Activation Of Various Transcription Factors Research Articles

HES6 knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.

Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models. Using bulk and scRNA-seq data, we show that HES6 is expressed during erythroid/megakaryocyte and pDC development, as well as in multipotent precursors and at specific stages of T- and B-cell development following preBCR and preTCR signalling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, pDCs, Band T-cells. In addition, HES6 knockdown HSPCs displayed reduced colony forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression impacts cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that impact these perturbations. Thus, our study uncovers new insights for a role of HES6 in human hematopoiesis.

Downregulation of miR-337-3p in hypoxia/reoxygenation neuroblastoma cells increases KCTD11 expression.

Neurodegeneration is linked to the progressive loss of neural function and is associated with several diseases. Hypoxia is a hallmark in many of these diseases, and several therapies have been developed to treat this disease, including gene expression therapies that should be tightly controlled to avoid side effects. Cells experiencing hypoxia undergo a series of physiological responses that are induced by the activation of various transcription factors. Modulation of microRNA (miRNA) expression to alter transcriptional regulation has been demonstrated to be beneficial in treating multiple diseases, and in this study, we therefore explored potential miRNA candidates that could influence hypoxia-induced nerve cell death. Our data suggest that in mouse neuroblasts Neuro-2a cells with hypoxia/reoxygenation (H/R), miR-337-3p is downregulated to increase the expression of Potassium channel tetramerization domain containing 11 (KCTD11) and subsequently promote apoptosis. Here, we demonstrate for the first time that KCTD11 plays a role in the cellular response to hypoxia, and we also provide a possible regulatory mechanism by identifying the axis of miR-337-3p/KCTD11 as a promising candidate modulator of nerve cell survival after H/R exposure.

ER stress mediated inflammation in cancer pathogenesis

Inflammation is a complex process which is associated with the initiation and progression of cancer. Prolonged Endoplasmic Reticulum (ER) stress triggers inflammation which is a key factor associated with cancer pathogenesis. ER stress also contributes to immune suppression in inflammatory and tumor microenvironment. It stimulates the production of pro-inflammatory cytokines by regulating the activation of various transcription factors and inflammatory signalling pathways. Targeting ER stress is an exciting possibility that can be used as a therapeutic strategy for cancer treatment. This mini review focuses on the emerging link between ER stress-induced inflammatory responses in cancer development.

FOXO1 regulates expression of Neurod4 in the pituitary gland

Pituitary gland function is regulated by the activity of various transcription factors that control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for normal somatotrope differentiation and function. Recent in vivo data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. In the current study, the somatotrope-like cell line GH3 was treated with a FOXO1 inhibitor, resulting in significant reduction in Neurod4 and Gh1 expression. Consistent with these findings, CRISPR/Cas9-mediated deletion of Foxo1 in GH3 cells significantly reduced expression of Gh1 and Neurod4. Chromatin immunoprecipitation sequencing identifies novel FOXO1 binding sites associated with the Neurod4, Gh1, and Pou1f1 genes. The FOXO1 binding site in the Neurod4 gene exhibits enhancer activity in somatotrope-like cells but not in gonadotrope-like cells. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation.

Effect of Diet and Oxidative Stress in the Pathogenesis of Lymphoproliferative Disorders.

Lymphomas are a heterogeneous group of pathologies that result from clonal proliferation of lymphocytes. They are classified into Hodgkin lymphoma and non-Hodgkin lymphoma; the latter develops as a result of B, T, or NK cells undergoing malignant transformation. It is believed that diet can modulate cellular redox state and that oxidative stress is implicated in lymphomagenesis by acting on several biological mechanisms; in fact, oxidative stress can generate a state of chronic inflammation through the activation of various transcription factors, thereby increasing the production of proinflammatory cytokines and causing overstimulation of B lymphocytes in the production of antibodies and possible alterations in cellular DNA. The purpose of our work is to investigate the results of in vitro and in vivo studies on the possible interaction between lymphomas, oxidative stress, and diet. A variety of dietary regimens and substances introduced with the diet that may have antioxidant and antiproliferative effects were assessed. The possibility of using nutraceuticals as novel anticancer agents is discussed; although the use of natural substances in lymphoma therapy is an interesting field of study, further studies are needed to define the efficacy of different nutraceuticals before introducing them into clinical practice.

Targeted Inhibition of Select Extracellular Signal-regulated Kinases 1 and 2 Functions Mitigates Pathological Features of Asthma in Mice.

ERK1/2 (extracellular signal-regulated kinases 1 and 2) regulate the activity of various transcription factors that contribute to asthma pathogenesis. Although an attractive drug target, broadly inhibiting ERK1/2 is challenging because of unwanted cellular toxicities. We have identified small molecule inhibitors with a benzenesulfonate scaffold that selectively inhibit ERK1/2-mediated activation of AP-1 (activator protein-1). Herein, we describe the findings of targeting ERK1/2-mediated substrate-specific signaling with the small molecule inhibitor SF-3-030 in a murine model of house dust mite (HDM)-induced asthma. In 8- to 10-week-old BALB/c mice, allergic asthma was established by repeated intranasal HDM (25 μg/mouse) instillation for 3 weeks (5 days/week). A subgroup of mice was prophylactically dosed with 10 mg/kg SF-3-030/DMSO intranasally 30 minutes before the HDM challenge. Following the dosing schedule, mice were evaluated for alterations in airway mechanics, inflammation, and markers of airway remodeling. SF-3-030 treatment significantly attenuated HDM-induced elevation of distinct inflammatory cell types and cytokine concentrations in BAL and IgE concentrations in the lungs. Histopathological analysis of lung tissue sections revealed diminished HDM-induced pleocellular peribronchial inflammation, mucus cell metaplasia, collagen accumulation, thickening of airway smooth muscle mass, and expression of markers of cell proliferation (Ki-67 and cyclin D1) in mice treated with SF-3-030. Furthermore, SF-3-030 treatment attenuated HDM-induced airway hyperresponsiveness in mice. Finally, mechanistic studies using transcriptome and proteome analyses suggest inhibition of HDM-induced genes involved in inflammation, cell proliferation, and tissue remodeling by SF-3-030. These preclinical findings demonstrate that function-selective inhibition of ERK1/2 signaling mitigates multiple features of asthma in a murine model.

Targeting HIF-1α by Natural and Synthetic Compounds: A Promising Approach for Anti-Cancer Therapeutics Development.

Advancement in novel target detection using improved molecular cancer biology has opened up new avenues for promising anti-cancer drug development. In the past two decades, the mechanism of tumor hypoxia has become more understandable with the discovery of hypoxia-inducible factor-1α (HIF-1α). It is a major transcriptional regulator that coordinates the activity of various transcription factors and their downstream molecules involved in tumorigenesis. HIF-1α not only plays a crucial role in the adaptation of tumor cells to hypoxia but also regulates different biological processes, including cell proliferation, survival, cellular metabolism, angiogenesis, metastasis, cancer stem cell maintenance, and propagation. Therefore, HIF-1α overexpression is strongly associated with poor prognosis in patients with different solid cancers. Hence, pharmacological targeting of HIF-1α has been considered to be a novel cancer therapeutic strategy in recent years. In this review, we provide brief descriptions of natural and synthetic compounds as HIF-1α inhibitors that have the potential to accelerate anticancer drug discovery. This review also introduces the mode of action of these compounds for a better understanding of the chemical leads, which could be useful as cancer therapeutics in the future.

112-LB: In Vitro DILI Risk Assessment of IDG16177, a Potent and Selective GPR40 Agonist, for the Treatment of Type 2 Diabetes

G protein-coupled receptor 40 (GPR40) , also known as free fatty acid receptor 1 (FFA1/FFAR1) , is expressed primarily on pancreatic β-cells and induces insulin secretion only at high glucose levels, making it a promising target for type 2 diabetes mellitus (T2DM) . Fasiglipam (TAK-875) , a partial agonist of GPR40, was discontinued in phase 3 due to hepatotoxicity despite its potent hypoglycemic effect. Previous studies of hepatic transporter inhibition, mitochondrial function, covalent protein binding and toxicity to human hepatocytes confirmed the lower drug-induced liver injury (DILI) potential of IDG16177 compared to fasiglifam, but bile acid (BA) analysis and transcription factor profiling were further performed. In a 3D human liver model, we indirectly evaluated the accumulation of BA in the liver by measuring the secretion of glycolic acid (GCA) , which is a major component of human BAs and has been identified as a significant increase in patients with DILI. Fasiglifam induced a significant accumulation of GCA at 4 μM lower than the human Cmax of 10 μM, whereas IDG16177 showed no significant accumulation at 1 μM higher than the predicted human Cmax of 0.3 μM. IDG16177 did not have a significant effect in quantitative evaluation of the activity of various transcription factors, whereas treatment with 10 uM fasiglifam for 24 hours significantly increased the activities of peroxisome proliferator activating receptor (PPAR) , activator protein 1 (AP-1) and nuclear respiratory factor 2 (NRF2) , which were highly correlated with liver disease pathogenesis/progression. Interestingly, treatment with 10 μM IDG16177 for 48 h increased the activity of FXR, a master regulator of BA metabolism that inhibits BA absorption system and BA synthesis and promotes BA enterohepatic circulation, by 1.58-fold. These results show that IDG16177 has the advantage of having a low DILI potential compared to fasiglifam in addition to its potent efficacy for the treatment of type 2 diabetes. Disclosure J. Yoon: None. H. Song: None. K. An: None. C. Hong: None. H. Kwak: None. H. Song: None.

Non-canonical vs. Canonical Functions of Heme Oxygenase-1 in Cancer.

Heme oxygenase-1 (HO-1) is a critical stress-responsive enzyme that has antioxidant and anti-inflammatory functions. HO-1 catalyzes heme degradation, which gives rise to the formation of carbon monoxide (CO), biliverdin, and iron. The upregulation of HO-1 under pathological conditions associated with cellular stress represents an important cytoprotective defense mechanism by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced. The same mechanism is hijacked by premalignant and cancerous cells. In recent years, however, there has been accumulating evidence supporting that the upregulation of HO-1 promotes cancer progression, independently of its catalytic activity. Such non-canonical functions of HO-1 are associated with its interaction with other proteins, particularly transcription factors. HO-1 also undergoes post-translational modifications that influence its stability, functional activity, cellular translocation, etc. HO-1 is normally present in the endoplasmic reticulum, but distinct subcellular localizations, especially in the nucleus, are observed in multiple cancers. The nuclear HO-1 modulates the activation of various transcription factors, which does not appear to be mediated by carbon monoxide and iron. This commentary summarizes the non-canonical functions of HO-1 in the context of cancer growth and progression and underlying regulatory mechanisms.

Isobavachalcone suppresses the TRIF-dependent signaling pathway of Toll-like receptors.

Toll-like receptors (TLRs) are integral membrane-bound receptors that are central to innate and adaptive immune responses. They are known to activate a cascade of downstream signals to induce the secretion of inflammatory cytokines, chemokines, and type I interferons. Dysregulated activation of TLR signaling pathways can induce the activation of various transcription factors, such as nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). TLRs act via MyD88- and TRIF-mediated pathways to induce inflammatory responses. To evaluate the therapeutic potential of isobavachalcone (IBC), a natural chalcone component of Angelica keiskei, we examined its effects on signal transduction via TLR signaling pathways. IBC inhibited the activation of NF-κB and IRF3 induced by TLR agonists and their target genes. IBC also inhibited the activation of NF-κB and IRF3 induced by overexpression of downstream signaling components of TLR signaling pathways. These results suggest that IBC can regulate both MyD88- and TRIF-dependent signaling pathways of TLRs, resulting in a dramatic increase of new therapeutic options for various inflammatory diseases involving TLRs.

Zmiz1a zebrafish mutants have defective erythropoiesis, altered expression of autophagy genes, and a deficient response to vitamin D

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits.To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

Curcumin Loaded Ethosomal Gel for Improved Topical Delivery: Formulation, Characterization and Ex-vivo Studies.

Curcumin is a curcuminoid, which is an active constituent of turmeric and is obtained from the rhizomes of Curcuma longa, family Zingiberaceae. Curcumin modulates the activity of various transcription factors and regulates the expression of inflammatory enzymes, cell survival proteins, adhesion molecules and cytokines by binding to a variety of proteins and inhibiting the activity of various kinases. Curcumin falls in the BCS class IV drug, with poor solubility and poor permeability which makes it very challenging to utilize the maximum therapeutic potential of this moiety Objective: The major aim of the study was to enhance transdermal penetration of curcumin via ethosomal gel and to overcome the barriers of poor permeability of transdermal drug delivery. Curcumin loaded ethosomes were prepared with varying quantities of ethanol and soya lecithin by the cold method and were optimised based on entrapment efficiency, vesicular size and Ex-vivo studies. Optimised ethosomal formulation was further incorporated into a gel and was evaluated. Ex-vivo studies were performed with the ethosomal gel of curcumin and was compared with simple drug solution. Prepared ethosomal system showed a vesicle size ranging from 211 to 320 nm with spherical, smooth surface and entrapment efficiency of 87 to 91%. Optimised ethosomal system (ET3) was incorporated into gel and was further evaluated. The findings of the research work suggested that the ethosomal gel holds excellent potential for transdermal delivery of curcumin.

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

PurposeNeuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).MethodsPharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter.ResultsGalangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.ConclusionGalangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.

Recognition of Candida albicans and Role of Innate Type 17 Immunity in Oral Candidiasis.

Candida albicans is an opportunistic pathogenic fungus considered to be a common member of the human microflora. Similar to some other opportunistic microbes, C. albicans can invade and benefit from its host when the immune status of that host is weakened. Most often this happens to immunocompromised individuals, leading to the infection of oral and vaginal mucosae or the systemic spread of the pathogen throughout the entire body. Oropharyngeal candidiasis (OPC) occurs in up to 90 percent of patients with acquired immunodeficiency syndrome (AIDS), making it the most frequent opportunistic infection for this group. Upon first signs of fungal invasion, a range of host signaling activates in order to eliminate the threat. Epithelial and myeloid type cells detect C. albicans mainly through receptor tyrosine kinases and pattern-recognition receptors. This review provides an overview of downstream signaling resulting in an adequate immune response through the activation of various transcription factors. The study discusses recent advances in research of the interleukin-17 (IL-17) producing innate cells, including natural T helper 17 (nTh17) cells, γδ T cells, invariant natural killer T (iNKT) cells and type 3 innate lymphoid cells (ILC3) that are involved in response to oral C. albicans infections.

Licochalcone B Ameliorates Liver Cancer via Targeting of Apoptotic Genes, DNA Repair Systems, and Cell Cycle Control.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a ubiquitous multifunctional protein required in the DNA base excision repair pathway and a noteworthy reducing-oxidizing factor that regulates the activity of various transcription factors. Cyclin-dependent kinases (CDKs) assume a key role in directing the progression of the cell- cycle. The present study evaluated the synergistic efficacy of using licochalcone B (LCB) and fullerene C60 (FnC60) nanoparticles against diethylnitrosamine (DEN)-induced hepatocarcinoma in rats and relevant signaling pathways, with APE1/Ref-1 and CDK-4, as novel anti-cancer- targeting. LCB alone and in combination with FnC60 significantly decreased DNA fragmentation, oxidative DNA damage (8-hydroxy-2′-deoxyguanosine levels), APE1/Ref-1, CDK-4, retinoblastoma, B- cell lymphoma-2 (Bcl-2), B-cell lymphoma-xL (Bcl-xL), and β-arrestin-2 mRNA expression, and APE1/Ref-1 and CDK-4 protein expression. In contrast, these treatments significantly increased the expression of protein 53 (p53), Bcl-2-associated X protein (Bax), and caspase-3. These data suggest that LCB either alone or in combination with FnC60 elicited significant protective effects against DEN-induced hepatocarcinogenesis, which may have occurred because of the regulation of enzymes involved in DNA repair and cell-cycle control at S phase progression as well as the induction of apoptosis at the gene and protein expression levels. Furthermore, FnC60 potentiated the effect of LCB at the molecular level, possibly through targeting of cancerous cells.

MON-483 FOXO1 Contributes to Somatotrope Differentiation through Regulation of Key Transcription Factors

Pituitary gland function is regulated by the activity of various transcription factors which control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for the proper timing of somatotrope differentiation and for somatotrope function, but the exact mechanism of action has yet to be elucidated. Recent data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. Previously, a mouse model with conditional deletion of Foxo1 from the developing pituitary gland displayed reduced Gh1 and Neurod4 transcripts as early as embryonic day 18.5. Additional data from adult animals with conditional deletion of both Foxo1 and Foxo3 from the pituitary gland have a similar reduction in Neurod4 and Gh1, as well as Pou1f1. In order to investigate the mechanism by which FOXO1 regulates pituitary gland gene expression and confirm in vivo findings, the somatotrope-like cell line MtT/S was treated with FOXO1 inhibitor, AS184256, for 24 hours at various concentrations. Neurod4 and Pou1f1 were the most severely affected genes with a dose-dependent reduction in transcript at inhibitor concentrations as low as 30 nM (p < 0.05). Gh1 transcripts were significantly reduced at 3 μM (p < 0.01). To ascertain whether FOXO1 binds to regulatory regions of key somatotrope genes, ChIP-seq was performed using the MtT/S cell line and anti-FOXO1 antibody. This study identified FOXO1 interaction with genomic regions associated with Neurod4, Pou1f1, and Gh1. Interestingly, the FOXO1 binding intervals associated with Neurod4 and Pou1f1 are highly conserved between rat and human, indicating these sequences are important for survival. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation. Investigation is also underway to identify proteins within the FOXO1 transcriptional complex. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was performed using the MtT/S cell line for this purpose. Proteins important for signal transduction and transcriptional regulation, including PKC, LRPPRC, and YBX1, were enriched in chromatin containing FOXO1, suggesting that they contribute to transcriptional control of FOXO1 targets. Overall, the emerging mechanism for FOXO1 control of somatotrope differentiation likely involves direct regulation of Neurod4, Pou1f1, and Gh1. These novel findings contribute to the much-needed understanding of pituitary cell fate decisions.

Structural mechanism of vaccinia virus protein C6 mediated inactivation of transcription factors IRF3 and IRF7 via inhibition of TBK1 mediated scaffold protein‐protein interactions

Interferons (IFNs) are signaling proteins with potent antiviral and immune stimulatory effects. IFN signaling is initiated after host cell pattern recognition receptors (PRRs) recognize pathogen‐associated molecular patterns (PAMPs) present in many viral proteins. This event in turn generates a complex signaling cascade involving numerous proteins, activation of various transcription factors (TF), such as IFN‐regulatory factors (IRF) 3 and 7. These TFs subsequently translocate to the nucleus to elevate the transcription of type I IFNs, cytokines, and chemokines. Viruses such as Vaccinia virus (VACV) have evolved many strategies to interfere with the IFNs or their mechanism of action. Specifically, VACV encodes a small 16kDa protein, C6, that inhibits the activation of IRF3/7 via binding with an upstream kinase, TBK1, and its adaptor proteins TANK, NAP1, and SINTBAD in order to interfere with the production of IFNβ. Binding with these adaptor proteins, C6 prevents TBK1/IKKɛ‐dependent activation of IRF3 and IRF7 allowing VACV to evade immune recognition. To elucidate the structural mechanism of how C6 mediated inhibition of these adaptor protein/TBK1 interactions can block the activation of IRF3 and IRF7, we have chosen C6 and TANK as canonical binding partners. We have identified the binding regions between C6 and TANK by utilizing co‐expression/co‐purification studies of C6 and TANK proteins, and co‐crystallization trials are ongoing. The immune evasive strategy exploited by VACV can be used to develop novel therapies, such as C6 mimetics to provide TBK1 inhibition. The development of therapeutics depends immensely on a detailed structural analysis of the interactions between C6 and adaptor proteins. Our structural studies of C6 and TBK1 adaptor proteins will provide useful insights in developing superior therapies against malignancies, infectious diseases, and autoimmune disorders.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms Underlying Tumor Suppressive Properties of Melatonin

There is considerable evidence that melatonin may be of use in the prevention and treatment of cancer. This manuscript will review some of the human, animal and cellular studies that provide evidence that melatonin has oncostatic properties. Confirmation that melatonin mitigates pathogenesis of cancer will be described from both direct study of its effects on carcinogenesis, and from indirect findings implicating disruption of the circadian cycle. A distinction is made between the role of melatonin in preventing the initiation of the tumorigenic pathway and the ability of melatonin to retard the progression of cancer. Melatonin appears to slow down the rate of advancement of established tumors and there is evidence that it constitutes a valuable complement to standard pharmacological and radiation treatment modalities. There are instances of the beneficial outcomes in cancer treatment which utilize a range of hormones and vitamins, melatonin being among the constituents of the mix. While these complex blends are empirically promising, they are only briefly mentioned here in view of the confounding influence of a multiplicity of agents studied simultaneously. The last section of this review examines the molecular mechanisms that potentially underlie the oncostatic effects of melatonin. Alterations in gene expression following activation of various transcription factors, are likely to be an important mediating event. These changes in gene activity not only relate to cancer but also to the aging process which underlies the onset of most tumors. In addition, epigenetic events such as modulation of histone acetylation and DNA methylation patterns throughout the lifespan of organisms need to be considered. The antioxidant and immunoregulatory roles of melatonin may also contribute to its cancer modulatory properties. Naturally, these mechanisms overlap and interact extensively. Nevertheless, in the interest of clarity and ease of reading, each is discussed as a separate topic section. The report ends with some general conclusions concerning the clinical value of melatonin which has been rather overlooked and understudied.

Abstract 5483: SIRT1 stabilization provides a therapeutic opportunity for reversing cachexia in pancreatic cancer

Abstract Cancer-associated cachexia is a complex metabolic syndrome which leads to excessive loss of skeletal muscle and adipose deposits. Up to 80% of pancreatic cancer patients suffer from cachexia and nearly one third die due to complexities related to the syndrome. Treatment of cachexia will not only improve the standard of living of pancreatic cancer patients but would also improve the patient survival. In the clinic, majority of the cancer patients are diagnosed at the refractory phase of cachexia, which cannot be reversed by dietary interventions. Till late therapies have not shown promising results in clinical trials. Hence, there is an urgent need to identify new targets to combat cancer cachexia. Sirtuins are protein deacetylases which affect the activity of various transcription factors and signaling components. The expression of all the sirtuins were analyzed after culturing mouse myotubes with tumor cell-conditioned media. The expression of SIRT1 decreased consistently upon treatment of myotubes with conditioned media from multiple human pancreatic cancer cell lines. Resveratrol, which is an activator of SIRT1 rescued the wasting phenotype in the myotubes. However, treatment of pancreatic cancer cells with resveratrol also decreased cell proliferation. Hence, we designed in vivo experiments to examine the effect of resveratrol on muscle wasting irrespective of its effects on tumor burden. We generated SIRT1 knockdown pancreatic cancer cell lines and orthotopically implanted them into the pancreas in athymic nude mice. The tumor-bearing mice were subjected to an oral delivery of resveratrol. These studies demonstrated that while resveratrol had limited effect on cancer cells due to SIRT1 knockdown, resveratrol treatment still protected the mice from muscle wasting by stabilizing SIRT1 in muscles. Hence, our results indicate that resveratrol-mediated stabilization of SIRT1 in muscles can revert wasting irrespective of its effects on tumor cells. Overall, our studies demonstrate the therapeutic potential of SIRT1 stabilization by resveratrol or related class of drugs in managing cancer-associated cachexia. Citation Format: Aneesha Dasgupta, Enza Vernucci, Surendra K. Shukla, Scott E. Mulder, Ryan J. King, Jaime Abrego, Nina V. Chaika, Kyla Buettner, Pankaj K. Singh, Pankaj K. Singh. SIRT1 stabilization provides a therapeutic opportunity for reversing cachexia in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5483.

Role of BASIC PENTACYSTEINE transcription factors in a subset of cytokinin signaling responses.

Cytokinin plays diverse roles in plant growth and development, generally acting by modulating gene transcription in target tissues. The type-B Arabidopsis response regulators (ARR) transcription factors have emerged as primary targets of cytokinin signaling and are required for essentially all cytokinin-mediated changes in gene expression. The diversity of cytokinin function is likely imparted by the activity of various transcription factors working with the type-B ARRs to alter specific sets of target genes. One potential set of co-regulators modulating the cytokinin response are the BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family of plant-specific transcription factors. Here, we show that disruption of multiple BPCs results in reduced sensitivity to cytokinin. Further, the BPCs are necessary for the induction of a subset of genes in response to cytokinin. We identified direct invivo targets of BPC6 using ChIP-Seq and found an enrichment of promoters of genes differentially expressed in response to cytokinin. Further, a significant number of BPC6 regulated genes are also direct targets of the type-B ARRs. Potential cis-binding elements for a number of other transcription factors linked to cytokinin action are enriched in the BPC binding fragments, including those for the cytokinin response factors (CRFs). In addition, several BPCs interact with a subset of type-A ARRs. Consistent with these results, a significant number of genes whose expression is altered in bpc mutant roots are also mis-expressed in crf1,3,5,6 and type-A arr3,4,5,6,7,8,9,15 mutant roots. These results suggest that the BPCs are part of a complex network of transcription factors that are involved in the response to cytokinin.