Lysine-specific Demethylase 1A Research Articles

FGL1: a novel biomarker and target for non-small cell lung cancer, promoting tumor progression and metastasis through KDM4A/STAT3 transcription mechanism

Non-small cell lung cancer (NSCLC) is characterized by a high incidence rate and poor prognosis worldwide. A deeper insight into the pathogenesis of NSCLC and identification of novel therapeutic targets are essential to improve the prognosis of NSCLC. In this study, we revealed that fibrinogen-like protein 1 (FGL1) promotes proliferation, migration, and invasion of NSCLC cells. Mechanistically, we found that Stat3 acts as a transcription factor and can be recruited to the FGL1 promoter, enhancing FGL1 promoter activity. Lysine-specific demethylase 4A (KDM4A) interacts with Stat3 and facilitates the removal of methyl groups from H3K9me3, thereby enhancing Stat3-mediated transcription of FGL1. Furthermore, we observed that Stat3 and KDM4A promote NSCLC cell proliferation, migration, and invasion partly by upregulating FGL1 expression. Additionally, the expression of FGL1 was significantly higher in cancer tissues (n = 90) than in adjacent non-cancerous tissues (n = 90). Furthermore, patients with high FGL1 expression had a shorter overall survival (OS) compared to those with low FGL1 expression. We measured the expression levels of FGL1 on circulating tumor cells (CTCs) in 65 patients and found that patients with a dynamic decrease in FGL1 expression on CTCs exhibited a better therapeutic response. These findings suggest that the dynamic changes in FGL1 expression can serve as a potential biomarker for predicting treatment efficacy in NSCLC. Overall, this study revealed the significant role and regulatory mechanisms of FGL1 in the development of NSCLC, suggesting its potential as a therapeutic target for patients with NSCLC. Future studies should provide more personalized and effective treatment options for patients with NSCLC to improve clinical outcomes.

Kdm4a is an activity downregulated barrier to generate engrams for memory separation

Memory engrams are a subset of learning activated neurons critical for memory recall, consolidation, extinction and separation. While the transcriptional profile of engrams after learning suggests profound neural changes underlying plasticity and memory formation, little is known about how memory engrams are selected and allocated. As epigenetic factors suppress memory formation, we developed a CRISPR screening in the hippocampus to search for factors controlling engram formation. We identified histone lysine-specific demethylase 4a (Kdm4a) as a negative regulator for engram formation. Kdm4a is downregulated after neural activation and controls the volume of mossy fiber boutons. Mechanistically, Kdm4a anchors to the exonic region of Trpm7 gene loci, causing the stalling of nascent RNAs and allowing burst transcription of Trpm7 upon the dismissal of Kdm4a. Furthermore, the YTH domain containing protein 2 (Ythdc2) recruits Kdm4a to the Trpm7 gene and stabilizes nascent RNAs. Reducing the expression of Kdm4a in the hippocampus via genetic manipulation or artificial neural activation facilitated the ability of pattern separation in rodents. Our work indicates that Kdm4a is a negative regulator of engram formation and suggests a priming state to generate a separate memory.

KDM3A knockdown regulates COMP, LOX, COL8A1 and ACOT1 genes in myocardial fibrosis

Cardiovascular disease (CVD) is one of the main causes of death in Saudi Arabia. Cardiac remodeling plays a critical role in the pathophysiology of heart failure. Major focus of our study was to identify crucial genes involved in the pathological remodeling of the heart caused by pressure overload. We utilized various in-silico tools to analyze and interpret microarray data obtained from the Gene Expression Omnibus (GEO) database (GSE120739), including GEO2R analysis, Metascape analysis, WebGestalt analysis, and IPA (Ingenuity pathway analysis). Our findings indicate that certain genes, including Cartilage Oligomeric Matrix Protein (COMP), collagen type VIII alpha 1 chain (COL8A1) and Lysyl Oxidase (LOX) under the influence caused by knockdown of KDM3A, were down regulated by the extracellular matrix pathway. Moreover, genes, such as Acyl-CoA Thioesterase 1 (ACOT1) were up regulated by the fatty acid metabolism pathway. Overexpression of lysine-specific demethylase 3A (KDM3A) leads to the up regulation of fibrosis-related genes COMP, COL8A1, and LOX and the down regulation of ACOT1, result in enhanced fibrosis and heart failure. Our results suggest that COMP, COL8A1, LOX, and ACOT1 warrant further investigation in the development of cardiac fibrosis and as potential biomarkers for causing heart failure.

LSD1 drives intestinal epithelial maturation and controls small intestinal immune cell composition independent of microbiota in a murine model

Postnatal development of the gastrointestinal tract involves the establishment of the commensal microbiota, the acquisition of immune tolerance via a balanced immune cell composition, and maturation of the intestinal epithelium. While studies have uncovered an interplay between the first two, less is known about the role of the maturing epithelium. Here we show that intestinal-epithelial intrinsic expression of lysine-specific demethylase 1A (LSD1) is necessary for the postnatal maturation of intestinal epithelium and maintenance of this developed state during adulthood. Using microbiota-depleted mice, we find plasma cells, innate lymphoid cells (ILCs), and a specific myeloid population to depend on LSD1-controlled epithelial maturation. We propose that LSD1 controls the expression of epithelial-derived chemokines, such as Cxcl16, and that this is a mode of action for this epithelial-immune cell interplay in local ILC2s but not ILC3s. Together, our findings suggest that the maturing epithelium plays a dominant role in regulating the local immune cell composition, thereby contributing to gut homeostasis.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme–substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Beta-elemene inhibits the growth of KDM6A-null bladder cancer cells by suppressing the epithelial-mesenchymal transition

BackgroundBladder cancer is a highly prevalent and lethal malignant tumor characterized by frequent mutations/deletions of lysine-specific demethylase 6A (KDM6A), which is suggested to be a key event in cancer progression and metastasis. Beta-elemene has been shown to inhibit metastasis and growth of various tumors, but its effect on KDM6A-null bladder cancer cells remains unknown. ObjectiveThis study aimed to investigate the potential and molecular mechanism of β-elemene in inhibiting the growth of KDM6A-null bladder cancer. MethodsThis study examined the migration ability and viability of RT-4 (KDM6A wild-type) and KU19-19 (KDM6A-null) cell lines using wound healing assay and CCK-8, respectively. The inhibitory effect of β-elemene on KU19-19 cell migration was evaluated using transwell and immunofluorescence assays, and the expression of transfer-related proteins and genes was analyzed through western blot and qRT-PCR, respectively. Molecular docking was performed to predict the targeting of β-elemene, and the effects were confirmed in KDM6A-knockdown RT-4 cells. Finally, the therapeutic effect of β-elemene on bladder cancer was tested in animal models. ResultsThe study observed that loss of KDM6A increased bladder cancer cell migration, with KU19-19 exhibiting significantly stronger migration than RT-4. Further investigation revealed that β-elemene effectively inhibited KU19-19 cell migration, likely through targeting EZH2 as determined by molecular docking. Overexpression of KDM6A inhibited KU19-19 metastasis, while knockdown of KDM6A in RT-4 cells enhanced cell migration, which was reversed by β-elemene treatment. Notably, in vivo testing revealed a significant suppression of KU19-19 cell growth with β-elemene administered at a dosage of 100 mg/kg. Conclusionβ-elemene has the potential to suppress the growth of KDM6A-null bladder cancer by inhibiting epithelial-mesenchymal transition (EMT), which could make it a promising therapeutic option for patients with KDM6A-null bladder cancer.

Evolution of lysine-specific demethylase 1 and REST corepressor gene families and their molecular interaction

Lysine-specific demethylase 1A (LSD1) binds to the REST corepressor (RCOR) protein family of corepressors to erase transcriptionally active marks on histones. Functional diversity in these complexes depends on the type of RCOR included, which modulates the catalytic activity of the complex. Here, we studied the duplicative history of the RCOR and LSD gene families and analyzed the evolution of their interaction. We found that RCOR genes are the product of the two rounds of whole-genome duplications that occurred early in vertebrate evolution. In contrast, the origin of the LSD genes traces back before to the divergence of animals and plants. Using bioinformatics tools, we show that the RCOR and LSD1 interaction precedes the RCOR repertoire expansion that occurred in the last common ancestor of jawed vertebrates. Overall, we trace LSD1-RCOR complex evolution and propose that animal non-model species offer advantages in addressing questions about the molecular biology of this epigenetic complex.

Lysine-specific Demethylase 6A Upregulates Cadherin-1 and Accelerates Gastric Cancer Growth.

Gastric cancer (GC) ranks fifth among all common malignancies globally. Genetic research has revealed several genes that are frequently dis-regulated in GC, such as lysine-specific demethylase 6A (KDM6A) and cadherin-1 (CDH1). This study aimed to examine the expression profile and role of KDM6A in GC, as well as the molecular pathway involved. The expression profile and overall survival data of KDM6A were retrieved from the TCGA database. Expression levels of KDM6A were also measured in GC patient samples and compared with those of healthy controls. Furthermore, stable silencing of KDM6A was introduced into the GC cell line NCI-N87, followed by assessments of cell proliferation, migration and invasion, in the xenograft mouse model. The metastatic status of mice injected with NCI-N87 cells was also analyzed. In patients diagnosed with GC, KDM6A was upregulated. Silencing KDM6A reduced the proliferation, migration and invasion of cells, as well as the growth of xenograft tumors. KDM6A knockdown also inhibited metastatic behaviors of injected NCI-N87 cells, as well as elevated CDH1 expression, leading to reversed epithelial-mesenchymal transition. KDM6A serves as an oncogene in GC and exerts its pro-tumor functions by repressing the expression of CDH1.

Spectroscopic investigations, quantum chemical, molecular docking and drug likeness studies of t-butyl-3,4,5-trimethyl-2-pyrrole carboxylate

The geometrical structure of t‑butyl‑3,4,5-trimethyl-2-pyrrole carboxylate (TBTPC) was computed by DFT calculations using the B3LYP method and the 6–311++G(d,p) basis set using the Gaussian 09 software. Vibrational wavenumbers were assigned using the VEDA 4.0 program. Natural bond orbital (NBO) analysis predicts the hyper-conjugative interaction and stabilization interaction of the TBTPC compound. HOMO, LUMO, and other molecular properties were calculated. The density of states (DOS) spectrum was also predicted. The ultraviolet-visible spectrum has been experimentally and theoretically validated and simulated. The electrophilic and nucleophilic reactive sites of the TBTPC molecule were determined using the Fukui function calculation and the molecular electrostatic potential (MEP) surface. Molecular docking analysis of the TBTPC ligand helps to investigate its inhibitory nature against Lysine-specific demethylase 1A (LSD1), α-amylase, BCL-1, and monoamine oxidase (MAO). From the molecular docking results, TBTPC has promising inhibition activity against gastric cancer.

Maternal Kdm2a-mediated PI3K/Akt signaling and E-cadherin stimulate the morula-to-blastocyst transition revealing crucial roles in early embryonic development

Histone methylation plays an essential role in oocyte growth and preimplantation embryonic development. The modification relies on histone methyl-transferases and demethylases, and one of these, lysine-specific demethylase 2a (Kdm2a), is responsible for modulating histone methylation during oocyte and early embryonic development. The mechanism of how Kdm2a deficiency disrupts early embryonic development and fertility remains elusive. To determine if maternally deposited Kdm2a is required for preimplantation embryonic development, the expression profile of Kdm2a during early embryos was detected via immunofluorescence staining and RT-qPCR. The Kdm2a gene in oocytes was specifically deleted with the Zp3-Cre/LoxP system and the effects of maternal Kdm2a loss were studied through a comprehensive range of female reproductive parameters including fertilization, embryo development, and the number of births. RNA transcriptome sequencing was performed to determine differential mRNA expression, and the interaction between Kdm2a and the PI3K/Akt pathway was studied with a specific inhibitor and activator. Our results revealed that Kdm2a was continuously expressed in preimplantation embryos and loss of maternal Kdm2a suppressed the morula-to-blastocyst transition, which may have been responsible for female subfertility. After the deletion of Kdm2a, the global H3K36me2 methylation in mutant embryos was markedly increased, but the expression of E-cadherin decreased significantly in morula embryos compared to controls. Mechanistically, RNA-seq analysis revealed that deficiency of maternal Kdm2a altered the mRNA expression profile, especially in the PI3K/Akt signaling pathway. Interestingly, the addition of a PI3K/Akt inhibitor (LY294002) to the culture medium blocked embryo development at the stage of morula; however, the developmental block caused by maternal Kdm2a loss was partially rescued with a PI3K/Akt activator (SC79). In summary, our results indicate that loss of Kdm2a influences the transcriptome profile and disrupts the PI3K/Akt signaling pathway during the development of preimplantation embryo. This can result in embryo block at the morula stage and female subfertility, which suggests that maternal Kdm2a is a potential partial redundancy with other genes encoding enzymes in the dynamics of early embryonic development. Our results provide further insight into the role of histone modification, especially on Kdm2a, in preimplantation embryonic development in mice.

Long noncoding RNAs with peptide-encoding potential identified in esophageal squamous cell carcinoma: KDM4A-AS1-encoded peptide weakens cancer cell viability and migratory capacity.

Currently, the knowledge of long noncoding RNA (lncRNA)-encoded peptides is quite lacking in esophageal squamous cell carcinoma (ESCC). In this study, we simultaneously identified six lncRNA open reading frames (ORFs) with peptide-coding abilities including lysine-specific demethylase 4A antisense RNA 1 (KDM4A-AS1) ORF by combining weighted gene co-expression network analysis (WGCNA) for ESCC clinical samples, ribosome footprints, ORF prediction, mass spectrometry (MS) identification, and western blotting. KDM4A-AS1 ORF-encoded peptide reduced ESCC cell viability and migratory ability. Co-immunoprecipitation and MS analysis revealed that KDM4A-AS1-encoded peptide specifically bound with 103 proteins in ESCC cells, and enrichment analysis suggested that peptide-bound proteins were related to fatty acid metabolism and redox process. Cell and molecular experiments demonstrated that KDM4A-AS1-encoded peptide inhibited stearoyl-CoA desaturase and fatty acid synthase expression, increased reactive oxygen species level, and reduced mitochondrial membrane potential in ESCC cells. In summary, multiple lncRNAs with translation potential were simultaneously identified by combining multiple approaches in ESCC, providing novel identificationstrategies for lncRNA-encoded peptides. Moreover, lncRNA KDM4A-AS1-encoded peptide weakened ESCC cell viability and migratory capacity and functioned in fatty acid metabolism and redox process.

Abstract 4733: Effect of KDM6A mutation in bladder cancer

Abstract As of 2020, bladder cancer is the tenth most common cancer in the world, with a heavier toll exerted on males. Its higher tumor recurrence and progression rates cause major treatment challenges. One of the most frequent mutations in bladder cancer is in lysine-specific demethylase 6A (KDM6A). This gene encodes for ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), which catalyzes the demethylation of di- and tri- methylated histones H3 lysine 27 and works as part of “COMPASS-LIKE” protein complexes to coordinate many transcriptional processes. The molecular basis for how KDM6A mutations promote carcinogenesis is still under investigation. In this study, we use single-cell RNA sequencing data from bladder cancers with and without KDM6A mutations in both human and mice to examine the effects of KDM6A mutations on the cellular composition and gene expression of tumor and normal bladders. In mice without tumors, KDM6A mutations seem to correlate with enrichment of macrophages in the bladder tissue microenvironment. The opposite is true, however, in the presence of tumors, as there were not only more macrophages but also more dendritic cells in wildtype mice. This pattern was also observed in human, suggesting differences in immune infiltrates in the tumor microenvironment are part of the phenotype of KDM6A mutation. Lastly, KDM6A mutations may also impact urothelial cell states at baseline, as KDM6A KO mouse bladders contain higher proportions of intermediate cell populations compared with their wildtype counterparts. Together, these observations contribute to a better understanding of the role KDM6A plays in bladder cancer. Citation Format: Ninh B. Le, Hong Qiu, Emily E. Fink, Surbhi Sona, Angela H. Ting, Byron H. Lee. Effect of KDM6A mutation in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4733.

Combinatorial targeting of epigenome-modifying enzymes with decitabine and RN-1 synergistically increases HbF

AbstractIncreased fetal hemoglobin (HbF) levels reduce the symptoms of sickle cell disease (SCD) and increase the lifespan of patients. Because curative strategies for bone marrow transplantation and gene therapy technologies remain unavailable to a large number of patients, the development of a safe and effective pharmacological therapy that increases HbF offers the greatest potential for disease intervention. Although hydroxyurea increases HbF, a substantial proportion of patients fail to demonstrate an adequate response. Pharmacological inhibitors of DNA methyltransferase (DNMT1) and lysine-specific demethylase 1A (LSD1), 2 epigenome-modifying enzymes associated with the multiprotein corepressor complex recruited to the repressed γ-globin gene, are powerful in vivo inducers of HbF. The hematological side effects of these inhibitors limit feasible clinical exposures. We evaluated whether administering these drugs in combination could reduce the dose and/or time of exposure to any single agent to minimize adverse effects, while achieving additive or synergistic increases in HbF. The DNMT1 inhibitor decitabine (0.5 mg/kg per day) and the LSD1 inhibitor RN-1 (0.25 mg/kg per day) administered in combination 2 days per week produced synergistic increases in F-cells, F-reticulocytes, and γ-globin messenger RNA in healthy baboons. Large increases in HbF and F-cells were observed in healthy, nonanemic, and anemic (phlebotomized) baboons. Combinatorial therapy targeting epigenome-modifying enzymes could thus be a useful strategy for producing larger increases in HbF to modify the clinical course of SCD.

Compound Danshen Dripping Pill inhibits hypercholesterolemia/atherosclerosis-induced heart failure in ApoE and LDLR dual deficient mice via multiple mechanisms.

Heart failure is the leading cause of death worldwide. Compound Danshen Dripping Pill (CDDP) or CDDP combined with simvastatin has been widely used to treat patients with myocardial infarction and other cardiovascular diseases in China. However, the effect of CDDP on hypercholesterolemia/atherosclerosis-induced heart failure is unknown. We constructed a new model of heart failure induced by hypercholesterolemia/atherosclerosis in apolipoprotein E (ApoE) and LDL receptor (LDLR) dual deficient (ApoE-/-LDLR-/-) mice and investigated the effect of CDDP or CDDP plus a low dose of simvastatin on the heart failure. CDDP or CDDP plus a low dose of simvastatin inhibited heart injury by multiple actions including anti-myocardial dysfunction and anti-fibrosis. Mechanistically, both Wnt and lysine-specific demethylase 4A (KDM4A) pathways were significantly activated in mice with heart injury. Conversely, CDDP or CDDP plus a low dose of simvastatin inhibited Wnt pathway by markedly up-regulating expression of Wnt inhibitors. While the anti-inflammation and anti-oxidative stress by CDDP were achieved by inhibiting KDM4A expression and activity. In addition, CDDP attenuated simvastatin-induced myolysis in skeletal muscle. Taken together, our study suggests that CDDP or CDDP plus a low dose of simvastatin can be an effective therapy to reduce hypercholesterolemia/atherosclerosis-induced heart failure.

Learning with uncertainty to accelerate the discovery of histone lysine-specific demethylase 1A (KDM1A/LSD1) inhibitors.

Machine learning including modern deep learning models has been extensively used in drug design and screening. However, reliable prediction of molecular properties is still challenging when exploring out-of-domain regimes, even for deep neural networks. Therefore, it is important to understand the uncertainty of model predictions, especially when the predictions are used to guide further experiments. In this study, we explored the utility and effectiveness of evidential uncertainty in compound screening. The evidential Graphormer model was proposed for uncertainty-guided discovery of KDM1A/LSD1 inhibitors. The benchmarking results illustrated that (i) Graphormer exhibited comparative predictive power to state-of-the-art models, and (ii) evidential regression enabled well-ranked uncertainty estimates and calibrated predictions. Subsequently, we leveraged time-splitting on the curated KDM1A/LSD1 dataset to simulate out-of-distribution predictions. The retrospective virtual screening showed that the evidential uncertainties helped reduce false positives among the top-acquired compounds and thus enabled higher experimental validation rates. The trained model was then used to virtually screen an independent in-house compound set. The top 50 compounds ranked by two different ranking strategies were experimentally validated, respectively. In general, our study highlighted the importance to understand the uncertainty in prediction, which can be recognized as an interpretable dimension to model predictions.

Olanzapine-induced decreases of FGF21 in brown adipose tissue via histone modulations drive UCP1-dependent thermogenetic impairment

Long-term olanzapine treatment has been associated with serious metabolism disorders, such as abnormal body weight gain, hyperglycemia, and dyslipidemia. Recently, accumulated evidence points to a link between the metabolic disorders caused by olanzapine and thermogenetic impairment. Fibroblast growth factor 21 (FGF21), a pleiotropic protein, is a potent stimulator of thermogenesis in brown adipose tissue (BAT). However, the relationship between autocrine FGF21 in BAT and thermogenetic impairment induced by olanzapine has not been investigated. In this study, C57BL/6 mice and C3H10T1/2 (a brown adipocyte cell line) were used to investigate the role of FGF21 in modulating thermogenetic impairments caused by olanzapine. Our data found a fall in BAT temperature, with a decrease in the protein levels of uncoupling protein 1 (UCP1) and FGF21 in olanzapine-treatment mice. Olanzapine-induced deficits of mitochondrial activity and the expression of UCP1 and related thermogenetic factors could be improved by FGF21-overexpression in brown adipocytes. Furthermore, ChIP-sequencing showed the H3K9me3 modification in Fgf21 was dramatically increased in BAT of mice with olanzapine treatment. Lysine-specific demethylase 4a (KDM4a), a histone demethylase responsible for site-specific erasure of H3K9me3, was decreased in olanzapine-treated C3H10T1/2 cells, whereas FGF21 and UCP1 expression and thermogenesis were upregulated in KMD2a-overexpressing brown adipocyte. We concluded that FGF21 was a crucial regulator mediating UCP1-dependent thermogenetic impairments by olanzapine-modulating histone methylations. Our results also provide novel insights into identifying a new therapeutic target for treating metabolic side effects caused by the antipsychotic drug.

Pharmacological Inhibition of Lysine-Specific Demethylase 1A Reduces Atherosclerotic Lesion Formation in Apolipoprotein E-Deficient Mice by a Mechanism Involving Decreased Oxidative Stress and Inflammation; Potential Implications in Human Atherosclerosis.

Dysregulated epigenetic mechanisms promote transcriptomic and phenotypic alterations in cardiovascular diseases. The role of histone methylation-related pathways in atherosclerosis is largely unknown. We hypothesize that lysine-specific demethylase 1A (LSD1/KDM1A) regulates key molecular effectors and pathways linked to atherosclerotic plaque formation. Human non-atherosclerotic and atherosclerotic tissue specimens, ApoE-/- mice, and in vitro polarized macrophages (Mac) were examined. Male ApoE-/- mice fed a normal/atherogenic diet were randomized to receive GSK2879552, a highly specific LSD1 inhibitor, or its vehicle, for 4 weeks. The mRNA and protein expression levels of LSD1/KDM1A were significantly elevated in atherosclerotic human carotid arteries, atherosclerotic aortas of ApoE-/- mice, and M1-Mac. Treatment of ApoE-/- mice with GSK2879552 significantly reduced the extent of atherosclerotic lesions and the aortic expression of NADPH oxidase subunits (Nox1/2/4, p22phox) and 4-hydroxynonenal-protein adducts. Concomitantly, the markers of immune cell infiltration and vascular inflammation were significantly decreased. LSD1 blockade down-regulated the expression of genes associated with Mac pro-inflammatory phenotype. Nox subunit transcript levels were significantly elevated in HEK293 reporter cells overexpressing LSD1. In experimental atherosclerosis, LSD1 mediates the up-regulation of molecular effectors connected to oxidative stress and inflammation. Together, these data indicate that LSD1-pharmacological interventions are novel targets for supportive therapeutic strategies in atherosclerosis.

Genome-Wide CRISPR-Cas9 Screen Identifies KDM6A As a Modulator of Daratumumab Sensitivity in Multiple Myeloma

Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38, which triggers both direct and immune-mediated cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) milieu. Importantly, Dara combination therapies have a high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse is commonly observed due to the development of Dara resistance. As an immune-targeting drug, the anti-tumor activity of Dara is closely associated with the expression of CD38, with nonresponders having a lower expression of CD38. To identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells, we performed a genome-wide CRISPR-Cas9 knockout (KO) screen in vitro. We identified 46 different sgRNAs that were positively correlated with MM cytotoxicity triggered by Dara and primary NK cells. Using the cells with the lowest expression of CD38 (bottom 5%), we performed a second round of screening and found that KDM6A was the top-ranked gene out of the set of overlapping genes from these two screenings. We confirmed that CD38 expression was significantly decreased (>2 times, p<0.01) by KDM6A KO and that re-introducing KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity. KDM6A encodes lysine-specific demethylase 6A, which demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. To confirm that CD38 expression was altered by the methylation status of H3K27, we compared the CD38 promoter area between KDM6A KO and control cells using ChIP-seq and ChIP qPCR and found that the H3K27me3 level was higher in KDM6A KO cells. H3K27 is methylated by EZH2, and we found that EZH2 inhibitor treatment significantly decreased the H3K27me3 level of the CD38 promoter area. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein expression in KDM6A KO cells. Importantly, EZH2 inhibitor also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity in vitro and in xenograft mouse model. We next re-introduced CD38 into KDM6A KO cells and found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment. This suggested that besides CD38 downregulation, there are other mechanisms underlying Dara resistance induced by KDM6A KO. We performed RNA-seq in KDM6A KO cells and found that CD48, an NK-activating ligand, was downregulated. Overexpression of CD48 in KDM6A KO cells significantly increased secretion of Granzyme B and Perforin, restoring the activity of NK cells and re-sensitizing KDM6A KO cells to Dara treatment. These data suggest that KDM6A not only regulates CD38 expression, but also NK cell activity by CD48 regulation, thereby modulating the efficacy of Dara. Taken together, our studies demonstrate that KDM6A regulates CD38 and CD48 expression in MM. Moreover, we validate that combination treatment with FDA-approved EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome.

Oocyte-Specific Knockout of Histone Lysine Demethylase KDM2a Compromises Fertility by Blocking the Development of Follicles and Oocytes.

The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17β-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.