DNA Transcriptional Activity Research Articles

Expression analysis of Inhibitor Of DNA Binding 1 (ID-1) gene in breast cancer.

ID-1 gene codes for a helix-loop-helix (HLH) protein that inhibits the DNA binding and transcriptional activation function of these proteins. We analyzed ID-1 expression in microarray and RNA Sequencing databases as well as 61 breast cancer tissues compared with adjacent non-cancerous tissues (ANCTs). Expression analysis of ID-1 gene in two microarray datasets and RNA sequencing data showed down-regulation of ID-1 in tumoral tissues compared with normal tissues. However, ID-1 expression analysis in tumoral tissues and ANCTs obtained from 61 patients revealed its over-expression in tumoral tissues. A negative association was detected between ID-1 expression levels and ER status. ID-1 expression may be implicated in the pathogenesis of breast cancer especially in patient with ER negative status.

DNA replication dynamics of vole genome and its epigenetic regulation

BackgroundThe genome of some vole rodents exhibit large blocks of heterochromatin coupled to their sex chromosomes. The DNA composition and transcriptional activity of these heterochromatin blocks have been studied, but little is known about their DNA replication dynamics and epigenetic composition.ResultsHere, we show prominent epigenetic marks of the heterochromatic blocks in the giant sex chromosomes of female Microtus cabrerae cells. While the X chromosomes are hypoacetylated and cytosine hypomethylated, they are either enriched for macroH2A and H3K27me3 typical for facultative heterochromatin or for H3K9me3 and HP1 beta typical for constitutive heterochromatin. Using pulse-chase replication labeling and time-lapse microscopy, we found that the heterochromatic block enriched for macroH2A/H3K27me3 of the X chromosome is replicated during mid-S-phase, prior to the heterochromatic block enriched for H3K9me3/HP1 beta, which is replicated during late S-phase. To test whether histone acetylation level regulates its replication dynamics, we induced either global hyperacetylation by pharmacological inhibition or by targeting a histone acetyltransferase to the heterochromatic region of the X chromosomes. Our data reveal that histone acetylation level affects DNA replication dynamics of the sex chromosomes’ heterochromatin and leads to a global reduction in replication fork rate genome wide.ConclusionsIn conclusion, we mapped major epigenetic modifications controlling the structure of the sex chromosome-associated heterochromatin and demonstrated the occurrence of differences in the molecular mechanisms controlling the replication timing of the heterochromatic blocks at the sex chromosomes in female Microtus cabrerae cells. Furthermore, we highlighted a conserved role of histone acetylation level on replication dynamics across mammalian species.

RUNX1 inhibits proliferation and induces apoptosis of t(8;21) leukemia cells via KLF4-mediated transactivation of P57.

RUNX1 is a key transcription factor in hematopoiesis and its disruption is one of the most common aberrations in acute myeloid leukemia. RUNX1 alterations affect its DNA binding capacity and transcriptional activities, leading to the deregulation of transcriptional targets, and abnormal proliferation and differentiation of myeloid cells. Identification of RUNX1 target genes and clarification of their biological functions are of great importance in the search for new therapeutic strategies for RUNX1-altered leukemia. In this study, we identified and confirmed that KLF4, a known tumor suppressor gene, as a direct target of RUNX1, was down-regulated in RUNX1-ETO leukemia. RUNX1 bound to KLF4 promoter in chromatin to activate its transcription, while the leukemogenic RUNX1-ETO fusion protein had little effect on this transactivation. KLF4 was also identified as a novel binding partner of RUNX1. RUNX1 interacted with KLF4 through Runt domain and further co-activated its target genes. However, RUNX1-ETO competed with RUNX1 to bind KLF4 through Runt and ETO domains, and abrogated transcription of KLF4. Finally, overexpression experiments indicated that RUNX1 inhibited proliferation and induced apoptosis of t(8;21) leukemia cells via KLF4-mediated upregulation of P57. These data suggest KLF4 dysregulation mediated by RUNX1-ETO enhances proliferation and retards apoptosis, and provides a potential target for therapy of t(8;21) acute myeloid leukemia.

Abstract P5-05-16: Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers

Abstract Estrogens, such as 17β-estradiol (E2), act through estrogen receptor alpha (ERα), a ligand-regulated transcription factor that binds across the genome to promote enhancer formation and regulate gene expression. ERα is expressed in approximately 70% of breast cancers, where it regulates the transcription of genes involved in mitogenic and inflammatory pathways. We are exploring the the acetylation of ERα on lysines 266 and 268, a modification that enhances the DNA binding and transcriptional activities of ERα. ERα acetylation is catalyzed by the lysine acetyltransferases p300 and CBP in an E2- and steroid receptor coregulator (SRC)-dependent manner. Acetylation-dependent activation of ERα has potential implications in breast cancers associated with enhanced coregulator interactions, such as SRC-3/Amplified in Breast (AIB1) gene amplifications and gain-of-function ERα mutations in endocrine resistant metastatic tumors, such as Y537S and D538G. Increased association of ERα with SRCs and p300/CBP leading to enhanced ERα acetylation may promote gain-of-function ERα activity. Our hypothesis is that acetylation of ERα alters its function by increasing E2-responsive gene transcription and signaling in breast cancers. Our current efforts are focused on investigating the role of ERα acetylation on chromatin binding and accessibility, enhancer activity, and target gene transcription. We are using the ER-positive MCF-7 breast cancer cell line with a knockdown/re-expression strategy with biochemical mimics of acetylated (K266/268Q) or unacetylated (K266/268R) ERα. Genomic ERα binding profiles using chromatin immunoprecipitation-sequencing (ChIP-seq) has defined overlapping, but unique, sets of transcriptional targets and recruitment kinetics for the ERα mutants. Current efforts are focused on defining global transcriptional responses associated with the unique binding activities using precision run-on sequencing (PRO-seq). Ultimately, our goal is to define how acetylation affects the binding of ERα to chromatin and its effect on the transcriptional activity of E2-target genes in breast cancers in the context of gain-of-function ERα mutations and coregulator amplifications. Supported in part by a postdoctoral fellowship from the Lalor Foundation to YMV, and grants from the NIDDK, NICHD, and CPRIT to WLK, as well as support from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment. Citation Format: Vasquez YM, Setlem R, Murakami S, Kraus WL. Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-16.

The cell-penetrating FOXM1 N-terminus (M1-138) demonstrates potent inhibitory effects on cancer cells by targeting FOXM1 and FOXM1-interacting factor SMAD3.

Transcription factor FOXM1 is involved in stimulating cell proliferation, enhancing DNA damage repair, promoting metastasis of cancer cells, and the inhibition of FOXM1 has been shown to prevent the initiation and progression of multiple cancers and FOXM1 is considered to be an effective target for tumor therapeutic drug development. The N-terminus of FOXM1 has been found to prevent transcriptional activities of FOXM1 and to mediate the interaction between FOXM1 and SMAD3.Methods: A recombinant FOXM1 N-terminal domain (1-138aa) fused with a nine arginine cell-penetrating peptide is produced with an E. coli expression system and named as M1-138. The effects of M1-138 on the proliferation, migration, and tumorigenic ability of cancer cells are analyzed in vitro with cell counting, transwell assays, and colony formation assays. Electrophoretic mobility shift assays (EMSAs) and Luciferase activity assays are used to test the DNA binding ability and transcriptional activity of transcription factors. The levels of mRNAs and proteins are measured by quantitative-PCR, Western blotting or Immunohistochemistry. The interactions among proteins are analyzed with Pull-down and Co-immunoprecipitation (Co-IP) assays. The nude mouse engrafted tumor models are used to test the inhibitory effects of M1-138 in vivo.Results: M1-138 diminishes the proliferation and migration abilities of cancer cells through binding to FOXM1 and FOXM1-interacting factor SMAD3, and consequently attenuating FOXM1 transcriptional activities from both direct and indirect FOXM1-promoter binding mechanisms and interfering with the interaction between FOXM1 and SMAD3. Treatment of M1-138 prevents tumorigenicity of cancer cells and inhibits tumor growth in nude mouse xenograft models with no obvious signs of toxicity.Conclusion: M1-138 is a promising drug candidate for the development of anti-cancer therapeutics targeting FOXM1 and SMAD3.

Quantitative PCR-Based Assessment of Vaccinia Virus RNA and DNA in Infected Cells.

Quantitative PCR-based methods have proven to be easy-to-use, cost-effective procedures for the quantification of viral gene expression and viral genome numbers. Quantitative PCR (qPCR) and quantitative reverse transcriptase-PCR (qRT-PCR) are rapid and sensitive approaches that can be used to pinpoint defects in viral DNA replication and transcriptional activity, respectively. Due to the significant nucleotide overlap between Poxviridae these methods can be employed across a wide range of viruses from this family. Here we provide methods for the quantification of vaccinia DNA replication by qPCR and quantification of the three classes of vaccinia gene transcription by qRT-PCR.

Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients

It has been proposed that serum hepatitis B core-related antigen (HBcrAg) reflects intrahepatic covalently closed circular (ccc)DNA levels. However, the correlation of HBcrAg with serum and intrahepatic viral markers and liver histology has not been comprehensively investigated in a large sample. We aimed to determine if HBcrAg could be a useful therapeutic marker in patients with chronic hepatitis B. HBcrAg was measured by chemiluminescent enzyme immunoassay in 130 (36 hepatitis B e antigen [HBeAg]+ and 94 HBeAg-) biopsy proven, untreated, patients with chronic hepatitis B. HBcrAg levels were correlated with: a) serum hepatitis B virus (HBV)-DNA, quantitative hepatitis B surface antigen and alanine aminotransferase levels; b) intrahepatic total (t)HBV-DNA, cccDNA, pregenomic (pg)RNA and cccDNA transcriptional activity (defined as pgRNA/cccDNA ratio); c) fibrosis and necroinflammatory activity scores. HBcrAg levels were significantly higher in HBeAg+ vs. HBeAg- patients and correlated with serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA and cccDNA levels, and transcriptional activity. Patients who were negative for HBcrAg (<3 LogU/ml) had less liver cccDNA and lower cccDNA activity than the HBcrAg+ group. Principal component analysis coupled with unsupervised clustering identified that in a subgroup of HBeAg- patients, higher HBcrAg levels were associated with higher serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA, cccDNA transcriptional activity and with higher fibrosis and necroinflammatory activity scores. Our results indicate that HBcrAg is a surrogate marker of both intrahepatic cccDNA and its transcriptional activity. HBcrAg could be useful in the evaluation of new antiviral therapies aiming at a functional cure of HBV infection either by directly or indirectly targeting the intrahepatic cccDNA pool. Hepatitis B virus causes a chronic infection which develops into severe liver disease and liver cancer. The viral covalently closed circular DNA (cccDNA) is responsible for the persistence of the infection in hepatocytes. To better manage patient treatment and follow-up, and to develop new antiviral treatments directly targeting the intrahepatic pool of cccDNA, serum surrogate markers reflecting the viral activity in the liver are urgently needed. In this work, we demonstrate that quantification of hepatitis B core-related antigen in serum correlates with cccDNA amount and activity and could be used to monitor disease progression.

FOXM1 and the NPM-ALK/STAT3 Axis Form a Novel Positive Feedback Loop in Promoting the Oncogenesis of ALK-Positive Anaplastic Large Cell Lymphoma

Peng Wang1, Moinul Haque2, Jing Li2,3, Yung-Hsing Huang2, Meaad Almowaled2, Carter Bargar4, Adam Karpf4, Will Chen2, Suzanne Turner5 and Raymond Lai2,61Division of Hematology, Dept of Medicine, University of Alberta, Edmonton, 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton Alberta, Canada; 3Electron Microscopy Center, Basic Medical Science College, Harbin Medical University, Harbin, Heilongjiang, China; 4Eppley Institute and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, USA; 5Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK;6Department of Oncology, University of Alberta, Edmonton, Alberta, Canada1. Backgrounds and AimsForkhead Box M1 (FOXM1) is a transcription factor implicated in the pathogenesis of solid tumors, and it has been shown to promote cell-cycle progression, stem cell renewal and chemotherapeutic resistance in cancer cells. Nonetheless, the biological significance of FOXM1 in hematologic malignancies has not been extensively studied. Here, we studied the expression and role of FOXM1 in ALK-positive anaplastic large cell lymphoma (ALK+ALCL).2 Methods and ResultsIn contrast with normal lymphocytes, FOXM1 was highly expressed in all ALK+ALCL cell lines (5/5), tumors from patients (6/6) and tumors arising from NPM-ALK transgenic mice. Experiments using nuclear/cytoplasmic fractionation, immunocytochemistry and reporter assays had provided evidence that FOXM1 is transcriptionally active in ALK+ALCL. Down-regulation of FOXM1 expression using shRNA and a pharmacologic agent (thiostrepton) resulted in a significant reduction in cell growth, colony formation in soft agar and cell-cycle arrest in ALK+ALCL cells. Further studies revealed that the oncogenic potential of FOXM1 is linked to substantial increases in the phosphorylation/activation status of NPM-ALK and STAT3, and the upregulations of a host of cytokines that have been previously shown to activate the NPM-ALK/STAT3 axis, including IGF-1, IL9 and IL21. Using co-immunoprecipitation, we found that NPM-ALK binds to FOXM1 in the nucleus of ALK+ALCL cells. Importantly, the binding of NPM-ALK to FOXM1 promotes the DNA binding ability and transcriptional activity of FOXM1, and functional inhibition of NPM-ALK using crizotinib or depletion of NPM-ALK using siRNA in ALK+ALCL cells significantly decreased the transcriptional activity of FOXM1.Conclusions:In conclusion, we have identified a novel oncogenic feedback loop involving FOXM1 and the NPM-ALK/STAT3 axis in ALK+ALCL. This study has revealed the first clear example in which NPM-ALK exerts important oncogenic functions in the nuclei of ALK+ALCL cells, by means of its binding to an oncogenic transcription factor so as to promote its DNA binding and transcription activity. DisclosuresNo relevant conflicts of interest to declare.

Targeted Delivery of CpG-Mir-146a Mimic Oligonucleotides As a Therapeutic Strategy to Reduce NF-Κb-Mediated Pathogenic Inflammation and Myeloid Leukemia Progression

NF-kB signaling plays central role in the regulation of immune cell activity. The microRNA-146a-5p (miR-146a) provides negative feedback inhibition of the NF-kB pathway to prevent either excessive immunity, such as cytokine release syndrome. Low expression of miR-146a is also implicated in certain types of leukemia, especially in del(5q)-syndrome myelodysplastic and acute myeloid leukemia (MDS/AML). While miR-146a is a potential therapeutic target, the lack of efficient miRNA delivery methods limits clinical translation. We previously developed a strategy for targeted delivery of oligonucleotide therapeutics, such as siRNA, into human and mouse myeloid cells and B cells, using partly or completely phophorothioated (PS) single-stranded oligodeoxynucleotides containing CpG motif (CpG ODN). Here, we demonstrate that similar strategy, using CpG ODN optimized for targeting human monocytes, can be employed for the delivery of functional miR-146a mimic. The CpG-miR146a mimic conjugate, but not unconjugated miR-146a, was quickly internalized by target human and mouse myeloid cells, such as dendritic cells, macrophages and leukemic cells. In vitro CpG-miR146a mimic reduced protein levels of downstream targets, Irak1 and Traf6, as well as the inhibition of DNA binding and transcriptional activity of NF-kB in myeloid cells. We further verified functional activity of CpG-miR146a mimic against several models of acute inflammation. The intravenous injections of this oligonucleotide reduced myeloproliferation and excessive cytokine response to bacterial endotoxin or to live Listeria challenge in miR-146a-/- mice. Furthermore, CpG-miR146a mimic had similar anti-inflammatory effect also in a human model of cytokine release syndrome induced by CD19 chimeric antigen receptor (CAR) T-cell activity. CpG-miR146a effectively abrogated CAR T-cell induced IL-6 production by human monocytes in vitro. Finally, our studies indicated that CpG-miR146a mimic can target del(5q) MDS and AML cells in vitro and in vivo resulting in growth inhibition of MDSL and HL-60 acute myeloid leukemia. Our results suggest that CpG-miR146a strategy can be provide a new-in-class immunomodulatory therapeutics for treatment of acute inflammatory disorders, myeloproliferative diseases and myeloid leukemia. DisclosuresWang:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Kortylewski:N/A: Patents & Royalties: I am an inventor on the US patent 9,976,147.

Activation of Haa1 and War1 transcription factors by differential binding of weak acid anions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Haa1 and War1 transcription factors are involved in cellular adaptation against hydrophilic weak acids and lipophilic weak acids, respectively. However, it is unclear how these transcription factors are differentially activated depending on the identity of the weak acid. Using a field-effect transistor (FET)-type biosensor based on carbon nanofibers, in the present study we demonstrate that Haa1 and War1 directly bind to various weak acid anions with different affinities. Haa1 is most sensitive to acetate, followed by lactate, whereas War1 is most sensitive to benzoate, followed by sorbate, reflecting their differential activation during weak acid stresses. We show that DNA binding by Haa1 is induced in the presence of acetic acid and that the N-terminal Zn-binding domain is essential for this activity. Acetate binds to the N-terminal 150-residue region, and the transcriptional activation domain is located between amino acid residues 230 and 483. Our data suggest that acetate binding converts an inactive Haa1 to the active form, which is capable of DNA binding and transcriptional activation.

Mitochondrial functions and melatonin: a tour of the reproductive cancers.

Cancers of the reproductive organs have a strong association with mitochondrial defects, and a deeper understanding of the role of this organelle in preneoplastic-neoplastic changes is important to determine the appropriate therapeutic intervention. Mitochondria are involved in events during cancer development, including metabolic and oxidative status, acquisition of metastatic potential, resistance to chemotherapy, apoptosis, and others. Because of their origin from melatonin-producing bacteria, mitochondria are speculated to produce melatonin and its derivatives at high levels; in addition, exogenously administered melatonin accumulates in the mitochondria against a concentration gradient. Melatonin is transported into tumor cell by GLUT/SLC2A and/or by the PEPT1/2 transporters, and plays beneficial roles in mitochondrial homeostasis, such as influencing oxidative phosphorylation and electron flux, ATP synthesis, bioenergetics, calcium influx, and mitochondrial permeability transition pore. Moreover, melatonin promotes mitochondrial homeostasis by regulating nuclear DNA and mtDNA transcriptional activities. This review focuses on the main functions of melatonin on mitochondrial processes, and reviews from a mechanistic standpoint, how mitochondrial crosstalk evolved in ovarian, endometrial, cervical, breast, and prostate cancers relative to melatonin's known actions. We put emphasis on signaling pathways whereby melatonin interferes within cancer-cell mitochondria after its administration. Depending on subtype and intratumor metabolic heterogeneity, melatonin seems to be helpful in promoting apoptosis, anti-proliferation, pro-oxidation, metabolic shifting, inhibiting neovasculogenesis and controlling inflammation, and restoration of chemosensitivity. This results in attenuation of development, progression, and metastatic potential of reproductive cancers, in addition to lowering the risk of recurrence and improving the life quality of patients.

Facilitated Diffusion Mechanisms in DNA Base Excision Repair and Transcriptional Activation.

Preservation of the coding potential of the genome and highly regulated gene expression over the life span of a human are two fundamental requirements of life. These processes require the action of repair enzymes or transcription factors that efficiently recognize specific sites of DNA damage or transcriptional regulation within a restricted time frame of the cell cycle or metabolism. A failure of these systems to act results in accumulated mutations, metabolic dysfunction, and disease. Despite the multifactorial complexity of cellular DNA repair and transcriptional regulation, both processes share a fundamental physical requirement that the proteins must rapidly diffuse to their specific DNA-binding sites that are embedded within the context of a vastly greater number of nonspecific DNA-binding sites. Superimposed on the needle-in-the-haystack problem is the complex nature of the cellular environment, which contains such high concentrations of macromolecules that the time frame for diffusion is expected to be severely extended as compared to dilute solution. Here we critically review the mechanisms for how these proteins solve the needle-in-the-haystack problem and how the effects of cellular macromolecular crowding can enhance facilitated diffusion processes. We restrict the review to human proteins that use stochastic, thermally driven site-recognition mechanisms, and we specifically exclude systems involving energy cofactors or circular DNA clamps. Our scope includes ensemble and single-molecule studies of the past decade or so, with an emphasis on connecting experimental observations to biological function.

S-nitrosylation of the zinc finger protein SRG1 regulates plant immunity

Nitric oxide (NO) orchestrates a plethora of incongruent plant immune responses, including the reprograming of global gene expression. However, the cognate molecular mechanisms remain largely unknown. Here we show a zinc finger transcription factor (ZF-TF), SRG1, is a central target of NO bioactivity during plant immunity, where it functions as a positive regulator. NO accumulation promotes SRG1 expression and subsequently SRG1 occupies a repeated canonical sequence within target promoters. An EAR domain enables SRG1 to recruit the corepressor TOPLESS, suppressing target gene expression. Sustained NO synthesis drives SRG1 S-nitrosylation predominantly at Cys87, relieving both SRG1 DNA binding and transcriptional repression activity. Accordingly, mutation of Cys87 compromises NO-mediated control of SRG1-dependent transcriptional suppression. Thus, the SRG1-SNO formation may contribute to a negative feedback loop that attenuates the plant immune response. SRG1 Cys87 is evolutionary conserved and thus may be a target for redox regulation of ZF-TF function across phylogenetic kingdoms.

Farnesoid X receptor‐α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Heat Shock Factor 1 Inhibits the Expression of Suppressor of Cytokine Signaling 3 in Cerulein-Induced Acute Pancreatitis.

Heat shock factor 1 (HSF1), an important transcriptional molecule in the heat shock process, can regulate the expression of a lot of inflammatory mediators in addition to heat shock proteins. This study evaluated the inhibitive function of HSF1 on the expression of suppressor of cytokine signaling 3 in cerulein-induced acute pancreatitis. After HSF1 mice, HSF1 mice, and AR42J cells were treated with cerulein, histopathological score, expression of SOCS3 mRNA, and protein levels were analyzed by using RT-PCR, quantitative real-time RT-PCR, and western blotting, respectively. DNA binding and transcription activity of HSF1 to the SOCS3 promoter were detected by chromatin immunoprecipitation and luciferase reporter assays. The histopathological scores of the pancreas decreased significantly in the cerulein-induced HSF1 mice compared with the cerulein-induced HSF1 mice. SOCS3 mRNA and protein level decreased in the pancreas of the unstimulated HSF1 and HSF1 mice, whereas increased in the pancreas of the cerulein-induced HSF1 and HSF1 mice, with higher in the pancreas of cerulein-induced HSF1mice. In the pcDNA3.1-transfected AR42J cells, SOCS3 protein decreased and was upregulated after the cerulein stimulation, whereas HSF1 overexpression inhibited the upregulation. In the scramble-transfected AR42J cells, SOCS3 protein decreased and was upregulated after the cerulein stimulation, whereas HSF1-RNAi further promoted the upregulation. EMSA and chromatin immunoprecipition showed that HSF1 could directly bind to SOCS3 promoter region. Reporter assays showed that HSF1 could inhibit the transcriptional activity on SOCS3 promoter. HSF1 can protect AR42J cells from cerulein-induced pancreatitis through inhibiting the expression of SOCS3.

Exposure of Barrett's and esophageal adenocarcinoma cells to bile acids activates EGFR-STAT3 signaling axis via induction of APE1.

The development of Barret’s esophagus (BE) and its progression to esophageal adenocarcinoma (EAC) is highly linked to exposure to acidic bile salts due to chronic gastroesophageal reflux disease (GERD). In this study, we investigated the role of Apurinic/apyrimidinic endonuclease 1 /redox effector factor-1 (APE-1/REF-1) in STAT3 activation in response to EAC. Our results indicate that APE1 is constitutively overexpressed in EAC whereas its expression is transiently induced in response to acidic bile salts in non-neoplastic BE. Using overexpression or shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using an APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. EGFR phosphorylation (Y1068) was directly associated with APE1 levels and redox function. Co-immunoprecipitation and proximity ligation assays indicated that APE-1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following acidic bile salts treatment. ChIP assays indicated that acidic bile salts treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. The induction of APE-1 - STAT3 axis in acidic bile salts conditions provided a survival advantage and promoted cellular proliferation. In summary, our study provides multiple pieces of evidence supporting a critical role for APE1 induction in activating the EGFR-STAT3 signaling axis in response to acidic bile salts, the main risk factors for Barrett’s carcinogenesis.

Abstract LB-396: Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma cells

Abstract Background: Barrett's esophagus (BE) is a premalignant condition where columnar metaplasia replaces the stratified squamous epithelium of the distal esophagus and is regarded as the main risk factor for esophageal adenocarcinoma (EAC). BE develops as a consequence of chronic gastroesophageal reflux disease (GERD), where acid and bile salts abnormally refluxate from the stomach into the distal region of esophagus. APE1/REF-1 is a multifunctional protein that plays an essential role in DNA repair activity and in redox-dependent transcriptional regulation. STAT3 signaling has been implicated in regulating cell survival, angiogenesis, and metastasis. EGFR is well known to facilitate the tyrosine phosphorylation of STAT3 and initiates downstream signaling cascades that facilitate proliferation, tumorigenesis, metastasis, and chemo resistance. In this study, we investigated the role of APE1 in activation of STAT3 in response to acidic bile salts (ABS) in EAC. Methodology: Non-neoplastic Barrett's (CPA, BAR10T and CPB) and esophageal adenocarcinoma (OE33, FLO-1) cells were exposed to acidic bile salts (pH4.0 and 5.5, 100 and 200uM) for 20 minutes. The mRNA and protein expression levels of APE-1, STAT3 and target genes were evaluated by real time PCR and immunoblotting. STAT3 transcription activity was studied by luciferase reporter assay. Immunofluorescence and cytoplasmic-nuclear fractionation were used to examine localization of APE-1, EGFR and STAT3 after exposure to acidic bile salts. Results: We demonstrate that APE1 is constitutively overexpressed in EAC whereas its expression is induced in non-neoplastic BE in response to ABS. Using overexpression and shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. Phosphorylation of EGFR (Y1068) was directly associated with APE1 levels and redox function. By using co-immunoprecipitation and proximity ligation assays, we found that APE1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in the mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following ABS treatment. ChIP assays indicated that ABS-treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL, following ABS treatment. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. In summary, our results provide evidence that bile reflux induced APE1 is involved in the activation of EGFR-STAT3 signaling axis in BE and EAC. Conclusion: The redox activity of APE1 mediates EGFR- STAT3 activation as a survival mechanism in response to acidic bile salts and oxidative stress. Constitutive activation of APE1 in EAC may present a therapeutic opportunity to targeting redox vulnerabilities in cancer cells. Citation Format: Ajaz A. Bhat, Heng Lu, Mohammad Suotto, Chen Zheng, Dunfa Peng, Mohammad Haris, Abbes Belkhiri, Wael El-Rifai. Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma cells [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 LB-396.

Reconstituting Arabidopsis CRY2 Signaling Pathway in Mammalian Cells Reveals Regulation of Transcription by Direct Binding of CRY2 to DNA

In response to blue light, cryptochromes photoexciteand interact with signal partners to transduce signalalmost synchronously in plants. The detailed mechanism of CRY-mediated light signaling remains unclear: the photobiochemical reactions of cryptochrome are transient and synchronous, thus making the monitoring and analysis of each step difficult in plant cells. In this study, we reconstituted the Arabidopsis CRY2 signaling pathway in mammalian cells and investigated the biological role of Arabidopsis CRY2 in this heterologous system, eliminating the interferences of other plant proteins. Our results demonstrated that, besides being the light receptor, Arabidopsis CRY2 binds to DNA directly and acts as a transcriptional activator in a blue-light-enhanced manner. Similar to classic transcription factors, we found that the transcriptional activity of CRY2 is regulated by its dimerization and phosphorylation. In addition, CRY2 cooperates with CIB1 to regulate transcription by enhancing the DNA affinity and transcriptional activity of CIB1 under blue light.

Abstract 2867: Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML

Abstract Binding of AML blasts to E-selectin activates cellular survival pathways leading to chemoresistance. GMI-1271 is a novel E-selectin antagonist that when used in combination with chemotherapy results in improved survival in mouse syngeneic and xenogeneic AML tumor models. GMI-1271 in combination with two chemotherapy regimens is in early clinical trials for the treatment of AML. Azacitidine (5-AC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. 5-AC is approved in Europe for the treatment of limited populations with AML. We evaluated GMI-1271 in combination with 5-AC in the KG1 AML tumor model to assess the potential for therapeutic benefit of the combination. NSG mice (10/group) received i.v. injections of KG1 cells, and were treated with saline, GMI-1271 alone, 5-AC alone, or the combination of GMI-1271 and 5-AC. The median survival time (MST) of mice treated with 5-AC was 88 days and statistically different (P&amp;lt;0.002) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). All mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. At study conclusion (Day 104 post tumor injection) 20% of mice treated with 5-AC remained alive. Importantly, the therapeutic activity of 5-AC was significantly enhanced when combined with GMI-1271 (MST&amp;gt;104 days, P=0.0140 compared to 5-AC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AC and that GMI-1271 attenuates this protection. To investigate the nature of the observed in vivo activity of GMI-1271 and 5-AC, KG1 cells were cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AC and the reactivity of the cells to HECA-452 (an antibody that recognizes an E-selectin carbohydrate ligand) and binding to E-selectin were determined by flow cytometry. Treatment with 5-AC resulted in a 28% increase in reactivity of cells to HECA-452 and a 32% increase in binding to E-selectin. Further in vitro assays of static adhesion revealed an increase in the adhesion of 5-AC-treated KG1 cells to E-selectin. Notably, the enhanced adhesion of KG-1 cells to E-selectin was reversed using GMI-1271. Collectively, these results demonstrate that 5-AC can lead to increased expression of E-selectin ligands on AML cells and that the therapeutic potential of 5-AC could be improved by combination with GMI-1271. Citation Format: Theodore A. Smith, William E. Fogler, Ji-Won Lee, John L. Magnani. Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML [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 2867.

Abstract 2994: Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents

Abstract Aberrant DNA hypermethylation within promoter regions and subsequent gene silencing are near universal hallmarks of human cancer. Reversal of DNA methylation by a hypomethylating agent, such as decitabine (Dacogen) or azacytidine (Vidaza), has shown clinical benefit for the treatment of heme malignancies. However, these agents have several limitations that preclude their full potential from being realized such as the requirement for IV administration, poor PK properties and a mechanism that requires incorporation into replicating DNA. This indirect, irreversible inhibition of the DNA methyltransferase (DNMT) family (DNMT1, 3a and 3b) and subsequent DNA damage induces significant dose-limiting toxicity thus preventing sufficient target engagement required for maximal demethylation. A drug discovery effort focused on DNMT1, the key family member responsible for maintaining the DNA methylation pattern, was initiated based on the compelling nature of the target coupled with the need for improved agents. A high-throughput screen identified a single dicyanopyridine (DCP) series of reversible, non-DNA incorporating, highly selective inhibitors for DNMT1 over DNMT3a or DNMT3b. Ensuing structure-activity relationship (SAR) optimization of the series led to the discovery of potent tool compounds that in cancer cells induced robust decreases in global DNA methylation, transcriptional activation of many silenced genes, and inhibition of cancer cell growth. In contrast to decitabine where most cells showed a cytotoxic response, our DNMT1 tool inhibitors primarily elicited a cytostatic response. Furthermore, studies in a mouse tumor model revealed decreased DNA methylation and a dose-dependent (1-45 mg/kg, BID) decrease in tumor growth with regression at the highest doses. In summary, a series of potent, selective DNMT1 inhibitors were discovered and refined delivering tool compounds capable of eliciting changes in DNA methylation, transcriptional activation, and tumor regression at well-tolerated doses. Thus demonstrating that selective, non-covalent inhibitors of DNMT1 may provide benefit over traditional DNA incorporating hypomethylating agents. Citation Format: Melissa B. Pappalardi, Mark Cockerill, Jessica L. Handler, Alexandra Stowell, Kathryn Keenan, Christian S. Sherk, Elisabeth A. Minthorn, Charles F. McHugh, Charlotte Burt, Kristen Wong, David T. Fosbenner, Mehul Patel, Jacques Briand, Helai Mohammad, Lourdes Rueda, Andrew Benowitz, Rab Prinjha, Dirk Heerding, Ryan G. Kruger, Ali Raoof, Allan Jordan, Bryan W. King, Michael T. McCabe. Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents [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 2994.