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Lactate dehydrogenase (LDH) catalyzes the reversible interconversion of pyruvate and lactate, coupled with the redox cycling of NADH and NAD+. While LDHA has been extensively studied as a therapeutic target, particularly in cancer, due to its role in the Warburg effect, LDHB remains underexplored, despite its involvement in the metabolic reprogramming of specific cancer types, including breast and lung cancers. Most known LDH inhibitors are designed against the LDHA isoform and act competitively at the active site. In contrast, LDHB exhibits distinct kinetic properties, substrate preferences, and structural features, warranting isoform-specific screening strategies. In this study, 115 natural compounds previously reported as LDHA inhibitors were systematically evaluated for LDHB inhibition using an integrated in silico and in vitro approach. Virtual screening identified 16 lead phytochemicals, among which luteolin and quercetin exhibited uncompetitive inhibition of LDHB, as demonstrated by enzyme kinetic assays. These findings were strongly supported by molecular docking analyses, which revealed that both compounds bind at an allosteric site located at the dimer interface, closely resembling the binding mode of the established LDHB uncompetitive inhibitor AXKO-0046. In contrast, comparative docking against LDHA confirmed their active-site binding and competitive inhibition, underscoring their isoform-specific behavior. Our findings highlight the necessity of assay conditions tailored to LDHB’s physiological role and demonstrate the application of a previously validated colorimetric assay for high-throughput screening. This work lays the foundation for the rational design of selective LDHB inhibitors from natural product libraries.

The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The ‘persistent’ CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein ( KLK ) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture. The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is regulated remains poorly understood. Here the authors find that there is a shared subset of CTCF-bound sites resistant to protein depletion in different cell lines, which are enriched at domain boundaries and chromatin loops constitutive to all cell types.

DNA replication timing is known to facilitate the establishment of the epigenome, however, the intimate connection between replication timing and changes to the genome and epigenome in cancer remain largely uncharacterised. Here, we perform Repli-Seq and integrated epigenome analyses and demonstrate that genomic regions that undergo long-range epigenetic deregulation in prostate cancer also show concordant differences in replication timing. A subset of altered replication timing domains are conserved across cancers from different tissue origins. Notably, late-replicating regions in cancer cells display a loss of DNA methylation, and a switch in heterochromatin features from H3K9me3-marked constitutive to H3K27me3-marked facultative heterochromatin. Finally, analysis of 214 prostate and 35 breast cancer genomes reveal that late-replicating regions are prone to cis and early-replication to trans chromosomal rearrangements. Together, our data suggests that the nature of chromosomal rearrangement in cancer is related to the spatial and temporal positioning and altered epigenetic states of early-replicating compared to late-replicating loci. The connection between DNA replication timing and changes that occur to the epigenome in cancer are still poorly understood. Here, the authors perform Repli-Seq and integrated epigenome analyses and find that genomic regions that undergo long-range epigenetic deregulation in prostate cancer also show concordant differences in replication timing.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer–promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer–promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth. Here, the authors provide mechanistic insights into how decitabine-induced DNA hypomethylation can potentially overcome endocrine resistance in ER+ breast cancer, by targeting the 3D epigenome to resolve gene deregulation and suppress tumor growth.

Introduction Androgen receptor (AR)-mediated transcription drives prostate cancer progression and remains a key therapeutic target. AR is activated by androgens, translocating to the nucleus to bind DNA regulatory elements (AREs) and initiate transcription. This process involves coregulatory proteins, pioneer factors like FOXA1, epigenetic modifications, and 3D chromatin interactions. However, the temporal coordination of AR and co-regulatory binding factors, epigenomic modifications, and 3D chromatin dynamics during AR-mediated transcription remains unclear. Results Using a time-course model of androgen stimulation in LNCaP prostate cancer cells, we integrated temporal multi-omics datasets into the MOFA+ framework to investigate AR-mediated transcriptional dynamics. Our analyses uncovered a critical role for rapid AR binding at gene promoters in driving nascent transcription of AR target genes. We demonstrated that AR binding is preceded by nucleosome H3K27 acetylation flanking androgen response elements, which are constitutively marked by FOXA1 binding. Notably, we identified a temporal early switch in nascent transcription post-androgen stimulation, from MYC to AR target genes, likely mediated by transient androgen-induced 3D chromatin interactions. Significance These findings reveal that androgen stimulation rapidly reorganizes 3D chromatin structure and differential MYC and AR transcription factor binding to trigger three distinct patterns of temporal epigenome states associated with the establishment of AR-mediated transcription in prostate cancer. These insights advance understanding of AR regulation in prostate cancer and contribute to a deeper understanding of the temporal relationship between MYC and AR transcriptional regulation in hormone-driven cancers.Competing Interest StatementThe authors have declared no competing interest.

Three-dimensional (3D) epigenome remodelling is an important mechanism of gene deregulation in cancer. However, its potential as a target to overcome therapy resistance remains largely unaddressed. Here we show that FDA-approved epigenetic therapy Decitabine (5-Aza-mC) suppresses tumour growth in preclinical metastatic ER+ breast tumour xenograft models. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher order chromatin structure and loss of topologically associated domain boundary insulation. Significant DNA hypomethylation at ER-enhancer elements was associated with gain in ER binding, creation of ectopic 3D enhancer-promoter interactions and concordant activation of ER-mediated transcription pathways. Importantly long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates how epigenetic therapy has potential to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions associated with suppression of tumour growth.

Replication timing is known to facilitate the establishment of epigenome, however, the intimate connection between DNA replication timing and changes to the genome and epigenome in cancer remain uncharted. Here, we perform Repli-Seq and integrated epigenome analysis and show that early-replicating loci are predisposed to hypermethylation and late-replicating loci to hypomethylation, enrichment of H3K27me3 and concomitant loss of H3K9me3. We find that altered replication timing domains correspond to long-range epigenetically deregulated regions in prostate cancer, and a subset of these domains are remarkably conserved across cancers from different tissue origins. Analyses of 214 prostate and 35 breast cancer genomes reveal that late-replicating DNA is prone to cis and early- replicating DNA to trans chromosomal rearrangements. We propose that differences in epigenetic deregulation related to spatial and temporal positioning between early and late replication potentiate the landscape of chromosomal rearrangements in cancer.

Background The Horvath epigenetic clock is widely used. It predicts age quite well from 353 CpG sites in the DNA methylation profile in unknown samples and has been used to calculate “age acceleration” in various tissues and environments. Results The model systematically underestimates age in tissues from older people. This is seen in all examined tissues but most strongly in the cerebellum and is consistently observed in multiple datasets. Age acceleration is thus age-dependent, and this can lead to spurious associations. The current literature includes examples of association tests with age acceleration calculated in a wide variety of ways. Conclusions The concept of an epigenetic clock is compelling, but caution should be taken in interpreting associations with age acceleration. Association tests of age acceleration should include age as a covariate.

In a new report, Benjamin Alman and his colleagues find that the morphogenic pathway activated by Hedgehog signaling is a key mediator of osteoarthritis, a condition that is marked by irreversible degeneration of the joints and with no current treatment. They also found that blockade of Hedgehog signaling prevented osteoarthritis in a mouse model, suggesting this pathway as a possible target to treat this devastating disease. Osteoarthritis is associated with the irreversible degeneration of articular cartilage. Notably, in this condition, articular cartilage chondrocytes undergo phenotypic and gene expression changes that are reminiscent of their end-stage differentiation in the growth plate during skeletal development 1 , 2 . Hedgehog (Hh) signaling regulates normal chondrocyte growth and differentiation 3 , 4 , 5 , 6 , 7 , 8 ; however, the role of Hh signaling in chondrocytes in osteoarthritis is unknown. Here we examine human osteoarthritic samples and mice in which osteoarthritis was surgically induced and find that Hh signaling is activated in osteoarthritis. Using several genetically modified mice, we found that higher levels of Hh signaling in chondrocytes cause a more severe osteoarthritic phenotype. Furthermore, we show in mice and in human cartilage explants that pharmacological or genetic inhibition of Hh signaling reduces the severity of osteoarthritis and that runt-related transcription factor-2 (RUNX2) potentially mediates this process by regulating a disintegrin and metalloproteinase with thrombospondin type 1 motif-5 (ADAMTS5) expression. Together, these findings raise the possibility that Hh blockade can be used as a therapeutic approach to inhibit articular cartilage degeneration.

Eosinophilic gastritis (EG) is a chronic inflammatory disease of the stomach characterized by eosinophil-predominant gastric mucosal inflammation and gastrointestinal symptoms. The aim of this study was to prospectively evaluate endoscopic features in a large series of children and adults with EG to better understand the endoscopic manifestations and develop a standardized instrument for investigations. Data were prospectively collected as part of the Consortium for Eosinophilic Gastrointestinal Disease Researchers, a national collaborative network. Endoscopic features were prospectively recorded using a system specifically developed for EG, the EG Endoscopic Reference System (EG-REFS). Correlations were made between EG-REFS and clinical and histologic features. Of 98 patients with EG, 65 underwent assessments using EG-REFS. The most common findings were erythema (72%), raised lesions (49%), erosions (46%), and granularity (35%); only 8% of patients with active histology (≥30 eosinophils/high-power field) exhibited no endoscopic findings. A strong correlation between EG-REFS scores and physician global assessment of endoscopy severity was demonstrated (Spearman r = 0.84, P < 0.0001). The overall score and specific components of EG-REFS were more common in the antrum than in the fundus or body. EG-REFS severity was significantly correlated with active histology, defined by a threshold of ≥30 eosinophils/high-power field (P = 0.0002). Prospective application of EG-REFS identified gastric features with a strong correlation with physician global assessment of endoscopic activity in EG. Endoscopic features demonstrated greater severity in patients with active histology and a predilection for the gastric antrum. Further development of EG-REFS should improve its utility in clinical studies.