Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis.

The histone lysine demethylase KDM5B is frequently up-regulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with the miR-140-3p level in primary human AML cells. Molecular studies showed that silencing KDM5B enhanced H3K4 trimethylation (H3K4me3) at the promoter of miR-140-3p, leading to high expression of miR-140-3p, which in turn inhibited B-cell CLL/lymphoma 2 (BCL2) expression. Finally, we demonstrate that the defective proliferation induced by KDM5B knockdown (KD) can be rescued with the miR-140-3p inhibitor or enhanced by combining KDM5B KD with a BCL2 inhibitor. Altogether, our data support the conclusion that KDM5B promotes tumorigenesis in human AML cells through the miR-140-3p/BCL2 axis. Targeting the KDM5B/miR-140-3p/BCL2 pathway may hold therapeutic promise for treating human AML.

The Intellectual Disability Risk Gene Kdm5b Regulates Long-Term Memory Consolidation in the Hippocampus.

The histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders, and heterozygous, protein-truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5b Δ ARID allele that lacks demethylase activity. Kdm5b Δ ARID/ Δ ARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon a learning stimulus compared with wild-type (WT) mice. A number of other learning-associated genes were also significantly dysregulated in the Kdm5b Δ ARID/ Δ ARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, WT mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity, and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms.

A PRC2-Kdm5b axis sustains tumorigenicity of acute myeloid leukemia.

Acute myeloid leukemias (AMLs) with the NUP98-NSD1 or mixed lineage leukemia (MLL) rearrangement (MLL-r) share transcriptomic profiles associated with stemness-related gene signatures and display poor prognosis. The molecular underpinnings of AML aggressiveness and stemness remain far from clear. Studies with EZH2 enzymatic inhibitors show that polycomb repressive complex 2 (PRC2) is crucial for tumorigenicity in NUP98-NSD1+ AML, whereas transcriptomic analysis reveal that Kdm5b, a lysine demethylase gene carrying "bivalent" chromatin domains, is directly repressed by PRC2. While ectopic expression of Kdm5b suppressed AML growth, its depletion not only promoted tumorigenicity but also attenuated anti-AML effects of PRC2 inhibitors, demonstrating a PRC2-|Kdm5b axis for AML oncogenesis. Integrated RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and Cleavage Under Targets & Release Using Nuclease (CUT&RUN) profiling also showed that Kdm5b directly binds and represses AML stemness genes. The anti-AML effect of Kdm5b relies on its chromatin association and/or scaffold functions rather than its demethylase activity. Collectively, this study describes a molecular axis that involves histone modifiers (PRC2-|Kdm5b) for sustaining AML oncogenesis.

Co-targeting SKP2 and KDM5B inhibits prostate cancer progression by abrogating AKT signaling with induction of senescence and apoptosis.

BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. METHODS: Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. RESULTS: SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated ß-galactosidase (SA-ß-Gal) staining. CONCLUSIONS: Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.

KDM5B promotes metastasis and epithelial-mesenchymal transition via Wnt/ß-catenin pathway in squamous cell carcinoma of the head and neck.

Metastasis determines clinical management decision and restricts the therapeutic efficiency in patients with squamous cell carcinoma of the head and neck (SCCHN). Epigenetic factor KDM5B serves as an oncogene in multiple cancers. However, its role in SCCHN metastasis remains unclear. Our previous study showed that KDM5B is significantly elevated in SCCHN tissue and is positively correlated with metastasis and recurrence. KDM5B overexpression predicted a poor prognosis in both disease-free survival and overall survival, which served as an independent prognostic factor in SCCHN patients. This study further investigates the exact impact of KDM5B in metastasis of SCCHN. We found that KDM5B knockdown significantly inhibits the migration and invasion of SCCHN cells both in vitro and in vivo. On the contrary, forced expression of KDM5B leads to enhanced migration and invasion, accompanied by canonical alterations of epithelial-mesenchymal transition (EMT). Mechanism investigations demonstrated that KDM5B activates Wnt/ß-catenin pathway, and inhibition of Wnt/ß-catenin pathway via a small molecule inhibitor iCRT-14 partially reverses the enhanced migratory and invasive ability caused by KDM5B in SCCHN cells. Together, our data indicate that KDM5B promotes EMT and metastasis via Wnt/ß-catenin pathway in SCCHN, suggesting that KDM5B may be a potential therapeutic target and prognosis biomarker in SCCHN.

Inhibition of KDM5B participates in immune microenvironment remodeling in pancreatic cancer by inducing STING expression.

OBJECTIVE: This study aims to investigate the effect of lysine demethylase 5B (KDM5B)-mediated dimethyl-lysine 4 histone H3 (H3K4me2) demethylation on immune microenvironment remodeling in pancreatic cancer. METHODS: Pan 02 mouse pancreatic cancer cell lines were cultured and used to establish tumor model in vivo. RT-qPCR and Western blot were used to detect the expression of stimulator of interferon gene (STING) and KDM5B in pancreatic cancer tissues and Pan 02 cells. The specific demethylation domain of KDM5B was detected by isothermal titration calorimetry binding assay. The regulatory roles of KDM5B in cell apoptosis and remodeling of immune microenvironment in vitro and in vivo were explored after loss-of functions in KDM5B. RESULTS: KDM5B was highly expressed but STING was poorly expressed in pancreatic cancer tissues and Pan 02 cells. After knockdown of KDM5B, CD8+ T cells recognized and killed Pan 02 cells, which promoted the infiltration of CD8+ T cells in Pan 02 cells, thus improving the anti-tumor ability. The PHD domain in KDM5B specifically bound to H3K4me2 peptide and inhibition of KDM5B induced STING expression. Knockdown of KDM5B up-regulated STING expression to promote apoptosis, thus regulating the immune microenvironment and inhibiting the growth of tumor in mice. Meanwhile, knockdown of KDM5B and STING simultaneously counteracted the knockdown effect of KDM5B. CONCLUSION: Inhibition of KDM5B can promote the expression of STING through H3K4me2 demethylation, which promoted the recognition and killing of Pan 02 cells by CD8+ T cells, thus improving the anti-tumor ability and regulating the immune microenvironment.

Lysine demethylase 5B (KDM5B): A key regulator of cancer drug resistance.

Chemoresistance, a roadblock in the chemotherapy process, has been impeding its effective treatment. KDM5B, a member of the histone demethylase family, has been crucial in the emergence and growth of malignancies. More significantly, KDM5B has recently been linked closely to cancer's resistance to chemotherapy. In this review, we explain the biological properties of KDM5B, its function in the emergence and evolution of cancer treatment resistance, and our hopes for future drug resistance-busting combinations involving KDM5B and related targets or medications.

Isorhamnetin Alleviates Mitochondrial Injury in Severe Acute Pancreatitis via Modulation of KDM5B/HtrA2 Signaling Pathway.

Severe acute pancreatitis (SAP), a widespread inflammatory condition impacting the abdomen with a high mortality rate, poses challenges due to its unclear pathogenesis and the absence of effective treatment options. Isorhamnetin (ISO), a naturally occurring flavonoid, demonstrates robust antioxidant and anti-inflammatory properties intricately linked to the modulation of mitochondrial function. However, the specific protective impact of ISO on SAP remains to be fully elucidated. In this study, we demonstrated that ISO treatment significantly alleviated pancreatic damage and reduced serum lipase and amylase levels in the mouse model of SAP induced by sodium taurocholate (STC) or L-arginine. Utilizing an in vitro SAP cell model, we found that ISO co-administration markedly prevented STC-induced pancreatic acinar cell necrosis, primarily by inhibiting mitochondrial ROS generation, preserving ATP production, maintaining mitochondrial membrane potential, and preventing the oxidative damage and release of mitochondrial DNA. Mechanistically, our investigation identified that high-temperature requirement A2 (HtrA2) may play a central regulatory role in mediating the protective effect of ISO on mitochondrial dysfunction in STC-injured acinar cells. Furthermore, through an integrated approach involving bioinformatics analysis, molecular docking analysis, and experimental validation, we uncovered that ISO may directly impede the histone demethylation activity of KDM5B, leading to the restoration of pancreatic HtrA2 expression and thereby preserving mitochondrial function in pancreatic acinar cells following STC treatment. In conclusion, this study not only sheds new light on the intricate molecular complexities associated with mitochondrial dysfunction during the progression of SAP but also underscores the promising value of ISO as a natural therapeutic option for SAP.

Knockdown of KDM5B Leads to DNA Damage and Cell Cycle Arrest in Granulosa Cells via MTF1.

KDM5B is essential for early embryo development, which is under the control of maternal factors in oocytes. Granulosa cells (GCs) play a critical role during oocyte mature. However, the role of KDM5B in GCs remains to be elucidated. In the current study, we found that KDM5B expressed highly in the ovaries and located in goat GCs. Using an RNA sequence, we identified 1353 differentially expressed genes in the KDM5B knockdown GCs, which were mainly enriched in cell cycle, cell division, DNA replication and the cellular oxidative phosphorylation regulation pathway. Moreover, we reported a decrease in the percentage of proliferated cells but an increase in the percentage of apoptotic cells in the KDM5B knockdown GCs. In addition, in the KDM5B knockdown GCs, the percentage of GCs blocked at the S phase was increased compared to the NC group, suggesting a critical role of KDM5B in the cell cycle. Moreover, in the KDM5B knockdown GCs, the reactive oxygen species level, the mitochondrial depolarization ratio, and the expression of intracellular phosphorylated histone H2AX (γH2AX) increased, suggesting that knockdown of KDM5B leads to DNA damage, primarily in the form of DNA double-strand breaks (DSBs). Interestingly, we found a down-regulation of MTF1 in the KDM5B knockdown GCs, and the level of cell proliferation, as well as the cell cycle block in the S phase, was improved. In contrast, in the group with both KDM5B knockdown and MTF1 overexpression, the level of ROS, the expression of γH2AX and the number of DNA DSB sites decreased. Taken together, our results suggest that KDM5B inhibits DNA damage and promotes the cell cycle in GCs, which might occur through the up-regulation of MTF1.

Lysine demethylase KDM5B down-regulates SIRT3-mediated mitochondrial glucose and lipid metabolism in diabetic neuropathy.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a common neurological complication of diabetes mellitus without efficient interventions. Both lysine demethylase 5B (KDM5B) and sirtuin-3 (SIRT3) have been found to regulate islet function and glucose homeostasis. KDM5B was predicted to bind to the SIRT3 promoter by bioinformatics. Here, we investigated whether KDM5B affected DPN development via modulating SIRT3. METHODS: The db/db mice and high glucose-stimulated Schwann cells (RSC96) were used as in vivo and in vitro models of DPN, respectively. Glucose level, glucose and insulin tolerance of mice were measured. Neurological function was evaluated by motor nerve conduction velocity (MNCV), tactile allodynia assay and thermal sensitivity assay. Adenosine triphosphate level, oxygen consumption rate, extracellular acidification rate, ß-oxidation rate, acetyl-CoA level, acetylation levels and activities of long-chain acyl CoA dehydrogenase (LCAD) and pyruvate dehydrogenase (PDH) were detected. Methyl thiazolyl tetrazolium assay was adopted to determine cell viability. Reactive oxygen species (ROS) production was detected by MitoSox staining. Western blotting for measuring target protein levels. Molecular mechanisms were investigated by co-immunoprecipitine (Co-IP), chromatin immunoprecipitation (ChIP) and luciferase reporter assay. RESULTS: KDM5B was up-regulated, while SIRT3 was down-regulated in DPN models. SIRT3 overexpression or AMPK activation ameliorated mitochondrial metabolism dysfunction and ROS overproduction during DPN. KDM5B overexpression triggered mitochondrial metabolism disorder and oxidative stress via directly transcriptional inhibiting SIRT3 expression by demethylating H3K4me3 or indirectly repressing AMPK pathway-regulated SIRT3 expression. CONCLUSION: KDM5B contributes to DPN via regulating SIRT3-mediated mitochondrial glucose and lipid metabolism. KDM5B inhibition may be an effective intervention for DPN.

Histone demethylase KDM5B catalyzed H3K4me3 demethylation to promote differentiation of bone marrow mesenchymal stem cells into cardiomyocytes.

BACKGROUND: Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported. OBJECTIVE: To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro. METHODS: Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection. RESULT: Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 and myocardial marker gene α-Actin and cTNT were significantly increased, and the Na+ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. CONCLUSIONS: It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels.

KDM5B promotes cell migration by regulating the noncanonical Wnt/PCP pathway in Hirschsprung's disease.

PURPOSE: We measured the expression of the histone demethylase lysine-specific demethylase 5B (KDM5B) in the bowels of patients with Hirschsprung's disease (HSCR) and investigated the molecular mechanism by which KDM5B promotes the migration of neuronal PC12 cells. METHODS: KDM5B expression was detected in the ganglionic and aganglionic colon of patients with HSCR (n = 10) and controls (n = 10). The expression and localization of KDM5B were assessed using immunohistochemical and immunofluorescence staining. Real-time PCR and Western blotting were performed to quantify KDM5B expression. The migration was determined using Transwell and wound-healing assays. G-LISA, GTPase pulldown and luciferase-based reporter gene assays were performed to evaluate the key components of Wnt/planar cell polarity (PCP) signaling in vitro. RESULTS: Our current study showed that KDM5B colocalized with neurons. KDM5B expression was reduced in HSCR specimens, while the aganglionic segments showed the greatest reduction. KDM5B knockdown inhibited the migration of PC12 cells. Moreover, inhibition of KDM5B decreased the expression of key genes in the Wnt/PCP pathway, and its inhibitory effect on PC12 cell migration was reversed by Wnt5a treatment. CONCLUSIONS: KDM5B promotes neuronal migration via the Wnt/PCP pathway. A potential role for KDM5B in altered enteric nervous system development in HSCR warrants further investigation.

Histone Lysine Methylases and Demethylases in the Landscape of Human Developmental Disorders.

Histone lysine methyltransferases (KMTs) and demethylases (KDMs) underpin gene regulation. Here we demonstrate that variants causing haploinsufficiency of KMTs and KDMs are frequently encountered in individuals with developmental disorders. Using a combination of human variation databases and existing animal models, we determine 22 KMTs and KDMs as additional candidates for dominantly inherited developmental disorders. We show that KMTs and KDMs that are associated with, or are candidates for, dominant developmental disorders tend to have a higher level of transcription, longer canonical transcripts, more interactors, and a higher number and more types of post-translational modifications than other KMT and KDMs. We provide evidence to firmly associate KMT2C, ASH1L, and KMT5B haploinsufficiency with dominant developmental disorders. Whereas KMT2C or ASH1L haploinsufficiency results in a predominantly neurodevelopmental phenotype with occasional physical anomalies, KMT5B mutations cause an overgrowth syndrome with intellectual disability. We further expand the phenotypic spectrum of KMT2B-related disorders and show that some individuals can have severe developmental delay without dystonia at least until mid-childhood. Additionally, we describe a recessive histone lysine-methylation defect caused by homozygous or compound heterozygous KDM5B variants and resulting in a recognizable syndrome with developmental delay, facial dysmorphism, and camptodactyly. Collectively, these results emphasize the significance of histone lysine methylation in normal human development and the importance of this process in human developmental disorders. Our results demonstrate that systematic clinically oriented pathway-based analysis of genomic data can accelerate the discovery of rare genetic disorders.

ACE2 Expression Is Increased in the Lungs of Patients With Comorbidities Associated With Severe COVID-19.

Patients who died from COVID-19 often had comorbidities, such as hypertension, diabetes, and chronic obstructive lung disease. Although angiotensin-converting enzyme 2 (ACE2) is crucial for SARS-CoV-2 to bind and enter host cells, no study has systematically assessed the ACE2 expression in the lungs of patients with these diseases. Here, we analyzed over 700 lung transcriptome samples from patients with comorbidities associated with severe COVID-19 and found that ACE2 was highly expressed in these patients compared to control individuals. This finding suggests that patients with such comorbidities may have higher chances of developing severe COVID-19. Correlation and network analyses revealed many potential regulators of ACE2 in the human lung, including genes related to histone modifications, such as HAT1, HDAC2, and KDM5B. Our systems biology approach offers a possible explanation for increased COVID-19 severity in patients with certain comorbidities.

KDM5B is a master regulator of the H3K4-methylome in stem cells, development and cancer.

Epigenetic regulation of chromatin plays a critical role in controlling stem cell function and tumorigenesis. The histone lysine demethylase, KDM5B, which catalyzes the demethylation of histone 3 lysine 4 (H3K4), is important for embryonic stem (ES) cell differentiation, and is a critical regulator of the H3K4-methylome during early mouse embryonic pre-implantation stage development. KDM5B is also overexpressed, amplified, or mutated in many cancer types. In cancer cells, KDM5B regulates expression of oncogenes and tumor suppressors by modulating H3K4 methylation levels. In this review, we examine how KDM5B regulates gene expression and cellular fates of stem cells and cancer cells by temporally and spatially controlling H3K4 methylation levels.

KDM5B promotes breast cancer cell proliferation and migration via AMPK-mediated lipid metabolism reprogramming.

Lysine demethylase 5B (KDM5B) is up-regulated in many cancers, including breast cancer. However, the underlying metabolic mechanisms of KDM5B on breast cancer progression are poorly understood. Here, we showed that KDM5B expression positively correlates with metastasis in breast cancer. Cell functional analyses were demonstrated that KDM5B knockdown and KDM5B inhibitor AS-8351 inhibited breast cancer cell proliferation and migration. Furthermore, we reported that KDM5B knockdown and AS-8351 reversed epithelial-mesenchymal transition (EMT) and decreased the protein levels of fatty acid synthase (FASN) and ATP citrate lyase (ACLY) in MCF-7 and MDA-MB-231 cells. Interestingly, we found that activation of AMP-activated protein kinase (AMPK) signaling pathway is involved in KDM5B-mediated EMT and lipid metabolism reprogramming in breast cancer cells. As a result, silencing of KDM5B-induced activation of AMPK signaling pathway inhibited breast cancer cell proliferation and migration. Taken together, our findings indicated that KDM5B was a novel regulator of lipid metabolism reprogramming, and it was suggested a new strategy to treat breast cancer.

Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells.

BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

Epigenetic Regulation of Angiogenesis by JARID1B-Induced Repression of HOXA5.

OBJECTIVE: Altering endothelial biology through epigenetic modifiers is an attractive novel concept, which is, however, just in its beginnings. We therefore set out to identify chromatin modifiers important for endothelial gene expression and contributing to angiogenesis. APPROACH AND RESULTS: To identify chromatin modifying enzymes in endothelial cells, histone demethylases were screened by microarray and polymerase chain reaction. The histone 3 lysine 4 demethylase JARID1B was identified as a highly expressed enzyme at the mRNA and protein levels. Knockdown of JARID1B by shRNA in human umbilical vein endothelial cells attenuated cell migration, angiogenic sprouting, and tube formation. Similarly, pharmacological inhibition and overexpression of a catalytic inactive JARID1B mutant reduced the angiogenic capacity of human umbilical vein endothelial cells. To identify the in vivo relevance of JARID1B in the vascular system, Jarid1b knockout mice were studied. As global knockout results in increased mortality and developmental defects, tamoxifen-inducible and endothelial-specific knockout mice were generated. Acute knockout of Jarid1b attenuated retinal angiogenesis and endothelial sprout outgrowth from aortic segments. To identify the underlying mechanism, a microarray experiment was performed, which led to the identification of the antiangiogenic transcription factor HOXA5 to be suppressed by JARID1B. Importantly, downregulation or inhibition of JARID1B, but not of JARID1A and JARID1C, induced HOXA5 expression in human umbilical vein endothelial cells. Consistently, chromatin immunoprecipitation revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. CONCLUSIONS: JARID1B, by suppressing HOXA5, maintains the endothelial angiogenic capacity in a demethylase-dependent manner.

Histone demethylase jumonji AT-rich interactive domain 1B (JARID1B) controls mammary gland development by regulating key developmental and lineage specification genes.

The JmjC domain-containing H3K4 histone demethylase jumonji AT-rich interactive domain 1B (JARID1B) (also known as KDM5B and PLU1) is overexpressed in breast cancer and is a potential target for breast cancer treatment. To investigate the in vivo function of JARID1B, we developed Jarid1b(-/-) mice and characterized their phenotypes in detail. Unlike previously reported Jarid1b(-/-) strains, the majority of these Jarid1b(-/-) mice were viable beyond embryonic and neonatal stages. This allowed us to further examine phenotypes associated with the loss of JARID1B in pubertal development and pregnancy. These Jarid1b(-/-) mice exhibited decreased body weight, premature mortality, decreased female fertility, and delayed mammary gland development. Related to these phenotypes, JARID1B loss decreased serum estrogen level and reduced mammary epithelial cell proliferation in early puberty. In mammary epithelial cells, JARID1B loss diminished the expression of key regulators for mammary morphogenesis and luminal lineage specification, including FOXA1 and estrogen receptor α. Mechanistically, JARID1B was required for GATA3 recruitment to the Foxa1 promoter to activate Foxa1 expression. These results indicate that JARID1B positively regulates mammary ductal development through both extrinsic and cell-autonomous mechanisms.