Twenty years ago, histone lysine demethylases (KDMs) were discovered. Since their discovery, they have been increasingly studied and shown to be important across species, development, and diseases. Considerable advances have been made toward understanding their (1) enzymology, (2) role as critical components of biological complexes, (3) role in normal cellular processes and functions, (4) implications in pathological conditions, and (5) therapeutic potential. This Review covers these key relationships related to the KDM field with the awareness that numerous laboratories have contributed to this field. The current knowledge coupled with future insights will shape our understanding about cell function, development, and disease onset and progression, which will allow for novel biomarkers to be identified and for optimal therapeutic options to be developed for KDM-related diseases in the years ahead.

Identifying the molecules that control neural stem cell (NSC) fate would revolutionize treatment strategies for neurodegenerative diseases. Histone lysine demethylase 1A (KDM1A) demethylates the mono- and di-methyl groups of histone 3 lysine 4 (H3K4) and H3K9 and plays an essential role in NSC proliferation. In this study, we investigated the effects of Seclidemstat (SP-2577), a reversible KDM1A inhibitor, and tranylcypromine (TCP), a monoamine oxidase inhibitor and recently known as an irreversible histone lysine demethylase 1A inhibitor, on NSCs. SP-2577 and TCP increased glial fibrillary acidic protein expression (GFAP), decreased βIII-tubulin (TUBB3) expression, and phosphorylated signal transducer and activator of transcription 3 (STAT3) in rat NSCs. SP-2577 and TCP enhanced the transcription of Gfap and reduced Tubb3 transcription. Furthermore, SP-2577 increased the transcription levels of interleukin-6 and leukemia inhibitory factor, while TCP induced the transcription level of fibroblast growth factor 2. Therefore, we show that the KDM1A inhibitors, SP-2577 and TCP, induce astrocytogenesis in rat NSCs. These findings suggest that KDM1A is a target for regulating NSCs fate and provide insights into the molecular mechanisms underlying neurodevelopmental processes and epigenetics.

Acute myeloid leukemia (AML) is a highly aggressive hematological malignancy with a significant unmet clinical need for new therapeutic agents. Lysine-specific demethylase 1 (LSD1), a key regulator of leukemia stem cell self-renewal, has emerged as a promising epigenetic target for AML treatment. Herein, we employed an innovative multi-step integrated screening protocol, encompassing pharmacophore modeling, docking screening, molecular dynamics simulation, and biological evaluation, to identify novel LSD1 inhibitors. This comprehensive approach led to the discovery of six potent LSD1 inhibitors (we named these inhibitors LTMs 1-6), with LTM-1 exhibiting the most pronounced inhibitory effects on LSD1 (IC50 = 2.11 ± 0.14 nM) and the highest selectivity for LSD1 over LSD2 (>2370-fold). Notably, LTM-1 demonstrated outstanding antitumor activity both in vitro and in vivo. In vitro, LTM-1 showed potent anti-proliferative effects against LSD1-addicted MV-4-11 leukemia cells (IC50 = 0.16 ± 0.01 μM). In vivo, LTM-1 treatment significantly reduced tumor growth in MV-4-11 xenografted mice. Moreover, LTM-1 did not induce significant changes in liver and kidney function indices, suggesting a favorable safety profile. These results indicate that LTM-1 is a highly promising preclinical candidate for AML treatment, offering a new strategy for the development of more effective and selective LSD1 inhibitors.

Histone methyltransferases and demethylases play crucial roles in gene regulation and are vital for proper functioning of multiple tissues. Lysine-specific histone demethylase 1A (Kdm1a), is responsible for the demethylation of specific lysines, namely K4 and K9, on histone H3. In this study, we investigated the functions of Kdm1a during mouse kidney development upon targeted deletion in renal progenitor cells. Loss of Kdm1a in Six2-positive nephron progenitors resulted in significant reduction in renal mass, tissue structural changes and impaired function. To further understand the molecular function of Kdm1a during kidney development, we conducted multi-omics analyses that included transcriptome profiling, Chromatin immunoprecipitation (ChIP) sequencing, and methylome assessments. These omic analyses identified Kdm1a as a critical gene regulator required for sustained expression of several nephron segment marker genes, as well as vast number of solute carrier (Slc) genes and a few imprinted genes. Absence of Kdm1a in kidneys led to an increase in global H3K9 methylation peaks, which correlated with the transcriptional downregulation of numerous genes. Among these were markers of nephron progenitors and presumptive tubular precursors. We also observed that specific gene bodies exhibited altered DNA methylation patterns at intragenic differentially methylated regions (DMRs) upon Kdm1a deletion, while the overall global levels of DNA methylation remained unchanged. Our data point to a key regulatory role for Kdm1a in the renal progenitor epigenome, influencing kidney specific gene expression in the developing nephrons. Together the study highlights an indispensable role for Kdm1a for proper development of mouse kidneys, and its absence leading to significant developmental and functional impairment.

Members of the DNA methyltransferase (DNMT) family have been recognized as major epigenetic regulators of altered gene expression during tumor development. They establish and maintain DNA methylation of the CpG island of promoter and non-CpG region of the genome. The abnormal methylation status of tumor suppressor genes (TSGs) has been associated with tumorigenesis, leading to genomic instability, improper gene silence, and immune evasion. DNMT1 helps preserve methylation patterns during DNA replication, whereas the DNMT3 family is responsible for de novo methylation, creating new methylation patterns. Altered DNA methylation significantly supports tumor growth by changing gene expression patterns. FDA-approved DNMT inhibitors reverse hypermethylation-induced gene repression and improve therapeutic outcomes for cancer. Recent studies indicate that combining DNMT inhibitors with chemotherapies and immunotherapies can have synergistic effects, especially in aggressive metastatic tumors. Improving the treatment schedules, increasing isoform specificity, reducing toxicity, and utilizing genome-wide analyses of CRISPR-based editing to create personalized epigenetic therapies tailored to individual patient needs are promising strategies for enhancing therapeutic outcomes. This review discusses the interaction between DNMT regulators and DNMT1, its binding partners, the connection between DNA methylation and tumors, how these processes contribute to tumor development, and DNMT inhibitors' advancements and pharmacological properties.

In the germ line and during early embryogenesis, DNA methylation (DNAme) undergoes global erasure and re-establishment to support germ cell and embryonic development. While DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin are unknown. Through conditional gene deletion of the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b, we observe that DNMT3A primarily safeguards against DNA hypomethylation in undifferentiated spermatogonia, while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm. To assess the impact of altered sperm chromatin in formatting embryonic chromatin, we measure H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a transposon-based tagging approach. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.

Post-translational modifications (PTMs) are implicated in many biological processes including receptor activation, signal transduction, transcriptional regulation and protein turnover. Lysine's side chain is particularly notable, as it can undergo methylation, acetylation, SUMOylation and ubiquitination. Methylation affects not only lysine but also arginine residues, both of which are implicated in epigenetic regulation. Beyond histone-tails as substrates, dynamic methylation of transcription factors has been described. The focus of this review is on these non-histone substrates providing a detailed discussion of what is currently known about methylation of hypoxia-inducible factor (HIF), P53, nuclear receptors (NRs) and RELA. The role of methylation in regulating protein stability and function by acting as docking sites for methyl-reader proteins and via their crosstalk with other PTMs is explored.

Epigenetic intervention has become an important therapeutic strategy for a variety of diseases, such as cancer. Although a small number of epigenetic drugs have been marketed, most of these inhibitors are limited by their poor efficacy, dose-dependent toxicity, poor selectivity, and drug resistance. The development of covalent inhibitors has progressed from questioning to resurgence. Its slow dissociation is expected to inject new vitality into epigenetic drugs. In this review, more than 40 covalent inhibitors of 29 epigenetic targets were collated, focusing on their design strategies, reaction mechanisms, covalent warheads and targeted amino acids, and covalent verification methods. Furthermore, this review presented new opportunities based on the current development of covalent inhibitors targeting epigenetic regulators. It is believed that epigenetic covalent inhibitors will lead to more breakthroughs.

Lysine-specific demethylase 1 (LSD1/KDM1A) is a pivotal epigenetic enzyme that contributes to several malignancies including malignant glioma. LSD1 is a flavin adenine dinucleotide dependent histone demethylase that specifically targets histone H3 lysine (K) 4 mono- (me1) and di-methylation (me2) and H3K9me1/2 for demethylation. Herein we report the development of an LSD inhibitor, S2172, which efficiently penetrates the blood-brain barrier. S2172 effectively suppresses LSD1 enzymatic activity, resulting in the depletion of cell growth both in vitro in glioma stem cells (GSCs) (mean half-maximal inhibitory concentration (IC50) of 13.8 μM) and in vivo in a GSC orthotopic xenograft mouse model. Treatment with S2172 robustly reduced the expression of the stemness-related genes MYC and Nestin in GSC cells. Consistent with this, chromatin immunoprecipitation-sequencing revealed a significant S2172-dependent alteration in H3K4me2/H3K4me3 status. Furthermore, we identified 284 newly acquired H3K4me2 peak regions after S2172 treatment, which were encompassed within super-enhancer regions. The altered H3K4me2/H3K4me3 status induced by S2172 treatment affected the expression of genes related to tumorigenesis. Our data suggest that targeting LSD1 with S2172 could provide a promising treatment option for glioblastomas, particularly due to targeting of GSC populations.

The dual inhibition of histone lysine-specific demethylase 1 (LSD1) and histone deacetylase (HDAC) has emerged as a promising strategy for cancer therapy. In this study, we report the discovery of novel 5-cyano-3-phenylindole-based LSD1/HDAC dual inhibitors, evaluated through both in vitro and in vivo assays. Among these inhibitors, compound 20c was identified as particularly potent, exhibiting high inhibitory activity against LSD1 (IC50 = 39.0 nM) and HDAC1/2/3/6/8 (IC50 = 1.4, 1.0, 1.3, 2.9, and 16.0 nM, respectively). Compound 20c effectively modulated the expression of biomarkers associated with LSD1 and HDAC inhibition and demonstrated superior antiproliferative activity compared to SAHA and 4SC-202 across multiple colorectal cancer cell lines. Following pharmacokinetic studies, 20c was further assessed in HCT-116 and HT-29 xenograft mouse models. It demonstrated significantly enhanced antitumor efficacy compared to SAHA, without causing observable toxicity. These findings highlight the potential of LSD1/HDAC dual inhibitors for the treatment of malignant cancers.

The action of Corepressor for Element Silencing Transcription Factor 1 (CoREST) is primarily related to neural fate decisions. However, the molecular mechanisms linking neuroinflammation to the histone modifying complex remain unclear. CoREST is a hub for several cofactors that play important roles in epigenetic remodeling and transcriptional regulation. It allows us to question their functions during the inflammatory response in the Central Nervous System. The impact of LPS-induced neuroinflammation on the transcriptional epigenetic control of members with CoRest repressive complex in the hippocampus was investigated. Characterizing the basal transcriptional profile in the hippocampus of members with CoREST repressive complex showed that the Rcor3 is the most expressed gene, and the Rcor2 is the least expressed one. It was also demonstrated that the levels of Lsd1, CoREST1 (Rcor1), and CoREST2 (Rcor2) transcripts increased in the hippocampus after LPS i.p. administration, while for CoREST3 (Rcor3), no significant difference was observed. A significant increase was noticed in the percentages of the 5-meC mark (Hypermethylation) for the Rcor1 and Lsd1 genes with a positive Pearson correlation between methylation and expression. However, the correlation was directly proportional, ruling out DNA methylation as the main mechanism in transcriptional control.

Gene therapy has shown significant potential in treating various diseases, particularly inherited blood disorders such as hemophilia, sickle cell disease, and thalassemia. Advances in understanding the regulatory network of disease-associated genes have led to the identification of additional therapeutic targets for treatment, especially for β-hemoglobinopathies. Erythroid regulatory factor BCL11A offers the most promising therapeutic target for β-hemoglobinopathies, and reduction of its expression using the commercialized gene therapy product Casgevy has been approved for use in the UK and USA in 2023. Notably, the emergence of innovative gene editing technologies has further broadened the gene therapy landscape, presenting possibilities for treatment. Intensive studies indicate that base editing and prime editing, built upon CRISPR technology, enable precise single-base modification in hematopoietic stem cells for addressing inherited blood disorders ex vivo and in vivo. In this review, we present an overview of the current landscape of gene therapies, focusing on clinical research and gene therapy products for inherited blood disorders, evaluation of potential gene targets, and the gene editing tools employed in current gene therapy practices, which provides an insight for the establishment of safer and more effective gene therapy methods for a wider range of diseases in the future.

The development of chicken ovarian follicles involves two key stages of primordial follicle recruitment and follicle selection that are tightly regulated by multiple reproductive hormones and cytokines. Our previous study revealed an estrogen-stimulated increase in the phosphorylation level of serine at position 54 of lysine demethylase 1A (LSD1Ser54p) in the theca cells of chicken hierarchical ovarian follicles (Post-TCs). In this study, we further found that the upregulation of LSD1Ser54p by estrogen was performed by glycogen synthase kinase 3 beta (GSK3β) and that GSK3β promoted LSD1Ser54p levels by directly binding to the SWIRM and AOL1 domains of LSD1. Upon estrogen stimulation, the phosphorylation level of tyrosine at position 216 of GSK3β (GSK3βTyr216p) increased, which enhanced the binding between LSD1 and GSK3β. The subsequent transcriptome sequencing on chicken Post-TCs treated with estrogen and CUT&RUN sequencing against the LSD1Ser54p protein revealed that the expression of the farnesyl-diphosphate farnesyltransferase 1 (FDFT1) gene was simultaneously upregulated by estrogen, GSK3β, and LSD1Ser54p. Moreover, the overexpression of FDFT1 further promoted cholesterol biosynthesis in chicken Post-TCs. In short, the findings of this study suggest that estrogen-induced tyrosine phosphorylation at position 216 of GSK3β can upregulate the level of LSD1Ser54p, leading to the activation of FDFT1 expression and subsequently promoting cholesterol biosynthesis in chicken Post-TCs, which may in turn enhance estrogen synthesis.

The translocation t(14;18) activates BCL2 and is considered the initiating genetic lesion in most follicular lymphomas (FL). Surprisingly, FL patients fail to respond to the BCL2 inhibitor, Venetoclax. We show that mutations and deletions affecting the histone lysine methyltransferase SETD1B (KMT2G) occur in 7% of FLs and 16% of diffuse large B cell lymphomas (DLBCL). Deficiency in SETD1B confers striking resistance to Venetoclax and an experimental MCL-1 inhibitor. SETD1B also acts as a tumor suppressor and cooperates with the loss of KMT2D in lymphoma development in vivo. Consistently, loss of SETD1B in human lymphomas typically coincides with loss of KMT2D. Mechanistically, SETD1B is required for the expression of several proapoptotic BCL2 family proteins. Conversely, inhibitors of the KDM5 histone H3K4 demethylases restore BIM and BIK expression and synergize with Venetoclax in SETD1B-deficient lymphomas. These results establish SETD1B as an epigenetic regulator of cell death and reveal a pharmacological strategy to augment Venetoclax sensitivity in lymphoma.

LSD1 (histone lysine-specific demethylase 1) has been gradually disclosed to act as an immunomodulator to enhance antitumor immune response. Despite the identification of numerous potent LSD1 inhibitors, there remains a lack of LSD1 inhibitors approved for marketing. Novel LSD1 inhibitors with different mechanisms are therefore needed. Herein, we reported a series of novel quinazoline-based LSD1 inhibitors. Among them, compound Z-1 exhibited the best LSD1 inhibitory activity (IC50 = 0.108 μM). Z-1 also acted as a selective and cellular active as an LSD1 inhibitor. Furthermore, Z-1 promoted response of gastric cancer cells to T-cell killing effect by decreasing PD-L1 expression and further attenuated the PD-1/PD-L1 interaction. In vivo, Z-1 exhibited significant suppression effect on the growth of gastric cancer cells without obvious toxicity. Therefore, Z-1 represents a potential novel immunomodulator that targets LSD1, providing a lead compound with new function mechanism for gastric cancer treatment.

Gastric cancer (GC) poses a significant global health challenge, ranking fifth in incidence and third in mortality among all malignancies worldwide. Its insidious onset, aggressive growth, proclivity for metastasis, and limited treatment options have contributed to its high fatality rate. Traditional approaches for GC treatment primarily involve surgery and chemotherapy. However, there is growing interest in targeted therapies and immunotherapies. This comprehensive review highlights recent advancements in GC targeted therapy and immunotherapy. It delves into the mechanisms of various strategies, underscoring their potential in GC treatment. Additionally, the review evaluates the efficacy and safety of relevant clinical trials. Despite the benefits observed in numerous advanced GC patients with targeted therapies and immunotherapies, challenges persist. We discuss pertinent strategies to overcome these challenges, thereby providing a solid foundation for enhancing the clinical effectiveness of targeted therapies and immunotherapies.

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

The primary reason for high mortality rates among cancer patients is metastasis, where tumor cells migrate through the bloodstream from the original site to other parts of the body. Recent advancements in technology have significantly enhanced our comprehension of the mechanisms behind the bloodborne spread of circulating tumor cells (CTCs). One critical process, DNA methylation, regulates gene expression and chromosome stability, thus maintaining dynamic equilibrium in the body. Global hypomethylation and locus-specific hypermethylation are examples of changes in DNA methylation patterns that are pivotal to carcinogenesis. This comprehensive review first provides an overview of the various processes that contribute to the formation of CTCs, including epithelial-mesenchymal transition (EMT), immune surveillance, and colonization. We then conduct an in-depth analysis of how modifications in DNA methylation within CTCs impact each of these critical stages during CTC dissemination. Furthermore, we explored potential clinical implications of changes in DNA methylation in CTCs for patients with cancer. By understanding these epigenetic modifications, we can gain insights into the metastatic process and identify new biomarkers for early detection, prognosis, and targeted therapies. This review aims to bridge the gap between basic research and clinical application, highlighting the significance of DNA methylation in the context of cancer metastasis and offering new avenues for improving patient outcomes.

Trophoblasts play a crucial role in embryo implantation and in interacting with the maternal uterus. The trophoblast lineage develops into a substantial part of the placenta, a temporary extra-embryonic organ, capable of undergoing distinctive epigenetic events during development. The critical role of trophoblast-specific epigenetic signatures in regulating placental development has become known, significantly advancing our understanding of trophoblast identity and lineage development. Scientific efforts are revealing how trophoblast-specific epigenetic signatures mediate stage-specific gene regulatory programming during the development of the trophoblast lineage. These epigenetic signatures have a significant impact on blastocyst formation, placental development, as well as the growth and survival of embryos and fetuses. In evolution, DNA hypomethylation in the trophoblast lineage is conserved, and there is a significant disparity in the control of epigenetic dynamics and the landscape of genomic imprinting. Scientists have used murine and human multipotent trophoblast cells as in vitro models to recapitulate the essential epigenetic processes of placental development. Here, we review the epigenetic signatures of the trophoblast lineage and their biological functions to enhance our understanding of placental evolution, development, and function.

Lysine-specific demethylase 1 (LSD1), a highly sophisticated epigenetic regulator, orchestrates a range of critical cellular processes, holding promising therapeutic potential for treating diverse diseases. However, the clinical research progress targeting LSD1 is very slow. After 20 years of research, only one small-molecule drug, BEA-17, targeting the degradation of LSD1 and CoREST has been approved by the U.S. Food and Drug Administration. The primary reason for this may be the lack of abundant structural data regarding its intricate functions. To gain a deeper understanding of its conformational dynamics and guide the drug design process, we conducted molecular dynamics simulations to explore the conformational states of LSD1 in the apo state and under the influence of cofactors of flavin adenine dinucleotide (FAD) and CoREST. Our results showed that, across all states, the substrate binding pocket exhibited high flexibility, whereas the FAD binding pocket remained more stable. These distinct dynamical properties are essential for LSD1's ability to bind various substrates while maintaining efficient demethylation activity. Both pockets can be enlarged by merging with adjacent pockets, although only the substrate binding pocket can shrink into smaller pockets. These new pocket shapes can inform inhibitor design, particularly for selectively FAD-competitive inhibitors of LSD1, given the presence of numerous FAD-dependent enzymes in the human body. More interestingly, in the absence of FAD binding, the united substrate and FAD binding pocket are partitioned by the conserved residue of Tyr761, offering valuable insights for the design of inhibitors that disrupt the crucial steric role of Tyr761 and the redox role of FAD. Additionally, we identified pockets that positively or negatively correlate with the substrate and FAD binding pockets, which can be exploited for the design of allosteric or concurrent inhibitors. Our results reveal the intricate dynamical properties of LSD1 as well as multiple novel conformational states, which deepen our understanding of its sophisticated functions and aid in the rational design of new inhibitors.

Modulation of histone methylation status is regarded as an important mechanism of epigenetic regulation and has substantial clinical potential for the therapy of diseases, including cancer and other disorders. The present study aimed to provide a comprehensive introduction to the enzymology of histone demethylases, as well as their cancerous roles, molecular mechanisms, therapeutic possibilities, and challenges for targeting them, in order to advance drug design for clinical therapy and highlight new insight into the mechanisms of these enzymes in cancer. A series of clinical trials have been performed to explore potential roles of histone demethylases in several cancer types. Numerous targeted inhibitors associated with immunotherapy, chemotherapy, radiotherapy, and targeted therapy have been used to exert anticancer functions. Future studies should evaluate the dynamic transformation of histone demethylases leading to carcinogenesis and explore individual therapy.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

The enzymes performing protein post-translational modifications (PTMs) form a critical post-translational regulatory circuitry that orchestrates literally all cellular processes in the organism. In particular, the balance between cellular stemness and differentiation is crucial for the development of multicellular organisms. Importantly, the fine-tuning of this balance on the genetic level is largely mediated by specific PTMs of histones including lysine methylation. Lysine methylation is carried out by special enzymes (lysine methyltransferases) that transfer the methyl group from S-adenosyl-L-methionine to the lysine residues of protein substrates. Set7/9 is one of the exemplary protein methyltransferases that however, has not been fully studied yet. It was originally discovered as histone H3 lysine 4-specific methyltransferase, which later was shown to methylate a number of non-histone proteins that are crucial regulators of stemness and differentiation, including p53, pRb, YAP, DNMT1, SOX2, FOXO3, and others. In this review we summarize the information available to date on the role of Set7/9 in cellular differentiation and tissue development during embryogenesis and in adult organisms. Finally, we highlight and discuss the role of Set7/9 in pathological processes associated with aberrant cellular differentiation and self-renewal, including the formation of cancer stem cells.

Proteasome-mediated degradation of chromatin-bound NF-κB is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of the RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we find that two structurally similar substrate-recognizing components of Cullin-RING E3 ligases, WSB1 and WSB2, can recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. We showed that WSB1/2 negatively regulated a subset of NF-κB target genes via associating with chromatin where they targeted methylated RelA for ubiquitination, facilitating the termination of NF-κB-dependent transcription. WSB1/2 specifically interacted with methylated lysines (K) 314 and 315 of RelA via their N-terminal WD-40 repeat (WDR) domains, thereby promoting ubiquitination of RelA. Computational modeling further revealed that a conserved aspartic acid (D) at position 158 within the WDR domain of WSB2 coordinates K314/K315 of RelA, with a higher affinity when either of the lysines is methylated. Mutation of D158 abolished WSB2's ability to bind to and promote ubiquitination of methylated RelA. Together, our study identifies a novel function and the underlying mechanism for WSB1/2 in degrading chromatin-bound methylated RelA and preventing sustained NF-κB activation, providing potential new targets for therapeutic intervention of NF-κB-mediated inflammatory diseases.

The Wnt/β-Catenin pathway plays a key role in cell fate determination during development and in adult tissue regeneration by stem cells. These processes involve profound gene expression and epigenome remodeling and linking Wnt/β-Catenin signaling to chromatin modifications has been a challenge over the past decades. Functional studies of the lysine demethylase LSD1/KDM1A converge to indicate that this epigenetic regulator is a key regulator of cell fate, although the extracellular cues controlling LSD1 action remain largely unknown. Here we show that β-Catenin is a substrate of LSD1. Demethylation by LSD1 prevents β-Catenin degradation thereby maintaining its nuclear levels. Consistently, in absence of LSD1, β-Catenin transcriptional activity is reduced in both MuSCs and ESCs. Moreover, inactivation of LSD1 in mouse muscle stem cells and embryonic stem cells shows that LSD1 promotes mitotic spindle orientation via β-Catenin protein stabilization. Altogether, by inscribing LSD1 and β-Catenin in the same molecular cascade linking extracellular factors to gene expression, our results provide a mechanistic explanation to the similarity of action of canonical Wnt/β-Catenin signaling and LSD1 on stem cell fate.

EBV-infected lymphoma has a poor prognosis and various treatment strategies are being explored. Reports suggesting that B cell lymphoma can be induced by epigenetic regulation have piqued interest in studying mechanisms targeting epigenetic regulation. Here, we set out to identify an epigenetic regulator drug that acts synergistically with doxorubicin in EBV-positive lymphoma. We expressed the major EBV protein, LMP1, in B-cell lymphoma cell lines and used them to screen 100 epigenetic modifiers in combination with doxorubicin. The screening results identified TCP, which is an inhibitor of LSD1. Further analyses revealed that LMP1 increased the activity of LSD1 to enhance stemness ability under doxorubicin treatment, as evidenced by colony-forming and ALDEFLUOR activity assays. Quantseq 3' mRNA sequencing analysis of potential targets regulated by LSD1 in modulating stemness revealed that the LMP1-induced upregulation of CHAC2 was decreased when LSD1 was inhibited by TCP or downregulated by siRNA. We further observed that SOX2 expression was altered in response to CHAC2 expression, suggesting that stemness is regulated. Collectively, these findings suggest that LSD1 inhibitors could serve as promising therapeutic candidates for EBV-positive lymphoma, potentially reducing stemness activity when combined with conventional drugs to offer an effective treatment approach.

Kdm1a is a histone demethylase linked to intellectual disability with essential roles during gastrulation and the terminal differentiation of specialized cell types, including neurons, that remains highly expressed in the adult brain. To explore Kdm1a's function in adult neurons, we develop inducible and forebrain-restricted Kdm1a knockouts. By applying multi-omic transcriptome, epigenome and chromatin conformation data, combined with super-resolution microscopy, we find that Kdm1a elimination causes the neuronal activation of nonneuronal genes that are silenced by the polycomb repressor complex and interspersed with active genes. Functional assays demonstrate that the N-terminus of Kdm1a contains an intrinsically disordered region that is essential to segregate Kdm1a-repressed genes from the neighboring active chromatin environment. Finally, we show that the segregation of Kdm1a-target genes is weakened in neurons during natural aging, underscoring the role of Kdm1a safeguarding neuronal genome organization and gene silencing throughout life.

Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD) dependent monoamine oxidase (MAO) that erases the mono-, and dimethylation of histone 3 lysine 4 (H3K4), resulting in the suppression of target gene transcriptions. Besides, it can also demethylate some nonhistone substrates to regulate their biological functions. As reported, LSD1 is widely upregulated and plays a key role in several kinds of cancers, pharmacological or genetic ablation of LSD1 in cancer cells suppresses cell aggressiveness by several distinct mechanisms. Therefore, numerous LSD1 inhibitors, including covalent and noncovalent, have been developed and several of them have entered clinical trials. Herein, we systemically reviewed and discussed the biological function of LSD1 in tumors, lymphocytes as well as LSD1-targeting inhibitors in clinical trials, hoping to benefit the field of LSD1 and its inhibitors.

Lysine-specific demethylase 1 (LSD1) is a histone lysine demethylase that is significantly overexpressed or dysregulated in different cancers and plays important roles in cell growth, invasion, migration, immune escape, angiogenesis, gene regulation, and transcription. Therefore, it is a superb target for the discovery of novel antitumor agents. However, because of their innate and acquired resistance and low selectivity, LSD1 inhibitors are associated with limited therapeutic efficacy and high toxicity. Furthermore, LSD1 inhibitors synergistically improve the efficacy of additional antitumor drugs, which encourages numerous medicinal chemists to innovate and develop new-generation LSD1-based dual-target agents. This review discusses the theoretical foundation of the design of LSD1-based dual-target agents and summarizes their possible applications in treating cancers.

Lysine methylation is a major post-translational protein modification that occurs in both histones and non-histone proteins. Emerging studies show that the methylated lysine residues in non-histone proteins provide a proteolytic signal for ubiquitin-dependent proteolysis. The SET7 (SETD7) methyltransferase specifically transfers a methyl group from S-Adenosyl methionine to a specific lysine residue located in a methylation degron motif of a protein substrate to mark the methylated protein for ubiquitin-dependent proteolysis. LSD1 (Kdm1a) serves as a demethylase to dynamically remove the methyl group from the modified protein. The methylated lysine residue is specifically recognized by L3MBTL3, a methyl-lysine reader that contains the malignant brain tumor domain, to target the methylated proteins for proteolysis by the CRL4DCAF5 ubiquitin ligase complex. The methylated lysine residues are also recognized by PHF20L1 to protect the methylated proteins from proteolysis. The lysine methylation-mediated proteolysis regulates embryonic development, maintains pluripotency and self-renewal of embryonic stem cells and other stem cells such as neural stem cells and hematopoietic stem cells, and controls other biological processes. Dysregulation of the lysine methylation-dependent proteolysis is associated with various diseases, including cancers. Characterization of lysine methylation should reveal novel insights into how development and related diseases are regulated.

The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.

Epigenetic modifications are defined as heritable changes in gene activity that do not involve changes in the underlying DNA sequence. The oncogenic process is driven by the accumulation of alterations that impact genome's structure and function. Genetic mutations, which directly disrupt the DNA sequence, are complemented by epigenetic modifications that modulate gene expression, thereby facilitating the acquisition of malignant characteristics. Principals among these epigenetic changes are shifts in DNA methylation and histone mark patterns, which promote tumor development and metastasis. Notably, the reversible nature of epigenetic alterations, as opposed to the permanence of genetic changes, positions the epigenetic machinery as a prime target in the discovery of novel therapeutics. Our review delves into the complexities of epigenetic regulation, exploring its profound effects on tumor initiation, metastatic behavior, metabolic pathways, and the tumor microenvironment. We place a particular emphasis on the dysregulation at each level of epigenetic modulation, including but not limited to, the aberrations in enzymes responsible for DNA methylation and histone modification, subunit loss or fusions in chromatin remodeling complexes, and the disturbances in higher-order chromatin structure. Finally, we also evaluate therapeutic approaches that leverage the growing understanding of chromatin dysregulation, offering new avenues for cancer treatment.

A paradoxical increase of growth hormone (GH) following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants and somatic loss of heterozygosity of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism.

We aimed to analyze KDM1A gene sequence and KDM1A and GIPR expressions in somatotroph pituitary adenomas.

We conducted a cohort study at university hospitals in France and in Italy. We collected pituitary adenoma specimens from acromegalic patients who had undergone pituitary surgery. We performed targeted exome sequencing (gene panel analysis) and array-comparative genomic hybridization on somatic DNA derived from adenomas and performed droplet digital PCR on adenoma samples to quantify KDM1A and GIPR expressions.

One hundred and forty-six patients with sporadic acromegaly were studied; 72.6% presented unsuppressed classical GH response, whereas 27.4% displayed a paradoxical rise in GH after oral glucose load. We did not identify any pathogenic variant in the KDM1A gene in the adenomas of these patients. However, we identified a recurrent 1p deletion encompassing the KDM1A locus in 29 adenomas and observed a higher prevalence of paradoxical GH rise (P = .0166), lower KDM1A expression (4.47 ± 2.49 vs 8.56 ± 5.62, P < .0001), and higher GIPR expression (1.09 ± 0.92 vs 0.43 ± 0.51, P = .0012) in adenomas from patients with KDM1A haploinsufficiency compared with those with 2 KDM1A copies.

Unlike in GIP-dependent primary bilateral macronodular adrenal hyperplasia, KDM1A genetic variations are not the cause of GIPR expression in somatotroph pituitary adenomas. Recurrent KDM1A haploinsufficiency, more frequently observed in GIPR-expressing adenomas, could be responsible for decreased KDM1A function resulting in transcriptional derepression on the GIPR locus.

The smooth and precise transition from totipotency to pluripotency is a key process in embryonic development, generating pluripotent stem cells capable of forming all cell types. While endogenous retroviruses (ERVs) are essential for early development, their precise roles in this transition remains mysterious. Using cutting-edge genetic and biochemical techniques in mice, we identify MERVL-gag, a retroviral protein, as a crucial modulator of pluripotent factors OCT4 and SOX2 during lineage specification. MERVL-gag tightly operates with URI, a prefoldin protein that concurs with pluripotency bias in mouse blastomeres, and which is indeed required for totipotency-to-pluripotency transition. Accordingly, URI loss promotes a stable totipotent-like state and embryo arrest at 2C stage. Mechanistically, URI binds and shields OCT4 and SOX2 from proteasome degradation, while MERVL-gag displaces URI from pluripotent factor interaction, causing their degradation. Our findings reveal the symbiotic coevolution of ERVs with their host cells to ensure the smooth and timely progression of early embryo development.

Lysine specific demethylase 1 (LSD1), a transcriptional modulator that represses or activates target gene expression, is overexpressed in many cancer and causes imbalance in the expression of normal gene networks. Over two decades, numerous LSD1 inhibitors have been reported, especially some of which have entered clinical trials, including eight irreversible inhibitors (TCP, ORY-1001, GSK-2879552, INCB059872, IMG-7289, ORY-2001, TAK-418, and LH-1802) and two reversible inhibitors (CC-90011 and SP-2577). Most clinical LSD1 inhibitors demonstrated enhanced efficacy in combination with other agents. LSD1 multitarget inhibitors have also been reported, exampled by clinical dual LSD1/histone deacetylases (HDACs) inhibitors 4SC-202 and JBI-802. Herein, we present a comprehensive overview of the combination of LSD1 inhibitors with various antitumor agents, as well as LSD1 multitarget inhibitors. Additionally, the challenges and future research directionsare also discussed, and we hope this review will provide new insight into the development of LSD1-targeted anticancer agents.

Lysine-specific demethylase 1 (LSD1/KDM1A) has emerged as a promising therapeutic target for treating various cancers (such as breast cancer, liver cancer, etc.) and other diseases (blood diseases, cardiovascular diseases, etc.), owing to its observed overexpression, thereby presenting significant opportunities in drug development. Since its discovery in 2004, extensive research has been conducted on LSD1 inhibitors, with notable contributions from computational approaches. This review systematically summarizes LSD1 inhibitors investigated through computer-aided drug design (CADD) technologies since 2010, showcasing a diverse range of chemical scaffolds, including phenelzine derivatives, tranylcypromine (abbreviated as TCP or 2-PCPA) derivatives, nitrogen-containing heterocyclic (pyridine, pyrimidine, azole, thieno[3,2-b]pyrrole, indole, quinoline and benzoxazole) derivatives, natural products (including sanguinarine, phenolic compounds and resveratrol derivatives, flavonoids and other natural products) and others (including thiourea compounds, Fenoldopam and Raloxifene, (4-cyanophenyl)glycine derivatives, propargylamine and benzohydrazide derivatives and inhibitors discovered through AI techniques). Computational techniques, such as virtual screening, molecular docking and 3D-QSAR models, have played a pivotal role in elucidating the interactions between these inhibitors and LSD1. Moreover, the integration of cutting-edge technologies such as artificial intelligence holds promise in facilitating the discovery of novel LSD1 inhibitors. The comprehensive insights presented in this review aim to provide valuable information for advancing further research on LSD1 inhibitors.

Lysine-specific demethylase 1 (LSD1) is a promising therapeutic target for cancer therapy. So far, over 80 crystal structures of LSD1 in different complex states have been deposited in the Protein Data Bank, which are valuable resources for performing structure-based drug design. However, among all of the crystal structures of LSD1, the substrate binding pocket, which is the most efficient druggable site for designing LSD1 inhibitors at present, is very similar no matter whether LSD1 is in the apo or any holo forms, which is inconsistent with its versatile demethylase functions. To investigate whether the substrate binding pocket is rigid or exhibits other representative conformations different from the crystal conformations that are feasible for designing new LSD1 inhibitors, we performed funnel metadynamics simulations to study the conformation dynamics of LSD1 in the binding process of two effective LSD1 inhibitors (CC-90011 and 6X0, CC-90011 undergoing clinical trials). Our results showed that the entrance of the substrate binding pocket is very flexible. Two representative entrance conformations of LSD1 counting against binding with the substrate of histone H3 were detected, which may be used for structure-based LSD1 inhibitor design. Besides, alternative optimal binding modes and prebinding modes for both inhibitors were also detected, which depicted that the key interactions changed along with the binding process. Our results should provide great help for LSD1 inhibitor design.