting EZH2's epigenetic silencing. Chromatin Immunoprecipitation reveals the disruption of EZH2 binding to the E-Cadherin promoter, which suppresses cell migration and EMT-like transition. Western blotting confirms upregulation of epithelial and downregulation of mesenchymal markers, with inhibited wound closure in SH-SY5Y cells. This nanotherapy disrupts EZH2-E-Cadherin interaction, offering novel translational potential for NB treatment.

KMT2D is a critical regulator of chromatin accessibility and transcriptional landscapes in castration-resistant prostate cancer that drives both AR-dependent and AR-independent subtypes, highlighting KMT2D as a potential therapeutic target.

Alcohol use disorder (AUD) is a chronic, relapsing disease that is difficult to treat, and current therapies are not effective for many patients. Clinical studies show that stress increases heavy alcohol consumption and that stress is a predominant factor in rates of relapse. As such, pharmacotherapies that reduce stress-related alcohol drinking might be beneficial clinically. We recently showed that viral vector-mediated reduction of the epigenetic enzyme G9a (also known as euchromatic histone-lysine N-methyltransferase 2 or EHMT2) in the nucleus accumbens blocks stress-potentiated alcohol drinking. In addition, repeated systemic administration of the G9a inhibitor UNC0642 also reduces stress-potentiated alcohol drinking, suggesting that G9a inhibitors might be effective for treating AUD. Here, we further investigated the potential for UNC0642 to be used clinically to treat AUD. We first tested the effective dose of UNC0642 in multiple models of alcohol consumption in both male and female mice. We also tested half-life, oral bioavailability, maximum tolerated dose, and many measures of behavioral toxicity. We found that UNC0642 is effective at reducing stress-potentiated alcohol consumption in both male and female mice at lower doses than we previously reported, and we re-confirmed the anxiolytic-like effects of UNC0642. We also found that UNC0642 has a long half-life invivo, is orally bioavailable, is within an acceptable toxicity range for therapeutic use, and does not produce behavioral inhibition in many standard preclinical assessments, including locomotor activity, open field, Y-maze, rotarod, fear conditioning, sucrose self-administration, or sucrose-seeking. Together, our studies suggest that G9a inhibitors could be effective, safe, and novel AUD therapeutics that reduce stress and dependence-related high levels of alcohol drinking in AUD patients.

Set7 regulates ferroptosis in pulmonary arterial hypertension endothelial cells through the DPP4/NOX4 axis mediated by H3K4me1 modification.

Maternal carbohydrate-programming enhances carbohydrate utilization in zebrafish offspring by histone H3K9 methylation.

Histone methyltransferases SMYD2: the potential pharmacological target on cancers and aging-related diseases treatment.

Endometrial cancer (EC) incidence is increasing globally, highlighting the need for novel therapies targeting molecular drivers of malignancy. Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase implicated in tumor progression, is overexpressed in EC; however, its precise role and therapeutic potential remain unclear. In this study, we aimed to investigate EZH2 expression, its functional role, and the efficacy of EZH2 inhibitors in EC cell lines. EZH2 expression was analyzed in eight EC cell lines using western blotting and immunocytochemistry. The efficacy of five EZH2 inhibitors (CPI-1205, EI1, EPZ005687, EPZ-6438, and GSK126) was evaluated using drug sensitivity assays. Furthermore, functional analyses, including cell proliferation, colony formation, migration, and invasion assays, were performed following siRNA-mediated EZH2 knockdown in HEC-50B cells. Variable EZH2 expression was observed across EC cell lines, with high levels in HEC-50B and Ishikawa 3-H-12 cells. EZH2-high expressing cell lines were markedly more sensitive to EZH2 inhibitors, particularly GSK126, compared to EZH2-low expressing lines. EZH2 knockdown in HEC-50B cells reduced EZH2 expression and decreased sensitivity to EZH2 inhibitors, confirming target specificity, while also attenuating cell proliferation, colony formation, migration, and invasion. EZH2 plays a crucial role in promoting malignant phenotypes in EC, and its expression level correlates with cellular sensitivity to EZH2 inhibitors. These findings suggest that EZH2 could serve as a valuable therapeutic target and predictive biomarker for personalized medicine in EC.

The dysfunctional reconstitution of the intestinal barrier is pivotal in driving the initiation of inflammatory pathogenesis in Crohn's disease (CD), although the exact pathophysiology underlying this phenomenon has yet to be definitively characterised. This study aimed to investigate the role of the histone methyltransferase mixed lineage leukaemia 1 (MLL1) in the development of CD-like colitis and to elucidate the mechanism by which MLL1 promotes epithelial cell differentiation. Colonic tissue specimens from CD patients and TNBS-induced murine models were analysed to assess MLL1 expression dynamics. The functional impact of MLL1 on murine colitis modelling CD was systematically investigated through clinical symptom scoring, histopathological profiling and quantitative evaluation of intestinal barrier integrity. The role of MLL1 in promoting epithelial cell differentiation and repairing the intestinal barrier was investigated through immunofluorescence and western blotting. Additionally, potential mechanisms underlying the reparative effects of MLL1 on intestinal barrier function were explored. MLL1 expression was upregulated in colonic tissues from CD patients and TNBS-induced murine colitis models. In contrast, MLL1 suppression in the TNBS cohort attenuated mucosal inflammation and downregulated pro-inflammatory cytokine production (IL-1β, IL-6, TNF-α) within the colonic mucosa. Additionally, reduced MLL1 expression increased the differentiation capacity of intestinal epithelial cells, including goblet cells, absorptive cells and tuft cells, and promoted barrier function restoration in injured colons and lipopolysaccharide-stimulated colonic organoids. MLL1 downregulation activated the Gata4/Bmp4 signalling pathway, which may contribute to the reparative effects of MLL1 on intestinal barrier integrity. Downregulating MLL1 expression promotes intestinal epithelial cell differentiation by activating the Gata4/Bmp4 pathway. These findings elucidate a pathophysiological mechanism wherein MLL1 suppression potentiates intestinal barrier restoration, thereby attenuating colitis severity in murine models. The observed therapeutic efficacy positions MLL1 inhibition presents a novel strategy for CD management.

Histone variants along with their associated chaperones have been considered as one of the major complexes to provide versatility in organizing chromatin structure. Post-translational modifications (PTMs) of H3 variants serve as very important factors in promoting heterochromatin assembly, protecting telomere stability, and suppressing transposon activity. However, the precise mechanism by which specific PTMs on H3 variants suppress transposons remains elusive. Here, by monitoring retrotransposon mobilization during Drosophila hindgut development, we identified the DNA synthesis-coupled (DSC) H3.2K9me2 deposition pathway as a pivotal mechanism for transposon suppression. Depleting the factors in the DSC H3.2 complex, but not in the DNA synthesis-independent (DSI) H3.3 chaperone pathway, unleashed massive retrotransposon activation. DSC chaperones specifically establish dimethylation at the H3.2K9 site in heterochromatic regions by directly interacting with and recruiting the histone methyltransferase, G9a. Intriguingly, the cross-talk between DSC H3.2K9me2 and DSI H3.3K9me3 in heterochromatin is dynamically regulated and properly balanced. Although DSI H3.3K9me3 could efficiently be incorporated into transposon loci when the DSC H3.2K9me2 deposition pathway was disrupted, H3.3K9me3 alone was insufficient to establish functional heterochromatin required for transposon silencing during development. Altogether, our discoveries provide a framework to understand how cells employ specific histone variant modifications to construct and maintain heterochromatin, thereby ensuring transposon repression and safeguarding genome integrity.

The histone methyltransferase SMYD3 (SET and MYND domain containing 3) is critical for vascular homeostasis and may be implicated in pathological angiogenesis. However, its mechanism remains elusive, and targeted inhibitors are in early-stage development. We aim to clarify whether SMYD3 regulates angiogenesis and develop novel molecules targeting SMYD3. Expression profile of SMYD3 under pro-angiogenic conditions was characterized by bioinformatics analysis and endothelial cell (EC) validation. The effects of SMYD3 knockdown, knockdown-overexpression, and overexpression on angiogenesis were validated, including Matrigel neovascularization, aortic ring sprouting, and EC tube formation. Regarding molecule development, virtual screening, structural modifications, molecular docking, inhibition screening of EC proliferation, and SMYD3 enzyme activity and cellular thermal shift assays were employed. A novel molecule ZYZ329 was identified and evaluated in angiogenesis. Mechanistic studies involved genetic approaches and the mitochondrial reactive oxygen species (mROS) scavenger MitoQ (Mitoquinone mesylate). Finally, a murine hindlimb ischemia model and rat skin healing model were established to evaluate ZYZ329 on pathological and physiological angiogenesis. SMYD3 was upregulated in endothelial cells under ischemic, hypoxic, and VEGF-stimulated conditions. And SMYD3 gene knockdown, overexpression after knockdown, and overexpression regulated the angiogenesis capacity of endothelial cells. After virtual screening and structural modifications, the novel molecule ZYZ329 was developed for dual inhibition of EC proliferation (IC50 = 6.147 μM) and SMYD3 enzymatic function (IC50 = 0.419 μM). The ZYZ329 engaged SMYD3 in cells, and exhibited a certain degree of selectivity for SMYD3. Moreover, ZYZ329 significantly attenuated pathological angiogenesis in a dose-dependent manner. Mechanistically, genetic or pharmacological inhibition of SMYD3 impaired HIF-1α stabilization and downstream VEGFA production. SMYD3 genetic perturbation modulated mROS without affecting apoptosis in hypoxic endothelial cell. MitoQ intervention confirmed that moderate SMYD3-driven mROS elevation regulates the HIF-1α/VEGFA axis in angiogenesis. In hindlimb ischemia model, ZYZ329 significantly impaired blood flow recovery and post-ischemic angiogenesis, demonstrating superior efficacy to EPZ031686. However, ZYZ329 had no significant effect on physiological angiogenesis. SMYD3 drives angiogenesis partially via the mROS/HIF-1α/VEGFA axis. We developed a novel molecule ZYZ329, which potently suppresses pathological angiogenesis. This offers potential therapeutic target and lead structures for treating angiogenesis-related diseases.

Histone lysine methylation regulates the expressions of mitochondrial function-related genes, which presents a "nucleus-to-mitochondria" signal communication, playing a key role in aging control. However, the underlying mechanisms remain elusive due to the complexity of histone lysine methylation in transcription modulation. In this study, using C. elegans and mouse C2C12 cell-differentiated myotubes as research models, we found that histone H3K36me2 methyltransferase SET-18/SMYD2 were responsible for the increase of mitochondrial reactive oxygen species (mtROS) accumulation during aging. Mechanistically, SET-18/SMYD2-mediated H3K36me2 modification upregulated the expression of NADase tir-1 isoform d (tir-1d)/sarm1 to decrease NAD+ level. Consequently, mtROS level was elevated, which resulted in shortened worm lifespan as well as accelerated mouse myotubes atrophy (a hallmark of muscle aging). These findings proposed that mtROS generation is actively regulated other than passively accumulated in aging process, and revealed a "H3K36me2-NADase-mtROS" signaling axis of "nucleus-to-mitochondria" communication to modulate aging, which is conserved from C. elegans to mammals.

Aspergillus flavus, along with its notorious secondary metabolite aflatoxin B1 (AFB1), seriously endangers human health. Histone methyltransferase complex COMPASS (complex of proteins associated with Set1) plays a crucial role in regulating aflatoxin biosynthesis and virulence of A. flavus, but the underlying mechanism is unclear. Here, we find that Bre2, the key subunit of COMPASS, regulates AFB1 biosynthesis, fungal morphogenesis, and virulence through modulation of H3K4 methylation. ChIP-seq and biochemical analyses reveal that chromatin remodeling factor (CRF) Arp9 is directly targeted by Bre2, and Arp9 exerts bio-functions through interacting with the other CRFs such as RSC8, Arp7, and Sth1. ATAC-seq results indicate that Arp9 contributes to fungal pathogenicity by modulating chromatin conformation of genes that are involved in secondary metabolism, morphogenesis, and virulence. The study reveals an epigenetic signaling pathway mediated by chromatin remodeler Arp9 and provides a potential strategy for the control of pathogenic fungi and mycotoxins.

Histone lysine methyltransferases such as SETD1B regulate chromatin structure and gene transcription. Ketone bodies, including butyrate, act as histone deacetylase inhibitors. We report a 4-year-old boy with SETD1B-related absence epilepsy, refractory to conventional medications, who achieved sustained > 90% seizure reduction on the Modified Atkins ketogenic diet. Single-cell RNA sequencing of 25,159 peripheral mononuclear cells across 3 samples: baseline, 3 months on-diet and age-matched control, revealed widespread dysregulation of the patient's chromatin, ribosomal, immune and mitochondrial pathways at baseline, which were reversed with ketogenic therapy. These findings suggest that the ketogenic diet can improve gene regulation in chromatin-mediated brain disorders.