Phosphorylation Of Histone H3 Research Articles

The Multikinase Inhibitor AD80 Induces Mitotic Catastrophe and Autophagy in Pancreatic Cancer Cells.

Significant advances in understanding the molecular complexity of the development and progression of pancreatic cancer have been made, but this disease is still considered one of the most lethal human cancers and needs new therapeutic options. In the present study, the antineoplastic effects of AD80, a multikinase inhibitor, were investigated in models of pancreatic cancer. AD80 reduced cell viability and clonogenicity and induced polyploidy in pancreatic cancer cells. At the molecular level, AD80 reduced RPS6 and histone H3 phosphorylation and induced γH2AX and PARP1 cleavage. Additionally, the drug markedly decreased AURKA phosphorylation and expression. In PANC-1 cells, AD80 strongly induced autophagic flux (consumption of LC3B and SQSTM1/p62). AD80 modulated 32 out of 84 autophagy-related genes and was associated with vacuole organization, macroautophagy, response to starvation, cellular response to nitrogen levels, and cellular response to extracellular stimulus. In 3D pancreatic cancer models, AD80 also effectively reduced growth independent of anchorage and cell viability. In summary, AD80 induces mitotic aberrations, DNA damage, autophagy, and apoptosis in pancreatic cancer cells. Our exploratory study establishes novel targets underlying the antineoplastic activity of the drug and provides insights into the development of therapeutic strategies for this disease.

Histone H3K79 methylation by DOT1L promotes Aurora B localization at centromeres in mitosis

Centromere localization of the chromosome passenger complex (CPC) is paramount for achieving accurate sister chromosome segregation in mitosis. Although it has been widely recognized that the recruitment of CPC is directly regulated by two histone codes, phosphorylation of histone H3 at threonine 3 (H3T3ph) and phosphorylation of histone H2A at threonine 120 (H2AT120ph), the regulation of CPC localization by other histone codes remains elusive. We show that dysfunction of disruptor of telomeric silencing 1 like (DOT1L) leads to mislocation of the CPC in prometaphase, caused by disturbing the level of H3T3ph and its reader Survivin. This cascade is initiated by over-dephosphorylation of H3T3ph mediated by the phosphatase RepoMan-PP1, whose scaffold RepoMan translocalizes to chromosomes, while the level of H3K79me2/3 is diminished. Together, our findings uncover a biological function of DOT1L and H3K79 methylation in mitosis and give insight into how genomic stability is coordinated by different histone codes.

BI6727 and GSK461364A, potent PLK1 inhibitors induce G2/M arrest and apoptosis against cholangiocarcinoma cell lines

Polo-like kinase 1 (PLK1) is an essential mitotic checkpoint protein that plays a key role in cell cycle division. Overexpression of PLK1 has been associated with poor prognosis in various cancers. Cholangiocarcinoma (CCA) is a lethal bile duct cancer and the current treatments in inoperable patients have not been satisfactory. In order to develop novel targeted therapies, we investigated the efficacy of BI6727 (volasertib) and GSK461364A, polo-like kinase 1 (PLK1) inhibitors in KKU-100 and KKU-213A CCA cell lines. PLK1 expression was significantly up-regulated in CCA cases compared with normal tissues based on the results derived from GEPIA. Western blot results exhibited PLK1 protein expression in both CCA cell lines. Molecular dynamics simulations and free energy calculations based on MM/GBSA method revealed that BI6727-PLK1 and GSK461364A-PLK1 complexes were stable in an aqueous environment, and their complexation was mainly driven by Van der Waals interaction. BI6727 and GSK461364A clearly suppressed CCA cell proliferation and induced G2/M arrest, accompanied with upregulation of cyclin B1 and phosphorylated Histone H3 at Ser10 (pS10H3), specific markers of mitosis. Furthermore, both compounds triggered mitotic catastrophe followed by cell apoptosis via activation of PARP and Caspase 3, as well as downregulation of Mcl-1 anti-apoptotic protein in both CCA cell lines. In conclusion, pharmacologic PLK1 inhibition by BI6727 and GSK461364A blocked survival of CCA cells by several mechanisms. Our study provides evidence that BI6727 and GSK461364A could be alternative drugs and have potential implications at the clinical level for CCA therapy.

Astragaloside IV Blunts Epithelial-Mesenchymal Transition and G2/M Arrest to Alleviate Renal Fibrosis via Regulating ALDH2-Mediated Autophagy.

Previous studies show that astragaloside IV (ASIV) has anti-renal fibrosis effects. However, its mechanism remains elusive. In this study, we investigated the anti-fibrosis mechanisms of ASIV on chronic kidney disease (CKD) in vivo and in vitro. A CKD model was induced in rats with adenine (200 mg/kg/d, i.g.), and an in vitro renal fibrosis model was induced in human kidney-2 (HK-2) cells treated with TGF-β1. We revealed that ASIV significantly alleviated renal fibrosis by suppressing the expressions of epithelial-mesenchymal transition (EMT)-related proteins, including fibronectin, vimentin, and alpha-smooth muscle actin (α-SMA), and G2/M arrest-related proteins, including phosphorylated p53 (p-p53), p21, phosphorylated histone H3 (p-H3), and Ki67 in both of the in vivo and in vitro models. Transcriptomic analysis and subsequent validation showed that ASIV rescued ALDH2 expression and inhibited AKT/mTOR-mediated autophagy. Furthermore, in ALDH2-knockdown HK-2 cells, ASIV failed to inhibit AKT/mTOR-mediated autophagy and could not blunt EMT and G2/M arrest. In addition, we further demonstrated that rapamycin, an autophagy inducer, reversed the treatment of ASIV by promoting autophagy in TGF-β1-treated HK-2 cells. A dual-luciferase report assay indicated that ASIV enhanced the transcriptional activity of the ALDH2 promoter. In addition, a further molecular docking analysis showed the potential interaction of ALDH2 and ASIV. Collectively, our data indicate that ALDH2-mediated autophagy may be a novel target in treating renal fibrosis in CKD models, and ASIV may be an effective targeted drug for ALDH2, which illuminate a new insight into the treatment of renal fibrosis and provide new evidence of pharmacology to elucidate the anti-fibrosis mechanism of ASIV in treating renal fibrosis.

7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling.

Accumulation of 7-ketocholesterol (7KC) occurs in age-related macular degeneration (AMD) and was found previously to promote fibrosis, an untreatable cause of vision loss, partly through induction of endothelial-mesenchymal transition. To address the hypothesis that 7KC causes mesenchymal transition of retinal pigment epithelial cells (RPE), we exposed human primary RPE (hRPE) to 7KC or a control. 7KC-treated hRPE did not manifest increased mesenchymal markers, but instead maintained RPE-specific proteins and exhibited signs of senescence with increased serine phosphorylation of histone H3, serine/threonine phosphorylation of mammalian target of rapamycin (p-mTOR), p16 and p21, β-galactosidase labeling, and reduced LaminB1, suggesting senescence. The cells also developed senescence-associated secretory phenotype (SASP) determined by increased IL-1β, IL-6, and VEGF through mTOR-mediated NF-κB signaling, and reduced barrier integrity that was restored by the mTOR inhibitor, rapamycin. 7KC-induced p21, VEGF, and IL-1β were inhibited by an inhibitor of protein kinase C. The kinase regulates IQGAP1 serine phosphorylation. Furthermore, after 7KC injection and laser-induced injury, mice with an IQGAP1 serine 1441-point mutation had significantly reduced fibrosis compared to littermate control mice. Our results provide evidence that age-related accumulation of 7KC in drusen mediates senescence and SASP in RPE, and IQGAP1 serine phosphorylation is important in causing fibrosis in AMD.

In Silico Discovery of 5'-Modified 7-Deoxy-7-ethynyl-4'-thioadenosine as a HASPIN Inhibitor and Its Synergistic Anticancer Effect with the PLK1 Inhibitor.

Despite genetic perturbations resulting in embryo lethality for most mitotic kinases, loss of the histone H3 mitotic kinase HASPIN reveals no adverse effect in mice models, establishing HASPIN as a promising target for anticancer therapy. However, developing a HASPIN inhibitor from conventional pharmacophores poses a technical challenge as this atypical kinase shares slight similarities with eukaryotic protein kinases. Chemically modifying a cytotoxic 4'-thioadenosine analogue through high genotoxicity yielded several novel nongenotoxic kinase inhibitors. In silico apporoaches utilizing transcriptomic and chemical similarities with known compounds and KINOMEscan profiles unveiled the HASPIN inhibitor LJ4827. LJ4827's specificity and potency as a HASPIN inhibitor were verified through in vitro kinase assay and X-ray crystallography. HASPIN inhibition by LJ4827 reduced histone H3 phosphorylation and impeded Aurora B recruitment in cancer cell centromeres but not in noncancer cells. Through transcriptome analysis of lung cancer patients, PLK1 was determined as a druggable synergistic partner to complement HASPIN inhibition. Chemical or genetic PLK1 perturbation with LJ4827 effectuated pronounced lung cancer cytotoxicity in vitro and in vivo. Therefore, LJ4827 is a novel anticancer therapeutic for selectively impeding cancer mitosis through potent HASPIN inhibition, and simultaneous HASPIN and PLK1 interference is a promising therapeutic strategy for lung cancer.

Improving fine needle aspiration to predict the tumor biological aggressiveness in pancreatic neuroendocrine tumors using Ki-67 proliferation index, phosphorylated histone H3 (PHH3), and BCL-2

IntroductionSurgery is the only known cure for sporadic pancreatic neuroendocrine tumors (PNETs). Therefore, the prediction of the PNETs biological aggressiveness evaluated on endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has a significant impact on clinical management. The proliferation rate of Ki-67 in PNETs can help to predict the biological aggressiveness of the tumor. In addition, there is a relatively new proliferation marker called phosphorylated histone H3 (PHH3) that can identify and quantify dividing cells in tissue samples, which is a marker highly specific to mitotic figures. Other markers such as BCL-2 also contribute to tumorigenesis and may be involved in the differentiation of neuroendocrine cells. Materials and methodsA retrospective observational study was performed on patients undergoing surveillance for PNETs from January 2010 to May 2021. Data collection included the patients' age, sex, tumor location, tumor size in the surgical specimen, and tumor grade in FNA. The 2019 World Health Organization (WHO) classification guideline was followed to diagnose PNETs, including grade and stage. Immunohistochemical stainings for Ki-67, PHH3 and BCL-2 in PNETs were performed. ResultsAfter excluding cell blocks containing fewer than 100 tumor cells, 44 patients with EUS-FNA and surgical resection specimens were included in this study. There were 19 cases of G1 PNETs, 20 cases of G2 PNETs, and 5 cases of G3 PNETs. The grade assigned based on the Ki-67 index was higher and more sensitive than that based on the mitotic count using H&E slides in some cases of G2 and G3 PNETs. However, there was no significant difference between the mitotic count using PHH3-positive tumor cells and the Ki-67 index to grade PNETs.All grade 1 tumors (19 cases) on surgical resection specimens were correctly graded on FNA (100 % concordance rate). Within the 20 G2 PNETs, 15 cases of grade 2 on surgical resection specimens were graded correctly on FNA based on the Ki-67 index only. Five cases of grade 2 PNETs on surgical resection specimens were graded as grade 1 on FNA when using only the Ki-67 index. Three of five grade 3 tumors on surgical resection specimens were graded as grade 2 on FNA based on the Ki-67 index only. Using only FNA Ki-67 to predict PNET tumor grade, the concordance (accuracy) rate was 81.8 % in total. However, all these eight cases (5 cases of G2 PNETs and 3 cases of G3 PNETs) were graded correctly by using the Ki-67 index plus mitotic rate (using PHH3 IHC stains).Four of 18 (22.2 %) patients with PNETs were positive for BCL-2 stain. In these 4 cases positive for BCL-2 stains, 3 cases were G2 PNETs and one case was G3 PNETs. ConclusionGrade and the proliferative rate in EUS-FNA can be used to predict the tumor grade in surgical resection specimens. However, when using only FNA Ki-67 to predict PNET tumor grade, about 18 % of cases were downgraded by one level. To solve the problem, immunohistochemical staining for BCL-2 and especially PHH3 would be helpful. Our results demonstrated that the mitotic count using PHH3 IHC stains not only improved the accuracy and precision of PNET grading in the surgical resection specimens, but also could reliably be used in routine scoring of mitotic figures of FNA specimens.

AURKB Enhances Chromosomal Remodeling of Telomeric Genes and Accelerates Tumorigenesis of Uveal Melanoma.

Uveal melanoma (UM) is the most common intraocular malignancy in adults developing liver metastases, threatening a patient's life. Current therapeutics failed to significantly improve the survival of patients with UM. Thus, the discovery of potent drugs is imminent. Integrated bioinformatic analysis of The Cancer Genome Atlas and immunohistochemistry staining of patients' tissues revealed the oncogenic role of aurora kinase B (AURKB) in UM. Drug sensitivity assays and an orthotopic intraocular animal model were used to test the efficacy of AURKB inhibitors. RNA sequencing and immunoblotting were performed to identify the downstream effector. A chromatin immunoprecipitation assay was conducted to elucidate AURKB's transcriptional regulation on the target gene. AURKB was found overexpressed in patients with UM, resulting in a poor prognosis. Luckily, the AURKB-specific inhibitor, hesperadin, achieved prominent pharmacological efficiency in UM in vitro and in vivo. Mechanically, hesperadin compromised phosphorylation of histone H3 at serine 10 (H3S10ph) at the promoter of telomerase reverse transcriptase, accompanied by methylation of histone H3 at lysine 9. This methylated status of the promoter region forced chromatin condensation and consequently halted the transcription of telomerase reverse transcriptase. Collectively, our data demonstrated that AURKB inhibitors decelerated UM tumorigenesis by epigenetically silencing the expression of oncogenic telomerase reverse transcriptase, indicating AURKB as a potential therapeutic target in UM.

Abstract 2770: Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform

Abstract Aurora kinases (Aur) are a family of serine/threonine kinases essential for genetic stability and cell division during mitosis. In humans, activity of the three paralogs (AurA, AurB, and AurC) is regulated throughout the cell cycle, with peak activity during mitosis. Each paralog shares a conserved C-terminal catalytic domain, but differs in substrate specificity, sub-cellular localization, and function. AurA mainly controls centrosome maturation and bipolar spindle assembly; AurB phosphorylates histone H3 (Ser10), which aids in chromatin condensation and separation; AurC, whose expression is restricted to germ cells of both genders, shares similar characteristics to AurB including substrate localization, specificity, and function during mitosis. Aberrant Aur expression leads to genomic instability or aneuploidy, and overexpression of the three paralogs is found in several cancer types, including lung, colon, and gliomas. Aur inhibition induces growth arrest and apoptosis in a variety of cancer cell types. Thus, design and development of Aur inhibitors as anti-tumor agents has been widely explored in recent years, and many are under preclinical and clinical investigation. We used the Eurofins Discovery OncoPanel™ cell-based profiling platform to investigate the specific effects of AurA, AurB, and AurC inhibitors against a select panel of human cancer cell lines. To determine the mechanistic effect of Aur inhibition in cells, we measured phospho-histone H3 (Ser10) by high-content analysis. OncoPanel human tumor cell lines arrested with nocodazole were treated with Aur inhibitors for 2 hours. Phospho-histone H3 (Ser10) levels were measured and the percentage of the DMSO vehicle-treated control cell signal was calculated for each test point. Ten-point dose-response curves were fitted using a 4-parameter log-logistic model with custom curve-fitting software to determine IC50 values. The inhibitors were also evaluated for their effect on tumor cell proliferation, apoptosis, and cell cycle arrest over the same dose-response range, after a 10-day incubation. Following exposure, the cells were fixed, stained with DAPI, anti-cleaved caspase-3, and anti-phospho-histone H3 (Ser28), and imaged. Cell proliferation dose-response curves were also fitted using the same model as above. Correlations were then made between observed mechanistic and functional activity. Activity of the AurA-selective inhibitor, MLN8054, was also evaluated in the Eurofins Discovery BioMAP® Diversity PLUS human primary cell phenotypic profiling service to give a broader view of its biological activity. These studies demonstrate the combined value of using the OncoPanel and BioMAP platforms to determine mechanistic, functional, and broader biological activity of therapeutic candidates in relevant cellular models for a better translational understanding of potential therapeutic capabilities and accelerate decision-making. Citation Format: Luciano Galdieri, Justin Lipner, Steven M. Garner, Jillian Krings, Emily Hoehn, Brendan Lahm, Kaleb Collver, Kaitlyne Powers, Alastair J. King. Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform [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 2770.

Repurposing picropodophyllin as a potential thyroid eye disease treatment via delaying mitotic clonal expansion through a patient‐derived preclinical platform

Repurposing picropodophyllin as a potential thyroid eye disease treatment via delaying mitotic clonal expansion through a patient‐derived preclinical platform

MP14-18 MODELING BLADDER CANCER IN DOWN SYNDROME ANIMALS FOR DISCOVERY OF NOVEL THERAPEUTICS

You have accessJournal of UrologyCME1 Apr 2023MP14-18 MODELING BLADDER CANCER IN DOWN SYNDROME ANIMALS FOR DISCOVERY OF NOVEL THERAPEUTICS Binh Vo, Erika Abbott, Carolyn Vivian, Xena Moore, Ganeshkumar Rajendran, Scott Weir, Katie Dennis, Benjamin Woolbright, and John Taylor Binh VoBinh Vo More articles by this author , Erika AbbottErika Abbott More articles by this author , Carolyn VivianCarolyn Vivian More articles by this author , Xena MooreXena Moore More articles by this author , Ganeshkumar RajendranGaneshkumar Rajendran More articles by this author , Scott WeirScott Weir More articles by this author , Katie DennisKatie Dennis More articles by this author , Benjamin WoolbrightBenjamin Woolbright More articles by this author , and John TaylorJohn Taylor More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Down syndrome (DS) is the most common chromosomal abnormality in humans and results in an increased risk for hematologic cancers, despite a paradoxically lower rate of adult solid tumors. Epidemiological evidence supports the idea that DS patients have a lower risk for bladder cancer (BCa) in spite of reduced environmental exposures. We sought to evaluate potential protective mechanisms against cancer formation in the Ts65Dn mouse model of DS, and hypothesized Ts65Dn mice would be protected against (BCa) formation and progression. METHODS: Tumors were initiated in Ts65Dn and WT mice using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Ts65Dn or WT animals were given 0.05% BBN for 16 weeks before sacrifice. Bladders were weighed at time of death. For Reactome pathways, Gene Set Enrichment Analysis (GSEA) was performed after RNA-seq. Proliferation was assessed using the hexosaminidase assay on HTB-9, HTB-5 and T24 BCa cells. HTB-9 cells (5×105) xenografts were implanted in the flank of nude mice in 50% Matrigel/saline and allowed to develop over two weeks. When mean tumor sizes per cage reached 50-100 mm3 determined using calipers, treatment with 25 mg/kg barasertib, i.p., Q1D for 5 days per week was started. Tumors were excised and weighed on the final day. RESULTS: After 16 weeks of 0.05% BBN exposure, Ts65Dn mice had smaller bladder tumors which trended towards lower stage tumors compared to WT mice. Although Ds mice and patients are known to have higher expression of anti-angiogenic proteins as such DSCR1, we did not see a decrease in angiogenesis in the Ds tumors, indicating potentially novel mechanisms of protection. RNA-sequencing indicated pathways associated with the cell cycle were most affected in the Ts65Dn mice, including downregulation of the cell cycle regulator Aurora Kinase B (AURKB). We evaluated drug sensitivity in a set of BCa cell lines using the AURKB specific inhibitor barasertib-HQPA. HTB-9 cells were highly susceptible to barasertib (IC50 ∼30-50 nM) whereas T24/HTB-5 cells had dramatically higher IC50 values (∼50 µM). Drug efficacy was assessed by evaluating phosphorylation of the AURKB substrate histone H3. Phospho-histone H3 levels were reduced in all cell lines and inhibition of a known transporter of barasertib using the compound valspodar minimally enhanced barasertib toxicity in T24 cells, indicating the potential for a novel mechanism of resistance. CONCLUSIONS: Our results indicate Ts65Dn mice have reduced tumor formation when exposed to BBN. Using Ts65Dn mice as a model may be a means for finding new drug targets as demonstrated by our results targeting AURKB. Source of Funding: Leo and Anne Albert Institute for Bladder Cancer and Institute for Advancing Medical Innovation at KUMC © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e189 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Binh Vo More articles by this author Erika Abbott More articles by this author Carolyn Vivian More articles by this author Xena Moore More articles by this author Ganeshkumar Rajendran More articles by this author Scott Weir More articles by this author Katie Dennis More articles by this author Benjamin Woolbright More articles by this author John Taylor More articles by this author Expand All Advertisement PDF downloadLoading ...

Histone H3 phospho-regulation by KimH3 in both interphase and mitosis

Histone H3 is phosphorylated at Ser10 by multiple kinases, and many of them are anti-cancer targets. Here, we report the first kinase that can phosphorylate H3Ser10 in both interphase and mitosis, which we named KimH3 (kinase of interphase and mitotic Histone H3). Meta-analysis indicates that KimH3 is upregulated in a broad spectrum of human cancers and its high expression is correlated with reduced the median survival time of cancer patients. In mitosis, CDK1 phosphorylates KimH3, which then phosphorylates H3Ser10 to regulate cell cycle procession. In interphase, EGF induces KimH3 activation and H3Ser10 phosphorylation, which is involved in MAPK-ERK1/2 signaling pathway to activate immediate-early genes transcription. Consequently, a small molecule inhibitor of KimH3 significantly inhibited tumor growth in mice. This is not only consistent with the dual roles of KimH3 in both interphase and mitotic Histone H3 phosphorylation, but also reveals it as an important potential anti-cancer target.

A30 EPITHELIAL FUNCTION OF THE CIRCADIAN CLOCK GENE, BMAL1, IS NECESSARY FOR COLONIC REGENERATION

Abstract Background The circadian clock is a self-sustained molecular oscillator which drives 24-hour physiological rhythms. It consists of the genes Bmal1 and Clock that positively regulate Cry and Per, their negative regulators, resulting in a 24-hour transcription/translation feedback loop. Shift work, which causes disruptions to 24-hour physiological rhythms, has been shown to lead to an increased incidence of inflammatory bowel disease (IBD). We have previously established that mice lacking the non-redundant circadian regulator, Bmal1, exhibit more severe colitis compared to controls. Purpose This study aims to investigate the epithelial function of Bmal1 in colonic regeneration during colitis. Method In order to assess the cell-specific role of the clock, we tested the regenerative effects of Bmal1 in intestinal epithelial tissue using Vil+/+;Bmal1flox/flox (control) and VilCre/+;Bmal1flox/flox (conditional mutant) mice. Dextran Sulfate Sodium (DSS) was applied to induce acute colitis. Disease progression was evaluated during colitis and during recovery upon removal of DSS treatment. We hypothesized that the absence of a functional circadian clock disrupts effective proliferation and regeneration of intestinal epithelial cells during colitis remission. Result(s) Vil +/+ ;Bmal1 flox/flox control and VilCre/+;Bmal1flox/flox conditional mutant mice exhibit no significant differences in disease severity or tissue histopathology during colitis. However, after the removal of DSS, VilCre/+;Bmal1flox/flox conditional mutants show increased total lesions and overall inflammation, decreased crypt density as well as a higher propensity of hyperplastic crypts in the tissue. Regenerative ability of the colon is decreased in conditional mutants: phosphorylated-histone H3 and Ki67 show persistent increases in mitosis and overall proliferation near ulcerated lesions. This suggests that, while controls complete the regenerative response, conditional mutants fail to recover from colitis and show inefficient regeneration when Bmal1 is disrupted in intestinal epithelial cells. Conclusion(s) Our results support a critical role for Bmal1 in intestinal epithelial cells during post-colitis regeneration and recovery. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; OGS, Crohn's & Colitis Canada Disclosure of Interest None Declared

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting

The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.

Mitogen- and stress-activated protein kinase (MSK1/2) regulated gene expression in normal and disease states.

The mitogen- and stress-activated protein kinases (MSK) are epigenetic modifiers that regulate gene expression in normal and disease cell states. MSK1 and 2 are involved in a chain of signal transduction events bringing signals from the external environment of a cell to specific sites in the genome. MSK1/2 phosphorylate histone H3 at multiple sites, resulting in chromatin remodeling at regulatory elements of target genes and the induction of gene expression. Several transcription factors (RELA of NF-κB and CREB) are also phosphorylated by MSK1/2 and contribute to induction of gene expression. In response to signal transduction pathways, MSK1/2 can stimulate genes involved in cell proliferation, inflammation, innate immunity, neuronal function, and neoplastic transformation. Abrogation of the MSK-involved signaling pathway is among the mechanisms by which pathogenic bacteria subdue the host's innate immunity. Depending on the signal transduction pathways in play and the MSK-targeted genes, MSK may promote or hinder metastasis. Thus, depending on the type of cancer and genes involved, MSK overexpression may be a good or poor prognostic factor. In this review, we focus on mechanisms by which MSK1/2 regulate gene expression, and recent studies on their roles in normal and diseased cells.

Retraction of 'Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy'.

Retraction of 'Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy'.

OLIG2 is an in vivo bookmarking transcription factor in the developing neural tube in mouse.

Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. These inheritable features maintain a unique gene expression pattern, underlying cellular memory. Because of the degradation or displacement of mitotic chromosomes, most transcription factors do not contribute to cellular memory. However, accumulating in vitro evidence indicates that some transcription factors can be retained in mitotic chromosomes called as bookmarking. Such transcription factors may contribute to a novel third mechanism of cellular memory. Since most findings of transcription factor bookmarking have been reported in vitro, little is currently known in vivo. In the neural tube of mouse embryos, we discovered that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, binds to chromatin through every cell cycle including M-phase. OLIG2 chromosomal localization coincides with mitotic cell features such as the phosphorylation of histone H3, KI67, and nuclear membrane breakdown. Chromosomal localization of OLIG2 is regulated by an N-terminus triple serine motif. Photobleaching analysis revealed slow OLIG2 mobility, suggesting a high affinity of OLIG2 to DNA. In Olig2 N-terminal deletion mutant mice, motoneurons and oligodendrocyte progenitor numbers are reduced in the neural tube, suggesting that the bookmarking regulatory domain is important for OLIG2 function. We conclude that OLIG2 is a de novo in vivo bookmarking transcription factor. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate a novel function of transcription factors.

Dual FLT3/haspin kinase inhibitor based on 3H-pyrazolo[4,3-f]quinoline scaffold with activities against acute myeloid leukemia.

The 3H-pyrazolo[4,3-f]quinoline core, a privileged fusion moiety from quinoline and indazole, facilely synthesized in a one flask multi-component Doebner-Povarov reaction, is a newly described kinase hinge binder. Previous works have demonstrated that the 3H-pyrazolo[4,3-f]quinoline moiety can be tuned, via judicious substitution patterns, to selectively inhibit cancer-associated kinases, such as FLT3 and haspin. A first generation 3H-pyrazolo[4,3-f]quinoline-based haspin inhibitor, HSD972, and FLT3 inhibitor, HSD1169, were previously disclosed as inhibitors of various cancer cell lines. Given the recent revelation that haspin is over-expressed and plays critical proliferative roles in many cancers, and compounds with dual activity against FLT3 and other important kinases are now being actively developed by many groups, we became interested in optimizing the 3H-pyrazolo[4,3-f]quinoline-based compounds to improve activity against both FLT3 and haspin. Herein, we report the discovery of new 3H-pyrazolo[4,3-f]quinoline-based dual FLT3/haspin inhibitor, HSK205. HSK205 has remarkable potencies against FLT3-driven AML cell lines, inhibiting proliferation with GI50 values between 2-25 nM. Western blot analyses of treated AML cells confirm that HSK205 inhibit the phosphorylation of both FLT3 and histone H3 (a haspin target) in cells. While multi-component reactions (MCRs) have been used to make many bioactive molecules, there are very few examples of using MCRs to make compounds that target protein kinases, which have emerged as one of the top drug candidates (especially in oncology). This work highlights our recent efforts to make ultrapotent protein kinase inhibitors using multi-component reactions (especially the Doebner-Povarov reaction).

Impact of Extended and Combined Exposure of Bisphenol Compounds on Their Chromosome-Damaging Effect─Increased Potency and Shifted Mode of Action.

Bisphenol (BP) compounds are important environmental pollutants and endocrine disruptors. BPs are capable of inducing DNA/chromosome breaks (clastogenesis, involved in carcinogenesis), which requires activation by human CYP1A1. We hypothesized that combined BPs and extended (from the standard two-cell cycle) exposure may enhance their genotoxicity via modulating CYP enzymes. In this study, individual and combined BPA/BPF/BPS/BPAF and a human hepatoma (HepG2) cell line were used for testing several genotoxicity end points. Exposing for a two-cell cycle period (48 h), each BP alone (0.625-10 μM) was negative in the micronucleus test, while micronucleus was formed under three- (72 h) and four-cell cycle (96 h) exposure; BP combinations further elevated the potency (with nanomolar thresholds). Immunofluorescence analysis of the centromere with formed micronucleus indicated that 48 h exposure produced centromere-negative micronucleus and phosphorylated histone H2AX (γ-H2AX) (evidencing clastogenesis), while extended (72 and 96 h) exposure formed centromere-positive micronucleus and phosphorylated histone H3 (p-H3) (indicating chromosome loss, i.e., aneugenesis); moreover, 1-aminotriabenzotriazole (CYP inhibitor) selectively blocked the formation of centromere-negative micronucleus and γ-H2AX, without affecting that of centromere-positive micronucleus and p-H3. This study suggests that the genotoxicity of BPs is potentiated by combined and extended exposure, the latter being specific for aneuploidy formation, a CYP activity-independent effect.

Anlotinib Suppressed Ovarian Cancer Progression via Inducing G2/M Phase Arrest and Apoptosis

Ovarian cancer remains the most common gynecologic malignancy, because of its chemotherapy resistance and relapse. Anlotinib, a new oral multi-targeted tyrosine kinase inhibitor, has shown encouraging antitumor activity in several preclinical and clinical trials, while its effect on ovarian cancer has not been reported. In this study, we investigated the antitumor activity and underlying mechanism of anlotinib in ovarian cancer. Cell viability was analyzed by Cell Counting Kit-8 assay. Migration was measured by wound-healing assay. The cell cycle distribution and cell apoptosis rate were detected by flow cytometry. In vivo antitumor effect was analyzed in mouse ovarian carcinoma peritoneal metastasis model. We found that anlotinib inhibited the proliferation of ovarian cancer cells in a dose- and time- dependent manner by inducing G2/M phase arrest and apoptosis. Moreover, anlotinib upregulated the the phosphorylation of Histone H3, and expression of p21 protein in vitro. In addition, anlotinib inhibited the migration of ovarian cancer cells in vitro. Furthermore, anlotinib inhibited tumor growth by inhibiting cell proliferation and suppressing ovarian cancer angiogenesis in vivo. This study demonstrated the extraordinary anti-ovarian cancer effect of anlotinib, which may provide a promising therapeutic strategy for ovarian cancer.