Lysine-specific Demethylase 1A Research Articles

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.

204 (PB084) - TAS1440, a novel reversible LSD1 inhibitor, modulates immunosuppressive population and potentiates the antitumor efficacy of anti-PD-1 antibody therapy

Background: Lysine-specific demethylase 1A (LSD1/KDM1A) is a flavin adenine dinucleotide (FAD)-dependent histone demethylase that specifically modifies histone H3 lysine 4 and lysine 9. In several cancers, LSD1 has been reported to play role in cancer cell survival and growth. Therefore, LSD1 is considered to be a promising drug target. Importantly, LSD1 inhibitors are undergoing clinical development. It has been recently reported that in cancer cells, LSD1 is associated with suppression of antitumor immunity, thereby contributing to cancer cell survival through immunosuppression.

KDM3A Attenuates Myocardial Ischemic and Reperfusion Injury by Ameliorating Cardiac Microvascular Endothelial Cell Pyroptosis.

Cardiac microvascular endothelial cell ischemia-reperfusion (CMEC I/R) injury occurs in approximately 50% of acute myocardial infarction patients subjected to successful revascularization therapy. This injury leads to cardiac microcirculatory system dysfunctions, which seriously affect cardiac functions and long-term prognostic outcomes. Previously, we elucidated the role of lysine-specific demethylase 3A (KDM3A) in protecting cardiomyocytes from I/R injury; however, its roles in CMEC I/R injuries have yet to be fully established. In this study, hypoxia/reoxygenation (H/R) treatment significantly impaired CMEC functions and induced their pyroptosis, accompanied by KDM3A downregulation. Then, gain- and loss-of-function assays were performed to investigate the roles of KDM3A in CMEC H/R injury in vitro. KDM3A knockout enhanced CMEC malfunctions and accelerated the expressions of pyroptosis-associated proteins, such as NLRP3, cleaved-caspase-1, ASC, IL-1β, GSDMD-N, and IL-18. Conversely, KDM3A overexpression developed ameliorated alternations in CMEC H/R injury. In vivo, KDM3A knockout resulted in the deterioration of cardiac functions and decreased the no-reflow area as well as capillary density. Mechanistically, KDM3A activated the PI3K/Akt signaling pathway and ameliorated I/R-mediated CMEC pyroptosis. In conclusion, KDM3A is a promising treatment target for alleviating CMEC I/R injury.

Inhibition of LSD1 with Bomedemstat Sensitizes Small Cell Lung Cancer to Immune Checkpoint Blockade and T-Cell Killing.

The addition of immune checkpoint blockade (ICB) to platinum/etoposide chemotherapy changed the standard of care for small cell lung cancer (SCLC) treatment. However, ICB addition only modestly improved clinical outcomes, likely reflecting the high prevalence of an immunologically "cold" tumor microenvironment in SCLC, despite high mutational burden. Nevertheless, some patients clearly benefit from ICB and recent reports have associated clinical responses to ICB in SCLC with (i) decreased neuroendocrine characteristics and (ii) activation of NOTCH signaling. We previously showed that inhibition of the lysine-specific demethylase 1a (LSD1) demethylase activates NOTCH and suppresses neuroendocrine features of SCLC, leading us to investigate whether LSD1 inhibition would enhance the response to PD-1 inhibition in SCLC. We employed a syngeneic immunocompetent model of SCLC, derived from a genetically engineered mouse model harboring Rb1/Trp53 inactivation, to investigate combining the LSD1 inhibitor bomedemstat with anti-PD-1 therapy. In vivo experiments were complemented by cell-based studies in murine and human models. Bomedemstat potentiated responses to PD-1 inhibition in a syngeneic model of SCLC, resulting in increased CD8+ T-cell infiltration and strong tumor growth inhibition. Bomedemstat increased MHC class I expression in mouse SCLC tumor cells in vivo and augmented MHC-I induction by IFNγ and increased killing by tumor-specific T cells in cell culture. LSD1 inhibition increased MHC-I expression and enhanced responses to PD-1 inhibition in vivo, supporting a new clinical trial to combine bomedemstat with standard-of-care PD-1 axis inhibition in SCLC.

Mechanisms of carcinogenic activity triggered by lysine-specific demethylase 1A

Epigenetics has emerged as a prime focus area in the field of cancer research. Lysine-specific demethylase 1A (LSD1), the first discovered histone demethylase, is mainly responsible for catalysing demethylation of histone 3 lysine 4 (H3K4) and H3K9 to activate or inhibit gene transcription. LSD1 is abnormally expressed in various cancers and participates in cancer proliferation, apoptosis, metastasis, invasion, drug resistance and other processes by interacting with regulatory factors. Therefore, it may serve as a potential therapeutic target for cancer. This review summarises the major oncogenic mechanisms mediated by LSD1 and provides a reference for developing novel and efficient anticancer strategies targeting LSD1.

Kdm4aa is required for reproduction and development of zebrafish

Lysine-specific demethylase 4A (KDM4A) catalyzes demethylation of histone lysine residues, which regulates chromatin state and transcription. In drosophila and mice, KDM4A plays an important role in multiple biological processes including development, aging, metabolism, and immunity, however the functions of KDM4A in fish are still unclear. There are two copies of the kdm4a gene in zebrafish, namely kdm4aa and kdm4ab, kdm4aa was edited using CRISPR/Cas9 technology in the present study, then homozygous kdm4aa mutants (kdm4aa−/−) were obtained, and loss of kdm4aa was confirmed by sequencing and increased H3K9me3. Whole-mount in situ hybridization showed that kdm4aa is widely expressed during the embryonic development of zebrafish. Compared with WT zebrafish, kdm4aa−/− zebrafish showed no significant difference in gamete formation and fertilization, but the survival rate of kdm4aa−/− embryos dramatically reduced to 21% at 26 hpf. Further observation showed that about 80% of survived kdm4aa−/− zebrafish experienced disruption in stripe formation, and 10% of survived kdm4aa−/− zebrafish underwent vertebral malformation. Alizarin red S staining demonstrated the abnormal spinal development in kdm4aa−/− zebrafish. These results indicated that kdm4aa is required for normal embryonic development of zebrafish, loss of kdm4aa function leads to decreased survival during the early stages of zebrafish development and morphological variation in adult zebrafish.

MicroRNA-27a-3p delivered by extracellular vesicles from glioblastoma cells induces M2 macrophage polarization via the EZH1/KDM3A/CTGF axis

Glioblastoma (GBM) cell-derived extracellular vesicles (EVs) have been demonstrated to modulate tumor microenvironment. In the present study, we attempted to discuss the role of hsa-microRNA-27a-3p (miR-27a-3p) delivered by GBM-EVs in M2 macrophage polarization. The isolated GBM-EVs were co-cultured with macrophages. After co-culture under normoxia/hypoxia, the effect of EV-derived hsa-miR-27a-3p on GBM cell biological processes was analyzed. Additionally, the target genes of hsa-miR-27a-3p were predicted. Moreover, the binding of enhancer of zeste homologue 1 (EZH1) to lysine-specific demethylase 3A (KDM3A) promoter region and the interaction between KDM3A and connective tissue growth factor (CTGF) were analyzed. GBM mouse models were established to verify the functions of EV-derived hsa-miR-27a-3p in vivo. We found increased hsa-miR-27a-3p in GBM tissues as well as GBM-EVs, which induced M2 polarization, thus promoting proliferative, migrative and invasive potentials of GBM cells. hsa-miR-27a-3p targeted EZH1 and promoted KDM3A expression to elevate the CTGF expression. GBM-EV-delivered hsa-miR-27a-3p promoted the KDM3A-upregulated CTGF by downregulating EZH1, thereby promoting M2 macrophage polarization and development of GBM in vivo. We demonstrated that EV-derived hsa-miR-27a-3p may promote M2 macrophage polarization to induce GBM.

Role of sevoflurane in myocardial ischemia-reperfusion injury via the ubiquitin-specific protease 22/lysine-specific demethylase 3A axis

ABSTRACT Myocardial ischemia-reperfusion injury (MIRI) represents a coronary artery disease, accompanied by high morbidity and mortality. Sevoflurane post-conditioning (SPC) is importantly reported in myocardial disease. Accordingly, the current study sought to evaluate the role of Sevo in MI/RI. Firstly, MI/RI models were established and subjected to SPC. Subsequently, pathological injury in the myocardium, myocardial infarction areas, H9c2 cell viability, apoptosis, and levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH) were all measured. Ubiquitin-specific peptidase (22USP22), lysine-specific demethylase 3A (KDM3A), and Yes1 associated transcriptional regulator (YAP1) were down-regulated in H9c2 cells using cell transfection to verify their roles. The interaction between USP22 and KDM3A and between KDM3A and YAP1 was further validated. USP 22, KDM3A, and YAP1 were found to be down-regulated in MI/RI and SPC protected MI/RI rats, as evidenced by up-regulated expressions of USP22, KDM3A, and YAP1, reduced pathological injury in the myocardium, myocardial infarction areas, apoptosis, and levels of CK-MB, cTnI, and LDH, and enhanced H9c2 cell viability; while the protective effects of Sevo were counteracted by silencing of USP22, KDM3A, and SPC upregulated USP22, which stabilized KDM3A protein levels via deubiquitination, and KDM3A inhibited histone 3 lysine 9 di-methylation (H3K9me2) levels in the YAP1 promoter to encourage YAP1 transcription, to reduce MI/RI.

MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER Ao Zhang, Hong Qiu, Timothy Chan, and Byron Lee Ao ZhangAo Zhang More articles by this author , Hong QiuHong Qiu More articles by this author , Timothy ChanTimothy Chan More articles by this author , and Byron LeeByron Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 6A (KDM6A) is a chromatin modifier that is frequently mutated in bladder cancer and causes epigenetic dysregulation. Recently immune check point inhibition has shown great clinical promise in bladder cancer. Because epigenetic signature has been shown to predict response to immunotherapy, here we assessed if loss of KDM6A leads to alterations of tumor immune microenvironment in a mouse bladder cancer model. METHODS: Kdm6a urothelium conditional knockout (cKO) mice were generated by crossing Kdm6aflox/flox mice with Upk3aCre mice that express Cre recombinase in the urothelium. Littermate control mice were of the same genetic background but intact Kdm6a gene. Mice at 8 weeks of age (three in each group) were fed with drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Bladders were harvested after 4 weeks and analyzed with H&E staining. Single cell RNA sequencing was performed on urothelium from control and Kdm6A cKO mice without BBN treatment. RESULTS: After treatment with BBN for 4 weeks, there was marked edema and inflammatory changes with increased infiltration of immune cells in H&E stained bladder sections in Kdm6a cKO mice compared to Kdm6a intact controls (Figure A). While urothelium from control mice maintained grossly unchanged tissue architecture, urothelium from cKO mice exhibited cytologic atypia with loss of organized cell layer. Comparison of single cell RNA sequencing profile revealed enrichment of immune cell population in cKO mice (Figure B, cluster 12), which was consistent with histological findings. CONCLUSIONS: Loss of KDM6A in the urothelium creates a distinct immune microenvironment in the BBN mouse bladder cancer model. Whether this immune cell-rich microenvironment affects response to immune check point inhibition warrants further investigation. Source of Funding: This work was supported in part by the 2021 Urology Care Foundation Residency Research Award Program and the Kahlert Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e80 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ao Zhang More articles by this author Hong Qiu More articles by this author Timothy Chan More articles by this author Byron Lee More articles by this author Expand All Advertisement PDF DownloadLoading ...

KDM3A Inhibition Ameliorates Hyperglycemia-Mediated Myocardial Injury by Epigenetic Modulation of Nuclear Factor Kappa-B/P65.

ObjectivesEven after the glucose level returns to normal, hyperglycemia-induced cardiac dysfunction as well as reactive oxygen species (ROS) generation, inflammatory responses, and apoptosis continued deterioration, showing a long-lasting adverse effect on cardiac function and structure. We aimed to unveil the molecular and cellular mechanisms underlying hyperglycemia-induced persistent myocardial injury and cardiac dysfunction.Methods and ResultsRecently, the accumulated evidence indicated epigenetic regulation act as a determining factor in hyperglycemia-induced continuous cardiovascular dysfunction. As an important histone demethylase, the expression of lysine-specific demethylase 3A (KDM3A) was continually increased, accompanied by a sustained decline of H3K9me2 levels in diabetic myocardium even if received hypoglycemic therapy. Besides, by utilizing gain- and loss-of-functional approaches, we identified KDM3A as a novel regulator that accelerates hyperglycemia-mediated myocardial injury by promoting ROS generation, aggregating inflammatory reaction, and facilitating cell apoptosis in vitro and in vivo. The KDM3A inhibition could significantly ameliorate the adverse effect of hyperglycemia in both diabetes model and diabetic intensive glycemic control model. Mechanically, our data uncovered that KDM3A could promote the expression and transcriptional activity of nuclear factor kappa-B (NF-κB/P65), and the succedent rescue experiments further verified that KDM3A regulates hyperglycemia-induced myocardial injury in an NF-κB/P65 dependent manner.ConclusionThis study revealed histone-modifying enzymes KDM3A drives persistent oxidative stress, inflammation, apoptosis, and subsequent myocardial injury in the diabetic heart by regulating the transcription of NF-κB/P65.

Plasma Proteome of Brain-Dead Organ Donors as a Predictor of Graft-Related Outcomes After Heart Transplantation

<h3>Purpose</h3> Most solid organ transplantations are performed using organs from brain-dead donors. The inflammatory, cardiovascular, hormonal, and metabolic changes related to brain death may affect the quality of the transplanted organs and expose the recipients to pathological changes. We aimed to systemically characterize the plasma proteome of brain-dead heart transplant donors and to search for donor plasma proteins that would be related to graft-related outcome. <h3>Methods</h3> Plasma samples from 53 human brain-dead donors were analyzed by label-free protein quantification using high-definition mass spectrometry. Uni-and multivariate analyses were applied to determine potential proteome differences between brain-dead donors and healthy controls (n=24), and the enriched biological pathways were analyzed. Individual donor protein levels were correlated with clinical transplant outcomes, and predictive donor scores using the significant proteins were constructed. <h3>Results</h3> Uni-and multivariate analyses revealed that brain-dead donors have a distinct plasma proteome, with the levels of 237 of 463 proteins being changed compared to healthy controls. Significant pathways included upregulated coagulation, gluconeogenesis and glycolysis, and downregulated complement, LXR/RXR and production of nitric oxide and reactive oxygen species in macrophages. Lysine-specific demethylase 3A was moderately correlated with any grade and severe PGD in point-biserial correlation. Myosin Va and proteasome activator complex subunit 2 were significantly associated with acute rejections with hemodynamic compromise within 30 days in univariate and multivariate Cox regression analyses. Furthermore, lysine-specific demethylase 3A and moesin were associated with worse graft-related 1-year survival in univariate analysis. <h3>Conclusion</h3> Our detailed analysis revealed that brain death significantly altered plasma proteome. We further discovered altered donor protein signatures that are related to pathological changes such as cardiomyocyte and endothelial dysfunction, inflammation, and vascular growth and arteriogenesis, and correlate with clinical post-transplant outcomes. The current study can be leveraged to discover predictive donor protein signatures in future cohorts, which may lead to biomarker discovery for better donor organ evaluation.

EP189: Insights into the phenotype of KDM1A-related neurodevelopmental disorder: A new chromatinopathy

Chromatinopathies are a molecular heterogeneous group of neurodevelopmental syndromes with typical abnormal face related to congenital chromatin remodelling defects. The lysine-specific demethylase 1A gene (KDM1A, OMIM*609132) encodes a histone demethylase which is involved in chromatin remodeling. Recently, de novo missense variants in KDM1A gene were linked to developmental delay/intellectual disability (DD/ID) with specific dysmorphisms, thus a new chromatinopathy is emerging, namely the Cleft Palate, Psychomotor Retardation and Distinctive Facial Features Syndrome (CPRF, OMIM#616728).

Intravesical delivery of KDM6A-mRNA via mucoadhesive nanoparticles inhibits the metastasis of bladder cancer

Lysine-specific demethylase 6A (KDM6A), also named UTX, is frequently mutated in bladder cancer (BCa). Although known as a tumor suppressor, KDM6A's therapeutic potential in the metastasis of BCa remains elusive. It also remains difficult to fulfill the effective up-regulation of KDM6A levels in bladder tumor tissues in situ to verify its potential in treating BCa metastasis. Here, we report a mucoadhesive messenger RNA (mRNA) nanoparticle (NP) strategy for the intravesical delivery of KDM6A-mRNA in mice bearing orthotopic Kdm6a-null BCa and show evidence of KDM6A's therapeutic potential in inhibiting the metastasis of BCa. Through this mucoadhesive mRNA NP strategy, the exposure of KDM6A-mRNA to the in situ BCa tumors can be greatly prolonged for effective expression, and the penetration can be also enhanced by adhering to the bladder for sustained delivery. This mRNA NP strategy is also demonstrated to be effective for combination cancer therapy with other clinically approved drugs (e.g., elemene), which could further enhance therapeutic outcomes. Our findings not only report intravesical delivery of mRNA via a mucoadhesive mRNA NP strategy but also provide the proof-of-concept for the usefulness of these mRNA NPs as tools in both mechanistic understanding and translational study of bladder-related diseases.

LncRNA HOX transcript antisense RNA mitigates cardiac function injury in chronic heart failure via regulating microRNA-30a-5p to target KDM3A.

Long noncoding RNA HOX transcript antisense RNA (HOTAIR) has been studied in multiple diseases, but the role of HOTAIR on chronic heart failure (CHF) through the regulation of microRNA (miR)‐30a‐5p and lysine‐specific demethylase 3A (KDM3A) remains unexplored. This research aims to probe the effects of HOTAIR on CHF progression via modulating miR‐30a‐5p to target KDM3A. CHF mouse model was established by intraperitoneal injection of doxorubicin. The CHF mice were then injected with high‐expressed HOTAIR, miR‐30a‐5p or KDM3A adenovirus vectors to determine the cardiac function, oxidative stress, inflammatory response, pathological change and cardiomyocyte apoptosis. HOTAIR, miR‐30a‐5p, KDM3A and Bcl‐2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) expression in CHF mice was detected. The binding relations among HOTAIR, miR‐30a‐5p and KDM3A were validated. HOTAIR and KDM3A were depleted, while miR‐30a‐5p was augmented in CHF mice. The elevated HOTAIR or KDM3A or could improve cardiac function, mitigate oxidative stress and pathological change, reduce inflammatory factor levels and cardiomyocyte apoptosis, while the increased miR‐30a‐5p exerted opposite effects. The miR‐30a‐5p elevation could reverse the effects of enriched HOTAIR, while BNIP3 reduction abrogated the effects of KDM3A on CHF. HOTAIR sponged miR‐30a‐5p that targeted KDM3A. HOTAIR improves cardiac injury in CHF via modulating miR‐30a‐5p to target KDM3A. This study provides novel therapeutic strategies for CHF treatment.

KDM1A Identified as a Potential Oncogenic Driver and Prognostic Biomarker via Multi-Omics Analysis.

Background Lysine-specific demethylase 1A (KDM1A) is a histone demethylation enzyme and a crucial epigenetic factor for multiple pathological pathways that mediate carcinogenesis and immunogenicity. Although increasing evidence supposes the association between KDM1A and cancers, no systematic multi-omics analysis of KDM1A is available. Methods We systematically evaluated the KDM1A expression of various cancer and normal tissues and the unique relationship between KDM1A expression and prognosis of cancer cases based on The Cancer Genome Atlas (TCGA), Genotype Tissue Expression (GTEx), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. The genetic variations, phosphorylation, and DNA methylation of KDM1A were analyzed via various tools. We further analyzed the correlation of KDM1A expression and fibroblasts and immune cell infiltration score of TCGA samples via TIMER2.0. Results KDM1A was highly expressed in 17 types of total 33 cancers, while it expressed low levels in only 4 cancers. High KDM1A expression was associated with worse survival status in various cancers. KDM1A expression was positively correlated with the cancer-associated fibroblasts and myeloid-derived suppressor cells infiltration levels in most cancer types. Additionally, KDM1A in most cancer types was negatively correlated with Th1 cell infiltration and positively correlated with Th2 cells. Moreover, spliceosome, cell cycle, and RNA transport pathways were involved in the functional mechanisms of KDM1A via enrichment analysis. Conclusions Our study describes the epigenetic factor KDM1A as an oncogene and prognostic biomarker. Our findings provide valuable guidance for further analysis of KDM1A function in pathogenesis and potential clinical treatment.

Plasma proteome of brain-dead organ donors predicts heart transplant outcome

The pathophysiological changes related to brain death may affect the quality of the transplanted organs and expose the recipients to risks. We probed systemic changes reflected in donor plasma proteome and investigated their relationship to heart transplant outcomes. Plasma samples from brain-dead multi-organ donors were analyzed by label-free protein quantification using high-definition mass spectrometry. Unsupervised and supervised statistical models were used to determine proteome differences between brain-dead donors and healthy controls. Proteome variation and the corresponding biological pathways were analyzed and correlated with transplant outcomes. Statistical models revealed that donors had a unique but heterogeneous plasma proteome with 237 of 463 proteins being changed compared to controls. Pathway analysis showed that coagulation, gluconeogenesis, and glycolysis pathways were upregulated in donors, while complement, LXR/RXR activation, and production of nitric oxide and reactive oxygen species in macrophages pathways were downregulated. In point-biserial correlation analysis, lysine-specific demethylase 3A was moderately correlated with any grade and severe PGD. In univariate and multivariate Cox regression analyses myosin Va and proteasome activator complex subunit 2 were significantly associated with the development of acute rejections with hemodynamic compromise within 30 days. Finally, we found that elevated levels of lysine-specific demethylase 3A and moesin were identified as predictors for graft-related 1-year mortality in univariate analysis. We show that brain death significantly changed plasma proteome signature Donor plasma protein changes related to endothelial cell and cardiomyocyte function, inflammation, and vascular growth and arteriogenesis could predict transplant outcome suggesting a role in donor evaluation.

Establishment of Bovine-Induced Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

Expression pattern and regulatory effect of lysine-specific demethylase 2A gene in clear cell renal cell carcinoma

BackgroundOur study aimed to explore the expression and the biological role of lysine-specific demethylase 2A (KDM2A) in clear cell renal cell carcinoma (ccRCC).MethodsIn vitro, KDM2A expression was measured by qRT-PCR and western blot. A total of 50 patients with ccRCC were included, and KDM2A expression in ccRCC tissues was assessed by qRT-PCR and immunohistochemistry. The effects of KDM2A expression on cell biological behavior were examined by cell counting kit-8 assay, transwell assay and flow cytometry, respectively. The prognostic value of KDM2A in ccRCC was evaluated by Kaplan–Meier method.ResultsThe KDM2A expression was significantly upregulated in ccRCC cell line (P < 0.05). Compared with para cancer tissues, ccRCC samples showed a higher KMD2A mRNA level and a larger proportion of high KDM2A expression (all P < 0.05). High KDM2A mRNA expression was more likely to occur in ccRCC tissues with tumor size > 7 cm (P = 0.005) and T3-T4 stage (P = 0.047). Knockdown of KDM2A significantly suppressed the proliferation and invasion, and promoted the apoptosis of ccRCC cells (all P < 0.05). Moreover, Kaplan–Meier survival analysis revealed that higher level of KDM2A expression in ccRCC patients was associated with lower survival rate (P = 0.004).ConclusionsOur findings demonstrated a vital role of KDM2A in the pathogenesis of ccRCC, which provides a new perspective to understand the underlying molecular mechanisms in ccRCC.

KDM1A Promotes Immunosuppression in Hepatocellular Carcinoma by Regulating PD-L1 through Demethylating MEF2D.

Background Immune checkpoint inhibitor therapy targeting antiprogrammed cell death-1 (anti-PD-1) or its ligand (anti-PD-L1) is effective in the treatment of some hepatocellular carcinomas (HCC). Hence, further identification of biological targets related to PD-L1 regulation in HCC is beneficial to improve the clinical efficacy of immunotherapy. Some HCC cells express lysine-specific demethylase 1A (KDM1A), which is implicated in the reduced survival time of patients. Here, we studied whether the level of PD-L1 and the immunosuppression are regulated by KDM1A and its miRNA in HCC cells. Methods In the present study, we studied clinical data from The Cancer Genome Atlas (TCGA) database. We performed qPCR and western blotting assays to measure the expression level of genes of interest. PD-L1 expression was also analyzed by FACS. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate gene knockout cells to investigate the relationships of genes of interest. We also developed a reporter gene assay (RGA) to explore the changes in T cell-induced antitumor immunity relative to PD-L1 expression in HCC cells. The binding between proteins and promoters or miRNAs and their target genes was explored by luciferase reporter assays. Results The results showed that PD-L1 and KDM1A were increased in HCC patients and cells, and KDM1A promoted the expression of PD-L1 in HCC cells. Our findings showed that the enhancement of PD-L1 expression was not attributed to mitochondrial dysfunction caused by increases in KDM1A in HCC cells. Furthermore, we observed a lower level of MEF2D methylation in HCC cells than in normal human liver cells. Demethylated MEF2D could bind to the promoter of PD-L1 and activate its expression, while KDM1A interacted with MEF2D and acted as a demethylase to reduce its methylation. Moreover, a new miRNA, miR-329-3p, targeting KDM1A was found to regulate the PD-L1 expression profile in HCC cells. In the xenograft model, the tumors treated with miR-329-3p showed growth inhibition. Conclusions Mechanistically, miR-329-3p inhibits tumor cellular immunosuppression and reinforces the response of tumor cells to T cell-induced cytotoxic effect by targeting KDM1A mRNA and downregulating its expression, which contributed to MEF2D demethylation and activation of PD-L1 expression.

Oral Administration of the LSD1 Inhibitor OG-S1335 Increases Fetal Hemoglobin in Humanized Transgenic Sickle Cell Disease Mice and in Baboons

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New and more effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Although BM transplantation, gene therapy, and gene editing technologies offer the promise of a cure, it is unlikely that these methodologies can be implemented to treat the large numbers of patients projected worldwide, including those in developing nations that could most easily be treated with an effective, orally administered drug therapy that increased HbF. The lysine-specific demethylase 1A (KDM1A, also referred to as LSD1), is a component of co-repressor complexes that repress γ-globin gene expression and a therapeutic target for HbF reactivation (Shi et al, Nat Med 19:291, 2013). Our laboratory has shown that subcutaneous administration of RN-1, a pharmacological inhibitor of LSD1, increased γ-globin expression in SCD transgenic mice (Rivers et al, Exp Hematol 43:546, 2015; Cui et al, Blood 126:386, 2015) and in baboons (Rivers et al, Haematol 101:688, 2016), widely acknowledged as the best animal model to test the activity of HbF-inducing drugs due to conservation of structure and developmental regulation of the β-globin locus among simian primates. The objective of this investigation was to test whether oral administration of a new LSD1 inhibitor, OG-S1335, could increase HbF in SCD transgenic mice and baboons. Oral administration of varying doses (0.25, 0.5, 1.0mg/kg/d; 4d) of OG-S1335 to SCD mice (3 mice per dose) increased γ-globin expression and the percentage of circulating erythrocytes and reticulocytes positive for HbF (F cells and F retics; Table 1). In baboons, dose escalation was initially performed by administering the drug to a single animal in cycles of 3 days, followed by 1-2 weeks of observation to measure effects on blood parameters. Increased levels of F retics were initially observed at a dose of 100μg/kg/d and subsequently 4 additional baboons were treated with OG-S1335 (by oral gavage) at a dose of either 100 or 200μg/kg/d (7 separate experiments in total). The data presented are in the mean+SD format, with “post-dose” indicating the time point when the highest variation from baseline levels was reported. Increased F retics (pre=8.4+8.9%, post=43.0+27.9%; p<0.01), γ-globin chain synthesis (pre=0.05+0.05 γ/γ+β, post=0.28+0.18 γ/γ+β; p<0.01), and γ-globin mRNA (pre=0.02 +0.02 γ/γ+β, post=0.20+0.11 γ/γ+β) were observed with maximal levels achieved 7 days after administration of the first dose (d8). A decrease in the absolute number of reticulocytes (pre=72.8+17.8 X104/μL, post=35.4+17.8 X 104/μL; p<0.05; nadir on d6-8), neutrophils (pre=4634+994/μL, post=2655+1257/μL; p<0.02; nadir on d13-17), and platelets (pre=406+86 X103/μL, post=168+98 X103/μL; p<0.01; nadir on d8) was also reported, together with increased monocytes (pre=200+68/μL, post=541+213/μL; p<0.01; peak on d10-15). A negative correlation between decreased platelet counts and F retics (r= -0.55), γ-globin chain synthesis (r= -0.92), and γ-globin mRNA (r= -0.80) suggests that variability in drug exposure in addition to possible genetic factors regulating γ-globin expression may be responsible for the observed variability between animals. Additional experiments were performed in two phlebotomized anemic baboons to test the effect of the drug in a model with increased relevance to sickle cell disease. OG-S1335 (100μg/kg/d; 3d) induced high levels of F retics (PA8695, pre-dose=24%, post=66.8%; PA8698: pre-dose=13%, post=93.6%), γ-globin chain synthesis (PA8695: pre-dose=0.07 γ/γ+β, post=0.20 γ/γ+β; PA8698: pre-dose=0.02 γ/γ+β, post=0.44 γ/γ+β) and γ-globin mRNA (PA8695: pre-dose=0.06 γ/γ+β, post=0.18 γ/γ+β; PA8698: pre-dose=0.03 γ/γ+β, post=0.33 γ/γ+β) in each animal. We conclude that oral administration of the LSD1 inhibitor OG-S1335 significantly increased F retics, γ-globin chain synthesis, and γ-globin mRNA in SCD mice and in baboons, supporting further efforts toward the development of this drug for SCD therapy. [Display omitted] DisclosuresCiceri:Oryzon Genomics SA: Employment. Cavalcanti:Oryzon Genomics SA: Employment.