Methyltransferase Inhibitor Research Articles

C-di-GMP inhibits rRNA methylation and impairs ribosome assembly in the presence of kanamycin.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial secondary messenger with diverse functions. A previous Escherichia coli proteome microarray identified that c-di-GMP binds to the 23S rRNA methyltransferases RlmI and RlmE. Here we show that c-di-GMP inhibits RlmI activity in rRNA methylation assays, and that it modulates ribosome assembly in the presence of kanamycin. Molecular dynamics simulation and mutagenesis studies reveal that c-di-GMP binds to RlmI at residues R64, R103, G114, and K201. Structural simulations indicate that c-di-GMP quenches RlmI activity by inducing the closure of the catalytic pocket. We also show that c-di-GMP promotes antibiotic tolerance through RlmI. Binding and methylation assays indicate that the inhibitory effect of c-di-GMP on RlmI is conserved across various pathogenic bacteria. Our data suggest an unexpected role for c-di-GMP in regulating ribosome assembly under stress through the inhibition of rRNA methyltransferases.

Verticillin A-Loaded Surgical Buttresses Prevent Local Pancreatic Cancer Recurrence in a Murine Model.

The fungal metabolite verticillin A is a potent and selective histone methyltransferase inhibitor. It regulates apoptosis, the cell cycle, and stress response, and displays potent activity in the suppression of tumor cell growth in several different in vivo models. Verticillin A sensitizes pancreatic cancer cells to anti-PD-1 immunotherapy by regulating PD-L1 expression. However, as with many natural products, delivery and systemic toxicity are challenges that must be overcome to advance their use as a chemotherapeutic. To both reduce systemic toxicity and improve delivery, we report a verticillin A-loaded surgical buttress, which is well-tolerated at a dose as high as 40 mg/kg. In contrast, free verticillin A administered systemically results in toxicity at a dose of 3 mg/kg. The verticillin A-loaded buttress suppresses tumor recurrence in vivo in a safe and dose-dependent manner against a highly aggressive and metastatic model of pancreatic cancer.

Exploring Epigenetic Complexity in Regulation of Hematopoietic Stem Cells Niche: A Mechanistic Journey from Normal to Malignant Hematopoiesis.

Epigenetic regulation in hematopoietic stem cells (HSCs) research has emerged as a transformative molecular approach that enhances understanding of hematopoiesis and hematological disorders. This chapter investigates the intricate epigenetic mechanisms that control HSCs function, including deoxyribonucleic acid (DNA) methylation, histone modifications, and chromatin remodeling. It also explores the role of non-coding ribonucleic acid (RNAs) as epigenetic regulators, highlighting how changes in gene expression can occur without alterations to the DNA sequence. Epigenetic mechanisms play a pivotal in regulating HSC self-renewal and differentiation, processes essential for maintaining a balanced hematopoietic system in which lineage-specific hematopoietic stem and progenitor cells (HSPCs) pool is sustained. Recent advancements in epigenetic mapping and sequencing technologies have illuminated the dynamic epigenetic landscapes that characterize HSCs and their progeny. Numerous studies have revealed that dysregulation of epigenetic pathways is a hallmark of various hematological malignancies, including leukemias, lymphomas, and myelodysplastic syndromes. This review highlights key findings that demonstrate the impact of epigenetic abnormalities on the disruption of HSPC niches and the progression of oncogenesis in hematological malignancies. Furthermore, this chapter explores the therapeutic potential of targeting epigenetic modifications that are critical in formation and progression of hematologic malignancies. It also discusses the latest developments in epigenetic therapies, including the use of DNA methyltransferase inhibitors, histone deacetylase inhibitors, and emerging drugs targeting other epigenetic regulators. These therapies represent a promising strategy for resetting aberrant epigenetic states, potentially restoring normal hematopoiesis. Conclusively, this chapter offers a thorough overview of the current landscape and future directions of epigenetic research related to the maintenance of the HSPC niches. The insights presented here aim to contribute significantly to the field, offering a reference point for molecular approaches that enhance our understanding of hematopoiesis and its associated hematological malignancies.

Stochastic demethylation and redundant epigenetic suppressive mechanisms generate highly heterogeneous responses to pharmacological DNA methyltransferase inhibition

BackgroundDespite promising preclinical studies, the application of DNA methyltransferase inhibitors in treating patients with solid cancers has thus far produced only modest outcomes. The presence of intratumoral heterogeneity in response to DNA methyltransferase inhibitors could significantly influence clinical efficacy, yet our understanding of the single-cell response to these drugs in solid tumors remains very limited.MethodsIn this study, we used cancer/testis antigen genes as a model for methylation-dependent gene expression to examine the activity of DNA methyltransferase inhibitors and their potential synergistic effect with histone deacetylase inhibitors at the single-cancer cell level. The analysis was performed on breast cancer patient-derived xenograft tumors and cell lines, employing a comprehensive set of techniques, including targeted single-cell mRNA sequencing. Mechanistic insights were further gained through DNA methylation profiling and chromatin structure analysis.ResultsWe show that breast cancer tumors and cell cultures exhibit a highly heterogenous response to DNA methyltransferase inhibitors, persisting even under high drug concentrations and efficient DNA methyltransferase depletion. The observed variability in response to DNA methyltransferase inhibitors was independent of cancer-associated aberrations and clonal genetic diversity. Instead, these variations were attributed to stochastic demethylation of regulatory CpG sites and the DNA methylation-independent suppressive function of histone deacetylases.ConclusionsOur findings point to intratumoral heterogeneity as a limiting factor in the use of DNA methyltransferase inhibitors as single agents in treatment of solid cancers and highlight histone deacetylase inhibitors as essential partners to DNA methyltransferase inhibitors in the clinic.

Biomedical Effects of Protein Arginine Methyltransferase Inhibitors.

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of arginine residues in eukaryotic proteins, playing critical roles in modulating diverse cellular processes. The importance of PRMTs in the incidence and progression of a wide range of diseases, particularly cancers, such as breast, liver, lung, colorectal cancer, lymphoma, leukemia, and acute myeloid leukemia (AML) is increasingly recognized. This underscores the critical need for the development of effective PRMT inhibitors as therapeutic intervention. The field of PRMT inhibitors is in the rapidly growing phase and it is necessary to conduct a summative review of how the so-far developed inhibitors impact PRMT functions and cellular physiology. Our review aims to summarize molecular action mechanisms of these PRMT inhibitors and particularly elaborate their triggered biomedical effects. We delineate the cellular phenotype consequences of select PRMT inhibitors across various disease models, thereby providing an understanding of the pharmacological mechanisms underpinning PRMT inhibition. The promising effects of PRMT5 inhibitors in targeted therapy of MTAP-deleted cancers is particularly highlighted. At last, we provide a perspective on the challenges and further opportunities of developing and applying novel PRMT inhibitors for clinical advancement.

Low dose DNA methyltransferase inhibitors potentiate PARP inhibitors in homologous recombination repair deficient tumors

BackgroundPoly (ADP-Ribose) polymerase inhibitors are approved for treatment of tumors with BRCA1/2 and other homologous recombination repair (HRR) mutations. However, clinical responses are often not durable and treatment may be detrimental in advanced cancer due to excessive toxicities. Thus we are seeking alternative therapeutics to enhance PARP-directed outcomes. In an effort to expand the clinical use of PARP inhibitors to HRR proficient tumors, several groups have tested combinations of DNA methyltransferase inhibitors and PARP inhibitors. While this approach attenuated tumor cell proliferation in preclinical studies, subsequent clinical trials revealed little benefit. We hypothesized that benefit for this drug combination would only be specific to HRR deficient tumors, due to their inability to enact high fidelity DNA repair with subsequent cell death.MethodsWe generated hypomorphic BRCA1 and BRCA2 variants of the HRR proficient triple negative breast cancer cell line MDA-MB-231. We compared therapeutic response features such as RAD51 focus formation, cell cycle fraction alterations, DNA damage accumulation, colony formation, and cell death of these and other cell lines with and without intrinsic BRCA1/2 mutations. Results were confirmed in BRCA1/2 intact and deficient xenografts and PDX.ResultsOur targeted variants and cells with intrinsic BRCA1/2 mutations responded to low dose combination therapeutic treatment by G2M stalling, compounded DNA damage, severely attenuated colony formation, and importantly, increased cell death. In contrast, the parental MDA-MB-231 cells and other HRR proficient cell lines produced smaller cell populations with short term treatment, but with much less cumulative DNA damage, and minimal cell death. In animal studies, our BRCA-engineered hypomorphs and several independent PDX models with clinically relevant BRCA mutations were acutely more vulnerable to this drug combination.ConclusionsWe conclude that low dose DNA methyltransferase inhibition can cooperate with low dose PARP inhibition to increase DNA damage predominantly in cells with HRR deficiencies, ultimately producing more cell death than in HRR proficient tumors. We predict that clinical benefit will more likely be apparent in patients with DNA repair defective tumors and are focusing clinical exploration of this drug combination in these patients, with the goals of enhancing tumor cell death at minimal toxicities.

ZNFX1 functions as a master regulator of epigenetically induced pathogen mimicry and inflammasome signaling in cancer.

DNA methyltransferase and poly (ADP-ribose) polymerase inhibitors (DNMTis, PARPis) induce a stimulator of interferon genes (STING)-dependent pathogen mimicry response (PMR) in ovarian and other cancers. Here, we showed that combining DNMTis and PARPis upregulates expression of the nucleic-acid sensor NFX1-type zinc finger-containing 1 protein (ZNFX1). ZNFX1 mediated induction of PMR in mitochondria, serving as a gateway for STING-dependent interferon/inflammasome signaling. Loss of ZNFX1 in ovarian cancer cells promoted proliferation and spheroid formation in vitro and tumor growth in vivo. In patient ovarian cancer databases, expression of ZNFX1 was elevated in advanced stage disease, and ZNFX1 expression alone significantly correlated with an increase in overall survival in a phase 3 trial for therapy-resistant ovarian cancer patients receiving bevacizumab in combination with chemotherapy. RNA-sequencing revealed an association between inflammasome signaling through ZNFX1 and abnormal vasculogenesis. Together, this study identified that ZNFX1 as a tumor suppressor that controls PMR signaling through mitochondria and may serve as a biomarker to facilitate personalized therapy in ovarian cancer patients.

A High-Throughput Screening Pipeline to Identify Methyltransferase and Exonuclease Inhibitors of SARS-CoV-2 NSP14.

SARS-CoV-2 infections led to a worldwide pandemic in 2020. As of 2024, therapeutics against SARS-CoV-2 have continued to be desirable. NSP14 is a dual-function methyltransferase (MTase) and exonuclease (ExoN) with key roles in SARS-CoV-2 genome propagation and host immune system evasion. In this work, we developed high-throughput screening (HTS) assays for NSP14 MTase and ExoN activities. We screened both activities against a collection of 40,664 compounds. A total of 1677 initial hit compounds were identified, cherrypicked, counterscreened for assay interference, and screened for off-target selectivity. We identified 396 and 174 high-quality hits against the MTase and ExoN activities, respectively. Along with inhibitors for individual activities, we identified dual-activity inhibitors, including a novel inhibitor that is not competitive with any substrate and interacts with a putative allosteric binding site. This study represents the largest published screen of SARS-CoV-2 NSP14 MTase and ExoN activities to date and culminates in a pipeline for the NSP14 drug discovery.

Proteomic Characterization of Liver Cancer Cells Treated with Clinical Targeted Drugs for Hepatocellular Carcinoma.

Background/Objectives: Hepatocellular carcinoma (HCC) remains a significant global health concern, primarily due to the limited efficacy of targeted therapies, which are often compromised by drug resistance and adverse side effects. Methods: In this study, we utilized a Tandem Mass Tag (TMT)-based quantitative proteomic approach to analyze global protein expression and serine/threonine/tyrosine (S/T/Y) phosphorylation modifications in HepG2 cells following treatment with three clinically relevant hepatocellular carcinoma-targeted agents: apatinib, regorafenib, and lenvatinib. Results: Utilizing KEGG pathway enrichment analysis, biological process enrichment analysis, and protein interaction network analysis, we elucidated the common and specific metabolic pathways, biological processes, and protein interaction regulatory networks influenced by three liver cancer therapeutics. The study additionally proposed potential combinational treatment strategies, highlighting a possible synergistic interaction between HCC-targeted drugs and the DNA methyltransferase inhibitor. Furthermore, through the integration of clinical phosphorylation site data, we identified several phosphorylation sites that exhibited higher abundance in tumor tissues compared to adjacent non-tumor tissues. These sites were associated with poor prognosis and elevated functional scores. Conclusions: In summary, this study conducted an in-depth analysis of the molecular alterations in proteins and phosphorylation modifications induced by clinical HCC-targeted drugs, predicting drug combination strategies and therapeutic targets.

Testing the potential of zebularine to induce heritable changes in crop growth and development

Key messageZebularine-treated wheat uncovered a phenotype with characteristics of an epigenetically regulated trait, but major chromosomal aberrations, not DNA methylation changes, are the cause, making zebularine unsuitable for epigenetic breeding.Breeding to identify disease-resistant and climate-tolerant high-yielding wheats has led to yield increases over many years, but new hardy, higher yielding varieties are still needed to improve food security in the face of climate change. Traditional breeding to develop new cultivars of wheat is a lengthy process taking more than seven years from the initial cross to cultivar release. The speed of breeding can be enhanced by using modern technologies including high-throughput phenomics, genomic selection, and directed mutation via CRISPR. Here we test the concept of modifying gene regulation by transiently disrupting DNA methylation with the methyltransferase inhibitor, zebularine (Zeb), as a means to uncover novel phenotypes in an elite cultivar to facilitate breeding for epigenetically controlled traits. The development and architecture of the wheat inflorescence, including spikelet density, are an important component of yield, and both grain size and number have been extensively modified during domestication and breeding of wheat cultivars. We identified several Zeb-treated plants with a dominant mutation that increased spikelet density compared to the untreated controls. Our analysis showed that in addition to causing loss of DNA methylation, Zeb treatment resulted in major chromosomal abnormalities, including trisomy and the formation of a novel telocentric chromosome. We provide evidence that increased copy number of the domestication gene, Q, is the most likely cause of increased spikelet density in two Zeb-treated plants. Collateral damage to chromosomes in Zeb-treated plants suggests that this is not a viable approach to epigenetic breeding.

Emergent properties of the lysine methylome reveal regulatory roles via protein interactions and histone mimicry.

Epigenomics has significantly advanced through the incorporation of Systems Biology approaches. This study aims to investigate the human lysine methylome as a system, using a data-science approach to reveal its emergent properties, particularly focusing on histone mimicry and the broader implications of lysine methylation across the proteome. We employed a data-science-driven OMICS approach, leveraging high-dimensional proteomic data to study the lysine methylome. The analysis focused on identifying sequence-based recognition motifs of lysine methyltransferases and evaluating the prevalence and distribution of lysine methylation across the human proteome. Our analysis revealed that lysine methylation impacts 15% of the known proteome, with a notable bias toward mono-methylation. We identified sequence-based recognition motifs of 13 lysine methyltransferases, highlighting candidates for histone mimicry. These findings suggest that the selective inhibition of individual lysine methyltransferases could have systemic effects rather than merely targeting histone methylation. The lysine methylome has significant mechanistic value and should be considered in the design and testing of therapeutic strategies, particularly in precision oncology. The study underscores the importance of considering non-histone proteins involved in DNA damage and repair, cell signaling, metabolism, and cell cycle pathways when targeting lysine methyltransferases.

Azacitidine and cytarabine induce sustained lymphopenia with abnormal differentiation of common lymphoid progenitors and prolonged suppression of Dnmt3a and Dnmt3b expression in mice.

Myelosuppression is a major side effect of chemotherapy. Although decreased blood cells are restored with the recovery of bone marrow cells, insufficient recovery of decreased lymphocytes was observed in mice given azacitidine (AZA), a DNA methyltransferase (DNMT) inhibitor, even following the restoration of bone marrow cells. To understand the mechanisms behind this sustained lymphopenia, we examined AZA's impact on the hematopoietic progenitor cells and the expression of Dnmts and differentiation-related genes. An antimetabolite of cytidine analog, cytarabine (Ara-C), was used as a reference compound. Decreases in almost all blood parameters and common lymphoid progenitors (CLPs) and the downregulation of Dnmts and differentiation-related genes in Lineage-Sca-1+c-kit+ (LSK) cells were observed in mice administered AZA or Ara-C for 7 d. In the posttreatment observation, all parameters, except for lymphocytes and monocytes, exhibited recovery within 3 wk after the final dosing in both treated groups. However, no recovery from the decreases in lymphocytes, especially B cells, and monocytes occurred even after 5 wk. The number of CLPs was elevated after 3 wk. There was a tendency toward recovery from the decreased expression of Dnmt1 and differentiation-related genes, but the expression levels of Dnmt3a and Dnmt3b did not fully recover even 5 wk after the final dosing. Taken together, the findings revealed that the mechanism of sustained lymphopenia observed in mice treated with AZA or Ara-C is associated, at least in part, with the abnormal differentiation of CLPs into B cells accompanied by the prolonged suppression of Dnmt3a and Dnmt3b expression on LSK cells.

Effect of decitabine on PD-L2 methylation in whole blood of iodine-induced autoimmune thyroiditis rats.

Excessive iodine intake can induce autoimmune thyroiditis (AIT), and the methylation of programmed death receptor 2 (PD-L2) may be involved in the development of iodine-induced AIT. Here, we investigated the immune role of methylation of the susceptibility gene PD-L2 in the occurrence of iodine-induced AIT using the DNA methyltransferase inhibitor Decitabine (Dec) in an experimental autoimmune thyroiditis (EAT) rat model. After injecting Dec intraperitoneally into EAT rats, we performed arsenic-cerium catalytic spectrophotometry, pathological hematoxylin and eosin staining, enzyme-linked immunosorbent assay, quantitative methylation-specific polymerase chain reaction (qMSP), and quantitative real-time polymerase chain reaction (qPCR) to determine the relevant indices. The results showed that compared with the control group, the urinary iodine, thyroid lymphocyte infiltration, thyroglobulin antibody (TgAb), interferon (IFN-γ), and interleukin (IL-23) levels of the EAT rats were significantly increased. The PD-L2 methylation levels were significantly decreased in EAT rats compared to control rats, and the mRNA expression of the PD-L2 was significantly increased. Following Dec intervention, the methylation level of the PD-L2 in rats increased and interferon and interleukin-23 levels decreased, albeit not significantly. However, the mRNA expression of PD-L2 decreased significantly after Dec intervention, and the thyroid function of EAT rats also showed a gradual improvement trend. In summary, hypomethylation of PD-L2 is closely related to the development of iodine-induced AIT. Pro-inflammatory cellular factors are also involved in iodine-induced AIT progression. Although Dec shows promise in the treatment of AIT, further evaluation of its safety is necessary.

The translational potential of epigenetic modulatory bioactive phytochemicals as adjuvant therapy against cancer.

In preclinical studies, bioactive phytochemicals have shown enormous potential therapeutic efficacy against various human malignancies. These natural compounds have been shown to possess an inherent potential to alter the molecular signaling pathways and epigenetic modulatory activity involved in multiple physiological functions. Recently, epigenetic therapy has emerged as an important therapeutic modality due to the reversible nature of epigenetic alterations. To date, epigenetic modulatory compounds, for example, DNA methyltransferase inhibitors 5-azacytidine and 5'-deoxyazacytidine, as well as histone deacetylase inhibitors Vorinostat, Romidepsin, and Belinostat (PXD101), have been clinically approved by the FDA for the treatment of patients of leukemia and myelodysplastic syndrome. However, these synthetic epigenetic inhibitors are not as effective against many of the solid tumors. Therefore, the epigenetic modulatory phytochemicals provide new hope for improving the treatment modality as neoadjuvant and adjuvant therapy. It has been established that targeting more than one protein in the transformed cells simultaneously, that is, the multi-targeted therapeutic approach, might invoke a better therapeutic response. Therefore, here, we are compiling diverse evidences of the translational potential of novel combinatorial approaches utilizing the epigenetic modulatory phytochemicals with available therapeutics in the course of cancer treatment.

Icariin Facilitates Osteogenic Differentiation and Suppresses Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Enhancing SOST Methylation in Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMO) is mainly concerned with the imbalance of bone resorption and bone formation. Icariin (ICA) plays a vital role in bone protection. This study investigated the mechanism of ICA in PMO rats. The rats were treated with ovariectomy (OVX) and ICA. Bone structure parameters were measured by Micro-CT. BMSCs were obtained from normal rats, OVX rats, and ICA-treated rats. BMSCs were infected with SOST overexpression lentivirus, and TWS119, an activator of Wnt pathway, was introduced for joint experiment. The binding of ERα to SOST promoter was verified. OVX/ICA rats were injected with DNA methyltransferase inhibitor 5-Aza-dC. ICA increased bone mass and decreased bone marrow fat content in OVX rats. ICA facilitated osteogenic differentiation and repressed adipogenic differentiation of BMSCs. Overexpressing SOST antagonized the effect of ICA, whereas TWS119 rescued the effect of overexpressing SOST. ICA reduced SOST expression by attenuating the effect of ERα. Methylation of SOST inhibited ERα binding to SOST promoter. Invivo experiments confirmed that ICA improved bone mass and reduced bone marrow fat content by enhancing SOST methylation. Overall, ICA upregulated SOST methylation and inhibited the binding of ERα to SOST promoter, thereby promoting osteogenic differentiation and repressing adipogenic differentiation of BMSCs.

A metal-phenolic nanotuner induces cancer pyroptosis for sono-immunotherapy.

Although ultrasound therapy is efficacious and safe in clinical oncology, its capacity to elicit an anti-tumor immune response is constrained by ultrasound-induced apoptosis. Pyroptosis, which releases immunogenic damage-associated molecular patterns (DAMPs), can significantly enhance immune activation. It necessitates robust Gasdermin E (GSDME) expression in cancer cells for caspase-3-mediated pyroptosis. An epigenetic strategy is introduced to induce cancer pyroptosis during sonotherapy using a nanocoordinator (HTA) constructed through metal-phenolic coordination involving Aza (a DNA methyltransferase inhibitor), TiO2 nanoparticles, and polyphenol-modified hyaluronic acid. While Aza restores GSDME expression, TiO2 generates reactive oxygen species (ROS) under ultrasound stimulation, activating caspase-3 and inducing pyroptosis via GSDME cleavage. In an orthotopic breast cancer model, HTA enhanced anti-tumor immunity and improved the efficacy of sonodynamic therapy (SDT). This approach presents a novel strategy for augmenting SDT through epigenetically induced pyroptosis.

Epigenetic therapies in cancer treatment: Opportunities and challenges

Epigenetic therapies offer a revolutionary approach to cancer treatment by targeting reversible modifications such as DNA methylation, histone modifications, and non-coding RNAs that regulate gene expression. FDA-approved drugs like DNA methyltransferase inhibitors and histone deacetylase inhibitors have demonstrated clinical efficacy, particularly in hematologic malignancies, while emerging agents like BET and EZH2 inhibitors show promise in addressing therapeutic gaps in solid tumors. This review delves into these advancements, highlighting the integration of epigenetic drugs with immunotherapy, chemotherapy, and targeted therapies to enhance therapeutic precision and overcome resistance. Moreover, it emphasizes opportunities such as biomarker-driven personalization and liquid biopsy technologies for real-time monitoring, alongside innovations like single-cell epigenomics and CRISPR-based tools that facilitate tumor heterogeneity studies and precise therapeutic interventions. Despite these developments, challenges such as off-target effects, delivery inefficiencies, and regulatory hurdles persist, necessitating further innovation. By integrating epigenetic therapies with RNA-based drugs, nanomedicine, personalized vaccines, 3D epigenomics, and artificial intelligence, this review underscores the transformative potential of these therapies in redefining oncology, offering personalized, durable, and precise solutions to the complexities of cancer.

Phenomics‐based Discovery of Novel Orthosteric Choline Kinase Inhibitors

Choline kinase alpha (CHKA) is a central mediator of cell metabolism linked to cancer and immune regulation. Cellular and clinical evaluation of CHKA has been hampered by challenges in the development of drug‐like choline kinase inhibitors. Here, we identify CHKA as an unexpected off‐target of histone methyltransferase inhibitors using an integrated phenomic approach. We confirm CHKA as a direct protein target of the aminoquinazolines UNC0638 and UNC0737 using a combination of chemoproteomic, biochemical, cellular, and metabolic profiling assays, possibly explaining the previously reported discrepancies observed for different G9a/GLP inhibitor scaffolds in cellular assays. Using primary human cell model systems, we discover that CHKA modulation impairs IgG secretion and B‐cell maturation consistent with the notion that choline metabolism plays an important role in immune signalling. Co‐crystal structures of UNC0638 and UNC0737 with CHKA unravel an unexpected binding mode and suggest the inhibitors as attractive starting points for the development of selective chemical tools to further explore the biological role of CHKA in cancer and immune metabolism.

Phenomics-Based Discovery of Novel Orthosteric Choline Kinase Inhibitors.

Choline kinase alpha (CHKA) is a central mediator of cell metabolism linked to cancer and immune regulation. Cellular and clinical evaluation of CHKA has been hampered by challenges in the development of drug-like choline kinase inhibitors. Here, we identify CHKA as an unexpected off-target of histone methyltransferase inhibitors using an integrated phenomic approach. We confirm CHKA as a direct protein target of the aminoquinazolines UNC0638 and UNC0737 using a combination of chemoproteomic, biochemical, cellular, and metabolic profiling assays, possibly explaining the previously reported discrepancies observed for different G9a/GLP inhibitor scaffolds in cellular assays. Using primary human cell model systems, we discover that CHKA modulation impairs IgG secretion and B-cell maturation consistent with the notion that choline metabolism plays an important role in immune signalling. Co-crystal structures of UNC0638 and UNC0737 with CHKA unravel an unexpected binding mode and suggest the inhibitors as attractive starting points for the development of selective chemical tools to further explore the biological role of CHKA in cancer and immune metabolism.

Enhancing HBV-specific T cell responses through a combination of epigenetic modulation and immune checkpoint inhibition.

Chronic HBV infection (CHB) exhausts HBV-specific T cells, develops epigenetic imprints that impair immune responses, and limits the effectiveness of immune checkpoint inhibitor (ICI) monotherapy, such as αPD-L1. This study aimed to determine whether the DNA methyltransferase inhibitor decitabine (DAC) could reverse these epigenetic imprints and enhance ICI efficacy in restoring HBV-specific T cell responses. We investigated HBV-specific T cell responses by 10-day in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with CHB. PBMCs were stimulated with HBV core-specific overlapping peptide pools and HLA-A*02-restricted peptides, core18 and pol455. The immunomodulatory effect of the DAC/αPD-L1 combination was assessed by flow cytometry, and our analysis included clinical characteristics, ex vivo DNA methylation of PBMCs, and IFNγ plasma levels. Treatment with DAC/αPD-L1 enhanced HBV-specific CD4+ T cell responses in a significant proportion of 53 patients, albeit with some variability. This effect was independent of the HBcrAg levels. Ex vivo DNA methylation revealed hypermethylation of key genes, such as IFNG among DAC-responders versus non-responders, supported by altered ex vivo IFNγ plasma levels. Further analysis of HBV-specific CD8+ T cell responses in 22 HLA-A*02-positive patients indicated distinct response patterns between core18 and pol455 stimulation, with pol455-specific CD8+ T cells showing increased susceptibility to DAC/αPD-L1, surpassing the αPD-L1 monotherapy response. The combination of DAC/αPD-L1 shows promise in improving HBV-specific T cell responses in vitro, highlighting the potential of remodeling exhaustion-associated epigenetic signatures to enhance HBV-specific T cell restoration and suggesting a novel immunotherapeutic avenue for CHB.