Consensus Binding Site Research Articles

Abstract 3534: FAT1: A potential target of NFkB (RelA) in GBM

Abstract FAT1 is a large transmembrane protein (502 kD), reportedly having dual role in different human cancers. Our lab has reported oncogenic role of FAT1 overexpression in glioblastoma multiforme (GBM, grade IV glioma) increasing migration-invasion as well as tumor inflammation and HIF1α activity. Morris et al. (2013), have reported that glioma patients with low FAT1-expressing tumors have significantly (p=0.037) longer survival (Rembrandt dataset), suggesting high FAT1 expression to have adverse effect on GBM patient survival. Since NFκB is a major transcription factor overexpressed in GBM and involve in regulating many oncognes at transcriptional level. There are no reports available on FAT1 upstream regulation. Therefore in this study we investigated the role of NFkB (RelA) in regulating FAT1 expression in GBM. In-silico analysis of FAT1 promoter predicted binding sites for several transcription factors including NFkB (RelA) having multiple binding motifs with high matrix score. We assessed the mRNA expression correlation of FAT1 and NFκB target gene (BCL2) in small cohort of GBM tumors (n=16). A significant positive correlation (0.647, p<0.01) was established between FAT1 and BCL2 in GBM tumors compared to normal brain (Clontech). In a sequential bioinformatics study, we analyzed TCGA GBM dataset and found significantly elevated expression of FAT1, RelA, IKBKB and RelA target genes (BCL2, MMP9 and Serpine) in GBM tumors (n=430) as compared to normal brain (n=10). In survival analysis of TCGA dataset {Grouped into high (n=100) and low FAT1 expression (n=100)}, low FAT1 expressing patients showed higher survival, corroborating the observation of Morris et al 2013. The role of NFkB (RelA) in FAT1 expression regulation was further analyzed in in-vitro in U87MG (GBM) cell line. We overexpressed and chemically inhibited NFkB using RelApBABE expression vector and PDTC respectively. There was increased FAT1expression in cells overexpressing NFkB and this effect could be reversed on NFkB inhibition, suggesting that RelA plays an important role in regulation of endogenous FAT1 expression. Furthermore, to confirm the regulatory role of NFκB (RelA) on FAT1 expression, 2-kb FAT1 promoter (pGLF2) and sequential 5’ deletion constructs (pGLF2A, pGLF2B, pGLF2C and pGLF2D) with different RelA binding sites were cloned into luciferase basic vector (pGL3B) and analyzed for their activity by in vitro luciferase assay. Among all constructs, 5’ deletion construct (pGLF2C) containing single consensus binding site for RelA and least no. of repressor sites, showed highest luciferase activity in U87MG. Furthermore, site directed mutagenesis of RelA consensus binding site decreased luciferase activities by three-fold as compared to the wild type construct, pGLF2C. The endogenous binding of RelA on FAT1 promoter was confirmed by ChIP (chromatin-immunoprecipitation) assay. Conclusively, our study for the first time identified NFkB (RelA) transcription factor as a potential regulator of FAT1. Citation Format: Chitrangda Srivastava, Khushboo Irshad, Parthaprasad Chattopadhyay, Chitra Sarkar, Ashish Suri, Subrata Sinha, Kunzang Chosdol. FAT1: A potential target of NFkB (RelA) in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3534. doi:10.1158/1538-7445.AM2017-3534

Molecular insight into protein binding orientations and interaction modes on hydrophobic charge-induction resin

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the functional ligand has been developed as a new technology for antibody purification. In the present work, molecular simulation methods were developed to investigate the interactions between the Fc fragment of IgG and a MEP ligand net. The MM/PBSA method was used to evaluate the binding energy for the MEP ligand net at different densities. It was found that ligand density had significant influence on the binding of Fc. Potential binding conformations were further analyzed by molecular dynamics simulation. It was found that the interaction between Fc and MEP ligand net is driven by self-adaptive conformation adjustment and multiple-site binding. Hydrophobic forces dominate the binding interaction, which appeared as the results of synergistic actions of binding sites located on CH2, CH3, and the consensus binding site (CBS) of the Fc fragment. At acidic pH, the electrostatic repulsion between the basic residues and the protonated pyridine ring group on MEP ligands is the main driving force for the detachment of the Fc fragment from the MEP net.

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia

AbstractSomatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5′-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy–induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Identification of an Enhancer Critical for the ephirn-A5 Gene Expression in the Posterior Region of the Mesencephalon.

Ephrin-A5 has been implicated in the regulation of brain morphogenesis and axon pathfinding. In this study, we used bacterial homologous recombination to express a LacZ reporter in various ephrin-A5 BAC clones to identify elements that regulate ephrin-A5 gene expression during mesencephalon development. We found that there is mesencephalon-specific enhancer activity localized to a specific +25.0 kb to +30.5 kb genomic region in the first intron of ephrin-A5. Further comparative genomic analysis indicated that two evolutionary conserved regions, ECR1 and ECR2, were present within this 5.5 kb region. Deletion of ECR1 from the enhancer resulted in disrupted mesencephalon-specific enhancer activity in transgenic embryos. We also found a consensus binding site for basic helix-loop-helix (bHLH) transcription factors (TFs) in a highly conserved region at the 3′-end of ECR1. We further demonstrated that specific deletion of the bHLH TF binding site abrogated the mesencephalon-specific enhancer activity in transgenic embryos. Finally, both electrophoretic mobility shift assay and luciferase-based transactivation assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific ephrin-A5 enhancer in vitro. Together, these results suggest that the bHLH TF binding site in ECR1 is involved in the positive regulation of ephrin-A5 gene expression during the development of the mesencephalon.

CDX2 upregulates SLC26A3 gene expression in intestinal epithelial cells.

SLC26A3 [downregulated in adenoma (DRA)] plays a key role in mammalian intestinal NaCl absorption, in that it mediates apical membrane Cl-/[Formula: see text] exchange. DRA function and expression are significantly decreased in diarrhea associated with inflammatory bowel disease. DRA is also considered to be a marker of cellular differentiation and is predominantly expressed in differentiated epithelial cells. Caudal-type homeobox protein-2 (CDX2) is known to regulate genes involved in intestinal epithelial differentiation and proliferation. Reduced expression of both DRA and CDX2 in intestinal inflammation prompted us to study whether the DRA gene is directly regulated by CDX2. Our initial studies utilizing CDX2 knockout (CDX2fV/fV;Cre+) mice showed a marked reduction in DRA mRNA and protein levels in proximal and distal colon. In silico analysis of the DRA promoter showed two consensus sites for CDX2 binding. Therefore, we utilized Caco-2 cells as an in vitro model to examine if DRA is a direct target of CDX2 regulation. siRNA-mediated silencing of CDX2 in Caco-2 cells resulted in a marked (~50%) decrease in DRA mRNA and protein levels, whereas ectopic overexpression of CDX2 upregulated DRA expression and also stimulated DRA promoter activity, suggesting transcriptional regulation. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated direct binding of CDX2 to one of the two putative CDX2 binding sites in the DRA promoter (+645/+663). In summary, our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2, implying that reduced expression of DRA in inflammatory bowel disease-associated diarrhea may, in part, be due to downregulation of CDX2 in the inflamed mucosa.NEW & NOTEWORTHY SLC26A3 [downregulated in adenoma (DRA)] mediates intestinal luminal NaCl absorption and is downregulated in inflammatory bowel disease-associated diarrhea. Since both DRA and caudal-type homeobox protein-2 (CDX2) are reduced in intestinal inflammation and the DRA promoter harbors CDX2 binding sites, we examined whether the DRA gene is regulated by CDX2. Our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2 via direct binding to the DRA promoter, suggesting that reduced expression of DRA in inflammatory bowel disease-associated diarrhea could, in part, be attributed to downregulation of CDX2.

Quantitative specificity of STAT1 and several variants.

The quantitative specificity of the STAT1 transcription factor was determined by measuring the relative affinity to hundreds of variants of the consensus binding site including variations in the length of the site. The known consensus sequence is observed to have the highest affinity, with all variants decreasing binding affinity considerably. There is very little loss of binding affinity when the CpG within the consensus binding site is methylated. Additionally, the specificity of mutant proteins, with variants of amino acids that interact with the DNA, was determined and nearly all of them are observed to lose specificity across the entire binding site. The change of Asn at position 460 to His, which corresponds to the natural amino acid at the homologous position in STAT6, does not change the specificity nor does it change the length preference to match that of STAT6. These results provide the first quantitative analysis of changes in binding affinity for the STAT1 protein, and several variants of it, to hundreds of different binding sites including different spacer lengths, and the effect of CpG methylation.

BRAF/MEK inhibitors promote CD47 expression that is reversible by ERK inhibition in melanoma.

The expression of CD47 on the cancer cell surface transmits “don’t eat me” signalling that not only inhibits phagocytosis of cancer cells by phagocytes but also impairs anti-cancer T cell responses. Here we report that oncogenic activation of ERK plays an important role in transcriptional activation of CD47 through nuclear respiratory factor 1 (NRF-1) in melanoma cells. Treatment with BRAF/MEK inhibitors upregulated CD47 in cultured melanoma cells and fresh melanoma isolates. Similarly, melanoma cells selected for resistance to the BRAF inhibitor vemurafenib expressed higher levels of CD47. The increase in CD47 expression was mediated by ERK signalling, as it was associated with rebound activation of ERK and co-knockdown of ERK1/2 by siRNA diminished upregulation of CD47 in melanoma cells after exposure to BRAF/MEK inhibitors. Furthermore, ERK1/2 knockdown also reduced the constitutive expression of CD47 in melanoma cells. We identified a DNA fragment that was enriched with the consensus binding sites for NRF-1 and was transcriptionally responsive to BRAF/MEK inhibitor treatment. Knockdown of NRF-1 inhibited the increase in CD47, indicating that NRF-1 has a critical role in transcriptional activation of CD47 by ERK signalling. Functional studies showed that melanoma cells resistant to vemurafenib were more susceptible to macrophage phagocytosis when CD47 was blocked. So these results suggest that NRF-1-mediated regulation of CD47 expression is a novel mechanism by which ERK signalling promotes the pathogenesis of melanoma, and that the combination of CD47 blockade and BRAF/MEK inhibitors may be a useful approach for improving their therapeutic efficacy.

Specificity-Determining DNA Triplet Code for Positioning of Human Preinitiation Complex

The notion that transcription factors bind DNA only through specific, consensus binding sites has been recently questioned. No specific consensus motif for the positioning of the human preinitiation complex (PIC) has been identified. Here, we reveal that nonconsensus, statistical, DNA triplet code provides specificity for the positioning of the human PIC. In particular, we reveal a highly nonrandom, statistical pattern of repetitive nucleotide triplets that correlates with the genomewide binding preferences of PIC measured by Chip-exo. We analyze the triplet enrichment and depletion near the transcription start site and identify triplets that have the strongest effect on PIC-DNA nonconsensus binding. Using statistical mechanics, a random-binder model without fitting parameters, with genomic DNA sequence being the only input, we further validate that the nonconsensus nucleotide triplet code constitutes a key signature providing PIC binding specificity in the human genome. Our results constitute a proof-of-concept for, to our knowledge, a new design principle for protein-DNA recognition in the human genome, which can lead to a better mechanistic understanding of transcriptional regulation.

Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn’s Disease

Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1× 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

P53 targets TSPAN8 to prevent invasion in melanoma cells

Cutaneous melanoma is a very deadly cancer because of its proclivity to metastasize. Despite the recent development of targeted and immune therapies, patient survival remains low. It is therefore crucial to enhance understanding of the molecular mechanisms underlying invasion. We previously identified tetraspanin 8 (TSPAN8) as an important modulator of melanoma invasiveness, and several of its transcriptional regulators, which affect TSPAN8 expression during melanoma progression toward an invasive stage. This study found that TSPAN8 promoter contains consensus-binding sites for p53 transcription factor. We demonstrated that p53 silencing was sufficient to turn on Tspan8 expression in non-invasive melanoma cells and that p53 acts as a direct transcriptional repressor of TSPAN8. We also showed that p53 modulated matrigel invasion in melanoma cells in a TSPAN8-dependent manner. In conclusion, this study reveals p53 as a negative regulator of Tspan8 expression. As TP53 gene is rarely mutated in melanoma, it was hitherto poorly studied but its role in apoptosis and growth suppression in melanoma is increasingly becoming clear. The study highlights the importance of p53 as a regulator of melanoma invasion and the concept that reactivating p53 could provide a strategy for modulating not only proliferative but also invasive capacity in melanoma treatment.

Suppression of FOXM1 Transcriptional Activities via a Single-Stranded DNA Aptamer Generated by SELEX

The transcription factor FOXM1 binds to its consensus sequence at promoters through its DNA binding domain (DBD) and activates proliferation-associated genes. The aberrant overexpression of FOXM1 correlates with tumorigenesis and progression of many cancers. Inhibiting FOXM1 transcriptional activities is proposed as a potential therapeutic strategy for cancer treatment. In this study, we obtained a FOXM1-specific single stranded DNA aptamer (FOXM1 Apt) by SELEX with a recombinant FOXM1 DBD protein as the target of selection. The binding of FOXM1 Apt to FOXM1 proteins were confirmed with electrophoretic mobility shift assays (EMSAs) and fluorescence polarization (FP) assays. Phosphorthioate-modified FOXM1 Apt (M-FOXM1 Apt) bound to FOXM1 as wild type FOXM1 Apt, and co-localized with FOXM1 in nucleus. M-FOXM1-Apt abolished the binding of FOXM1 on its consensus binding sites and suppressed FOXM1 transcriptional activities. Compared with the RNA interference of FOXM1 in cancer cells, M-FOXM1 Apt repressed cell proliferation and the expression of FOXM1 target genes without changing FOXM1 levels. Our results suggest that the obtained FOXM1 Apt could be used as a probe for FOXM1 detection and an inhibitor of FOXM1 transcriptional functions in cancer cells at the same time, providing a potential reagent for cancer diagnosis and treatment in the future.

Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic β-cells

ObjectiveThe transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure. MethodsIn order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. ResultsAlthough adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells. ConclusionsOur data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass.

The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein

The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function.

C/EBPβ Is Required for Survival of Ly6C- Monocytes after Committment to Monocyte Lineage through Upregulation of Csf1r

Monopoiesis is the process in which hematopoietic stem cells (HSCs) continuously give rise to monocytes. Accumulating evidence has identified cellular constituents of monopoiesis. Common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), macrophage-dendritic cell precursors (MDPs) and common monocyte progenitors (cMoPs) are the intermediates during the differentiation of HSCs into mature monocytes. In mice, CD11b+ CD115+ monocytes are further divided into two subsets based on the expression of Ly6C. Classical monocytes express Ly6C on their surface. By contrast, Ly6C− patrolling monocytes have been recently identified, and the molecular mechanisms which regulate the development and homeostasis of Ly6C−monocytes still remain elusive.C/EBPβ is a leucine zipper transcription factor which regulates stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Hayashi et al. Leukemia 2013). We have recently found that peripheral blood (PB) monocytes are significantly reduced in steady-state Cebpb−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year at this meeting, we have reported that cell death of Ly6C− monocytes was accelerated through reduced expression of Csf1r (encoding a receptor for M-CSF) in Cebpb−/− mice. Here in this study, we determined the precise developmental stage where C/EBPβ is mandatory for survival of Ly6C− monocytes, and investigated the mechanism of Csf1r regulation by C/EBPβ.A recent publication demonstrated that Mx1 is preferentially expressed by monocytes and a Mx1 promoter-mediated conditional system targets monocytes without inoculation of polyI:C (Hashimoto et al. Immunity, 2013), suggesting that Mx1-Cre Cebpbf/f mouse is ideal to evaluate the monocyte-specific requirement for C/EBPβ. We confirmed that upregulation of Cebpb mRNA during monopoiesis was significantly impaired after cMoP stage in Mx1-Cre+Cebpbf/f mice. In order to exclude the possible involvement of Cebpβ deficient microenvironment, bone marrow (BM) cells of Mx1-Cre+Cebpβf/f mice (CD45.2+) were transplanted into lethally irradiated CD45.1+ wild type mice. The frequencies of Ly6C− monocytes in the recipients of Mx1-Cre+Cebpbf/f BM cells were significantly reduced when compared to mice that received Mx1-Cre−Cebpbf/f BM cells (Figure). These results strongly suggest that C/EBPβ is specifically required after commitment to monocytes.In order to investigate the molecular mechanisms involved in the regulation of Csf1r by C/EBPβ, we utilized a combination of a promoter and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) for reporter assay (Pridans et al. Mol Ther Methods Clin Dev, 2014). These regulatory elements contain at least 2 consensus binding sites for C/EBPβ (one in the promoter and the other in the enhancer). C/EBPβ significantly enhanced the reporter activity of the regulatory elements in a dose-dependent manner, and introduction of mutations into either of the consensus binding sites abrogated the reporter activity. Next, we engineered EML cells, a mouse HSC line, to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significant increase of Csf1r mRNA and protein. Using these cells, we performed chromatin immunoprecipitation PCR. Upon treatment with tamoxifen, significant enrichment of C/EBPβ at the promoter region and the FIRE region was observed. These data indicated that C/EBPβ regulates Csf1r through direct binding to these regulatory elements.Collectively, these results demonstrate that C/EBPβ supports survival of Ly6C− monocytes after commitment to monocyte lineage through direct regulation of Csf1r, which is critical for survival and differentiation of monocytes. [Display omitted] DisclosuresHirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.

Critical Roles of Α-Type Lamins in Neutrophil Differentiation Revealed By Analyses of Lmna Trancriptional Regulation and Myeloid Models with Aberrant Lamin Α Expression

Neutrophils differentiate from common myeloid progenitors in the bone marrow where they form uniquely lobulated nuclei while acquiring multiple functions critical to their innate immune responses. We are investigating the roles of nuclear envelope (NE)-associated proteins, including the lamin B receptor (Lbr) and the nuclear lamina proteins lamin A/C, in regulating these nuclear changes, along with determining if these proteins are important to neutrophil functions including chemotaxis, phagocytosis, or reactive oxygen species (ROS) production. We previously reported that Lbr expression increases during neutrophil differentiation while lamin A/C expression decreases. We also used immortalized mouse myeloid progenitors to show that either loss of Lbr or increased lamin A/C expression caused nuclear hypolobulation similar to Pelger-Huët anomaly in humans, plus aberrant chemotaxis. Interestingly, cells lacking Lbr produced less ROS, whereas those overexpressing the lamins exhibited deficient phagocytosis. These studies suggest that the functions of these nuclear proteins go beyond simply supporting nuclear structural changes. Since our previous studies relied on the use of immortalized cell lines, which may not exhibit the full spectrum of normal neutrophil functional responses, we have focused on using techniques to derive and characterize neutrophils with aberrant NE protein expression from ex vivo cultured bone marrow. We also examined whether loss of both A-type lamins affects myeloid progenitor growth and differentiation.Here we show that functionally active neutrophils can be generated by ex vivo culture of bone marrow progenitors from cryopreserved stocks. Imaging flow cytometry, which combines the spatial resolution of fluorescence microscopy with the high throughput format of flow cytometry, was used to demonstrate that the ex vivo cultures at progressive stages of neutrophil maturation exhibit characteristic changes to cell surface marker expression. Customized gating and masking strategies were applied using the image analysis software to quantitatively measure phagocytosis of Escherichia coli particles and release of neutrophil extracellular traps (NETs). We also identified a method to evaluate NET release as a function of cell activation during phagocytosis.We next transduced our ex vivo cultured progenitors with retroviral vectors providing ectopic lamin A expression, and found that derived neutrophils exhibited hypolobulated nuclei morphologically resembling our in vitro models. We also knocked out Lmna expression in the immortalized myeloid progenitors by CRISPR-Cas9 mediated gene disruption, and identified defective growth of the progenitors plus aberrant ROS production in derived neutrophils, similar to the phenotypes observed in cells lacking Lbr.The transcriptional control of the Lmna gene during neutrophil differentiation also was investigated. In contrast to our previous report showing that the ETS factor GABP cooperates with PU.1 or C/EBPε to regulate the Lbr promoter, none of these factors affected background levels of Lmna promoter activities in COS cell reporter assays despite identified ETS and C/EBP consensus binding sites. However, addition of C/EBPα expression caused a greater than 50% decrease in Lmna promoter activities. Furthermore, quantitative ChIP assays demonstrated that C/EBPα most efficiently bound to identified C/EBP binding sequences in the Lmna promoter, indicating that C/EBPα inhibits Lmna expression in differentiating neutrophils.Combined our results demonstrate that precisely regulated levels of NE-associated proteins are critical to neutrophil nuclear maturation and their acquisition of certain functional responses. Our continued studies will utilize imaging flow cytometry to characterize the functions of ex vivo culture-derived neutrophils with aberrant A-type lamin expression, as well as identify roles of additional NE-associated proteins during neutrophil maturation. We are also poised to further characterize the control of genes encoding such NE proteins by various combinations of ETS and C/EBP transcription factors known to regulate myelopoiesis. We predict these studies will provide important insight into the molecular mechanisms governing neutrophil nuclear changes, and why aberrant lobulation is commonly associated with certain hematopoietic disorders. DisclosuresNo relevant conflicts of interest to declare.

Enhanced ETS-like Gene (ELK)-1 Expression Is a Predictor of Inferior Survival in Multiple Myeloma

Background:We have previously reported on the putative role of c-MET/ERK-1/2/ELK1/Proteassemblin (POMP) signaling axis in enhancing proteasome assembly and capacity, which in multiple myeloma (MM) treatment may reduce sensitivity to proteasome inhibitors (PIs) such ascarfilzomib orbortezomib [ASH 2014 MeetingAbstr. 274]. The transcription factor ELK1 was found to regulate POMP expression andcarfilzomib resistance via binding to transcription initiation sites of the POMP gene promoter containing two ELK-1 consensus binding sites. Analysis of the Millennium Pharmaceuticals gene expression profile (GEP) database of patients treated withbortezomib in the relapsed and relapsed/refractory settings showed that patients who had higher expression of MUC20 had superior overall survival (OS) and progression free survival (PFS) compared to those who had lower expression. However, similar outcome was not assessed for other downstream members of this signaling cascade, including the transcription factor ELK1.Methods:We performed statistical analysis on patient and GEP data bases downloaded from the Multiple Myeloma Research Foundation Researcher Gateway website (http://research.themmrf.org),with the objective ofassessing the effects of ELK1 gene expression (gene probe ENSG00000126767) on the OS and PFSin453 patients who had data available on both demographic and clinical characteristics, as well as GEP collected during their first-line therapy.Patients were characterized by clinical variables including age, gender, race, ethnicity, therapy received and international staging system (ISS).ELK1 gene expression was dichotomized into two groups:greater than or equal to median, or less than median expression of the ELK1 gene. The distributions of OS and PFS were estimated by the Kaplan-Meier method.Results:The median patient age was 64 years (range, 27- 93), and 60% of them were male. Of 453 patients, 350 (77%) were Caucasian, 75 (17%) African-American and 11 (2%) Asian. 420 (93%) were receiving first line of therapy, while 27 (6%) and 4 (1%) were receiving second or third line therapies, respectively. Two hundred and thirty nine (53%) patients received combinedbortezomib/immunomodulatory (IMiD) based therapy, 142 (31%) receivedbortezomib-based, and a further 33 (7%) receivedIMiD-based therapy alone, while 24 (5%) patients received combinedcarfilzomib/IMiD based therapy. One hundred and forty seven (33%) patients were ISS stage I,162 (37%) stage II, and 133 (30%) stage III. There were 39 deaths, of which 19 (49%) were due to disease progression. Patents who were greater than or equal to 64 years old had inferior OS compared to those less than 64 years of age (p=0.0266), patients who had ISS stage I or II had superior OS compared to those with stage III (p=0.00983), and after adjusting for stage III, patients who had less than median ELK1 gene expression had superior OS compared to those with greater than or equal to median ELK1 gene expression (Figure 1., p=0.00309). PFS was not affected.Conclusions: Our data continue to support a role for signaling through the c-MET/ERK-1/2/ELK1/POMP axis in enhancing proteasome assembly and capacity, thereby reducing sensitivity to proteasome inhibitors likecarfilzomib orbortezomib in MM. As such, the use of drugs suppressing c-MET signaling in early phase trial design, particularly in combination with other PIs, remains a potentially attractive strategy to overcome resistance to PIs in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HCK Transcription Is Regulated By AP1, NF-Kb and STAT3 Transcription Factors in MYD88 Mutated WM and ABC-DLBCL Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family of tyrosine kinases (SFKs). HCK transcription is aberrantly upregulated in Waldenström’s Macroglobulinemia (WM) and Activated B-cell (ABC) subtype Diffuse Large B-cell Lymphoma (DLBCL) in response to activating mutations in MYD88 (Yang et al, Blood 2016). To clarify the mechanism responsible for the aberrant upregulation of HCK transcription inMYD88 mutated cells, we analyzed the promoter sequence of HCK using PROMO and identified consensus binding sites for transcription factors (AP1, NF-kB, STAT3, and IRF1) that are regulated by mutated MYD88 (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013; Juilland et al, Blood 2016; Yang et al, Blood 2016). We performed Chromatin Immuno-precipitation (ChIP) assays using ChIP grade antibodies to JunB, c-Jun, NF-kB-p65, STAT3 and IRF1 in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1, OCI-Ly3) cells that highly express HCK transcripts, as well as wild type MYD88 expressing GCB DLBCL (OCI-Ly7, OCI-Ly19) cells that show low HCK transcription. Following ChIP, a HCK promoter specific quantitative PCR assay was used to detect HCK promoter sequences. These studies showed that JunB, NF-kB-p65 and STAT3 bound more robustly to the HCK promoter in MYD88 mutated WM and ABC-DLBCL cells versus MYD88 wild type GCB DLBCL cell lines, while c-Jun bound more abundantly to the HCK promoter sequence in all DLBCL cell lines, regardless of MYD88 mutation status. In contrast c-Jun binding was low in MYD88 mutated WM cells. IRF1 binding to the HCK promoter was similar in all cell lines, regardless of the MYD88 mutation status. To further investigate HCK regulation, we developed an HCK promoter driven luciferase reporter vector (WT) with mutated AP-1 binding (AP1-mu-1~6), NF-kB binding (NF-kB-mu-1~5), and STAT3 binding (STAT3-mu) sites and investigated their impact on HCK promoter activity in MYD88 mutated BCWM.1 cells. We observed that mutation of AP1-mu-1,4,5,6; NF-kB-mu-1,4,5, as well as STAT3-mu binding sites greatly reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 transcription factors in HCK gene expression in MYD88 mutated cells. To further clarify the importance of these transcription factors in aberrant HCK gene expression in MYD88 mutated cells, we treated BCWM.1, MWCL-1, TMD-8 and HBL-1 cells with the AP-1 inhibitor SR 11302; NF-kB inhibitor QNZ; and the STAT3 inhibitor STA-21. Treatment of cells for 2 hours with SR 11302, QNZ, and STA-21 at sub-EC50 concentrations resulted in decreased HCK expression in MYD88 mutated all cell lines. Lastly, we investigated the contribution of BCR signaling to HCK transcription. BCWM.1, MWCL-1, TMD-8, and HBL-1 cells were treated with the Syk kinase inhibitor R406, and HCK transcription levels were then assessed. Differences in HCK expression were observed between MYD88 mutated WM and ABC DLBCL cells following R406, supporting a contributing role for BCR signaling in ABC DLBCL but not WM cells to HCK expression. Our data provide critical new insights into HCK regulation, and a framework for targeting pro-survival HCK signaling in WM and ABC DLBCL cells dependent on activating MYD88 mutations. DisclosuresCastillo:Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding; Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.

PCTP (Phosphatidylcholine Transfer Protein) is Regulated By RUNX1 in Platelets/Megakaryocytes and is Associated with Adverse Cardiovascular Events

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5’ upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p < 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value < 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors.Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. DisclosuresNo relevant conflicts of interest to declare.

Dynamic Changes in Chromatin Accessibility Occur in CD8+ T Cells Responding to Viral Infection

In response to acute infection, naive CD8+ Tcells expand, differentiate into effector cells, and then contract to a long-lived pool of memory cells after pathogen clearance. During chronic infections or in tumors, CD8+ Tcells acquire an "exhausted" phenotype. Here we present genome-wide comparisons of chromatin accessibility and gene expression from endogenous CD8+ Tcells responding to acute and chronic viral infection using ATAC-seq and RNA-seq techniques. Acquisition of effector, memory, or exhausted phenotypes was associated with stable changes in chromatin accessibility away from the naive Tcell state. Regions differentially accessible between functional subsets invivo were enriched for binding sites of transcription factors known to regulate these subsets, including E2A, BATF, IRF4, T-bet, and TCF1. Exhaustion-specific accessible regions were enriched for consensus binding sites for NFAT and Nr4a family members, indicating that chronic stimulation confers a unique accessibility profile on exhausted cells.

Noncovalent Binding to DNA: Still a Target in Developing Anticancer Agents.

DNA-binding compounds are of extraordinary importance in medicine, accounting for a substantial portion of antitumor drugs in clinical usage. However, their mechanisms of action remain sometimes incompletely understood. This review critically examines two broad classes of molecules that bind noncovalently to DNA: intercalators and groove binders. Intercalators bind to DNA by inserting their chromophore moiety between two consecutive base pairs, whereas groove binders fit into the grooves of DNA. Noncovalent DNAinteractive drugs can recognize certain supramolecular DNA structures such as the Gquadruplexes found in telomeres and in numerous gene promoters, and they can act as topoisomerase I and II poisons. We discuss how DNA-binding compounds affect transcription and compete with protein factors for binding to consensus binding sites in gene promoters both in vitro and in cultured cancer cells. Moreover, we comment on the design of molecules that can tightly and specifically bind to any desired target DNA, such as various hairpin polyamides which efficacy as chemotherapeutic agents is being evaluated. At present, genome-wide studies, which provide details of events that may influence both cancer progression and therapeutic outcome, are a common way used to analyze the effects of DNA-binding compounds. A conclusive feature that emerges from reviewing the information on DNA-binding compounds is that both natural sources and chemical approaches can be productively used to obtain drugs to manipulate gene expression in cancer cells.