Ets-related Transcription Factor Research Articles

Current understanding of ELF4 deficiency: a novel inborn error of immunity.

ELF4 deficiency has been recently recognized as a novel disorder within the spectrum of inborn errors of immunity (IEIs), specifically categorized as a "disease of immune dysregulation." Cases of this condition, reported by our team and others, are very limited worldwide. As such, our current knowledge of this new disease remains preliminary.This review aims to provide a brief overview of the clinical manifestations, pathogenesis, and treatment strategies for this novel IEI. A comprehensive review was conducted after an extensive literature search in the PubMed/Medline database and websites concerning transcriptional factor ELF4 and reports concerning patients with ELF4 deficiency. Our search strategy was "ELF4 OR ETS-related transcription factor Elf-4 OR EL4-like factor 4 OR myeloid Elf-1-like factor" as of the time of manuscript submission. The current signature manifestations of ELF4 deficiency disorder are recurrent and prolonged oral ulcer, abdominal pain, and diarrhea in pediatric males. In some cases, immunodeficiency and autoimmunity can also be prominent. Targeted Sanger sequencing or whole exome sequencing can be used to detect variation in ELF4 gene. Western blotting for ELF4 expression of the patient's cells can confirm the pathogenic effect of the variant. To fully confirm the pathogenicity of the variant, further functional test is strongly advised. Glucocorticoid and biologics are the mainstream management of ELF4 deficiency disorder. Pediatric males presenting with recurring ulcerations in digestive tract epithelium with or without recurrent fever should be suspected of DEX. When atypical presentations are prominent, variations in ELF4 gene should be carefully evaluated functionally due to the complex nature of ELF4 function. Experience of treating DEX includes use of glucocorticoid and biologics and more precise treatment needs more patients to identify and further mechanistic study.

Lactate promotes neuronal differentiation of SH-SY5Y cells by lactate-responsive gene sets through NDRG3-dependent and -independent manners

Lactate serves as the major glucose alternative to an energy substrate in the brain. Lactate level is increased in the fetal brain from the middle stage of gestation, indicating the involvement of lactate in brain development and neuronal differentiation. Recent reports show that lactate functions as a signaling molecule to regulate gene expression and protein stability. However, the roles of lactate signaling in neuronal cells remain unknown. Here, we showed that lactate promotes the all stages of neuronal differentiation of SH-SY5Y and Neuro2A, human and mouse neuroblastoma cell lines, characterized by increased neuronal marker expression and the rates of neurites extension. Transcriptomics revealed many lactate-responsive genes sets such as SPARCL1 in SH-SY5Y, Neuro2A, and primary embryonic mouse neuronal cells. The effects of lactate on neuronal function were mainly mediated through monocarboxylate transporters 1 (MCT1). We found that NDRG family member 3 (NDRG3), a lactate-binding protein, was highly expressed and stabilized by lactate treatment during neuronal differentiation. Combinative RNA-seq of SH-SY5Y with lactate treatment and NDRG3 knockdown shows that the promotive effects of lactate on neural differentiation are regulated through NDRG3-dependent and independent manners. Moreover, we identified TEA domain family member 1 (TEAD1) and ETS-related transcription factor 4 (ELF4) are the specific transcription factors that are regulated by both lactate and NDRG3 in neuronal differentiation. TEAD1 and ELF4 differently affect the expression of neuronal marker genes in SH-SY5Y cells. These results highlight the biological roles of extracellular and intracellular lactate as a critical signaling molecule that modifies neuronal differentiation.

FLI1 accelerates leukemogenesis through transcriptional regulation of pyruvate kinase-L/R and other glycolytic genes.

In cancer cells, multiple oncogenes and tumor suppressors control glycolysis to sustain rapid proliferation. The ETS-related transcription factor Fli1 plays a critical role in the induction and progression of leukemia, yet, the underlying mechanism of this oncogenic event is still not fully understood. In this study, RNAseq analysis of FLI1-depleted human leukemic cells revealed transcriptional suppression of the PKLR gene and activation of multiple glycolytic genes, such as PKM1/2. Pharmacological inhibition of glycolysis by PKM2 inhibitor, Shikonin, significantly suppressed leukemic cell proliferation. FLI1 directly binds to the PKLR promoter, leading to the suppression of this inhibitor of glycolysis. In accordance, shRNA-mediated depletion of PKLR in leukemic HEL cells expressing high levels of FLI1 accelerated leukemia proliferation, pointing for the first time to its tumor suppressor function. PKLR knockdown also led to downregulation of the erythroid markers EPOR, HBA1, and HBA2 and suppression of erythroid differentiation. Interestingly, silencing of PKLR in HEL cells significantly increased FLI1 expression, which was associated with faster proliferation in culture. In FLI1-expressing leukemic cells, lower PKLR expression was associated with higher expression of PKM1 and PKM2, which promote aerobic glycolysis. Finally, injection of pyruvate, a known inhibitor of glycolysis, into leukemia mice significantly suppressed leukemogenesis. These results demonstrate that FLI1 promotes leukemia in part by inducing glycolysis, implicates PKLR in erythroid differentiation, and suggests that targeting glycolysis may be an attractive therapeutic strategy for cancers driven by FLI1 overexpression.

Abstract P3089: Etv2 Functions As A Pioneer Transcription Factor To Regulate Endothelial Lineage Development And Reprogramming

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic heart disease. The ETS-related transcription factor 2 (ETV2) is a transcription factor that is both necessary and sufficient for the development of endothelial and blood lineages. However, the molecular mechanism by which ETV2 promotes endothelial and blood development is not known and warrants further investigation. Here, we report that ETV2 functions as a novel pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. Pioneer factors are a subset of transcription factors that bind closed chromatin and promote chromatin relaxation in order to drive the development and reprogramming of cell lineages. By comparing embryonic stem cell differentiation and fibroblast reprogramming models with single cell RNA-seq, ATAC-seq, ChIP-seq and in-vitro nucleosomal binding techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We also determined that ETV2 executed a pioneering role by recruiting and directly interacting with the ATP-dependent chromatin remodeling enzyme BRG1 to remodel chromatin around endothelial genes to help maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results that define a novel pioneer factor and epigenetic regulatory pathways that govern the specification and differentiation of endothelial progenitor cells, will serve as a platform for targeted therapies to promote cardiovascular regeneration.

Abstract 3737: DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia

Abstract Infants and young children (> 3 yrs.) harboring ETO2-GLIS2 have the worst outcomes amongst all pediatric acute myeloid leukemia (AML) subtypes, with poor responses to induction therapy, high incidence of relapse, and dismal 5-year survival rates. Previous studies demonstrated that the ETO2-GLIS2 fusion leads to imbalance in the expression of ETS-related transcription factor ERG and GATA1 with neo-super enhancer (SE) activities in a single oncogenic hit (Thirant et al. 2017). ERG was found to co-localize with ETO2-GLIS2 fusion at 90% (372/419) of H3K27ac marked SE-regions and was strongly upregulated (9.44-fold). Herein, we aimed to investigate cooperativity of DNA methylation and chromatin marks on ERG overexpression in this AML subtype. We analyzed transcriptomic data from pediatric ETO2-GLIS2 AML patients (n=40) from dbGAP (phs000465.v19. p8) in comparison to normal bone marrow (NBM) (n=71) samples, confirming upregulation (median log FC: 2.4-fold) in ERG expression in the ETO2-GLIS2 subgroup compared to NBM. Next, we performed an unbiased genome-wide methylation array using Infinium MethylationEPIC 850K-arrays (Illumina). Differences in mean methylation (β-value) at individual CpG sites between NBM and AML samples were considered significant at differences greater than ±10%. We observed reduced methylation at 72% (5/7) CpGs at the ERG-promoter (1500bp upstream of transcription start site) in ETO2-GLIS2 subgroup compared to NBM. Overall median methylation across the promoter was reduced by 12% in the AML subgroup. In comparison, 22 differentially methylated CpGs were observed in the body, of which methylation increased at 68% (15/22) of the CpGs, with median methylation similarly increased by 15% in the AML patients. This represents a classic example of methylation-expression relation, having a promoter demethylation and body-hypermethylation, linked to upregulation of the gene-expression. Next, we investigated the overlaps between CpGs and the distribution of DNAse clusters and H3K27ac marks based on ENCODE data on myeloid leukemia cell line K562. We observed frequent overlaps between demethylated CpGs and DNAse clusters, while H3K27ac broad peaks were coincident with both demethylated CpGs at promoter or hypermethylated CpGs at body. Based on our findings, we thus conclude that DNA-methylation both at the ERG promoter and body is tightly linked to the overlapping chromatin marks of the gene. DNA-methylation might aid in opening of adjacent chromatin on ERG to possibly facilitate SE enrichment and over-expression the gene. Future studies will investigate targeted alterations of DNA-methylation on the gene to test the impact on ERG expression and leukemic growth. Citation Format: Samrat Roy Choudhury, Jordan T. Bird, Stephanie Byrum, Jason E. Farrar. DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3737.

Transcriptome Meta-Analysis Associated Targeting Hub Genes and Pathways of Drought and Salt Stress Responses in Cotton (Gossypium hirsutum): A Network Biology Approach.

Abiotic stress tolerance is an intricate feature controlled through several genes and networks in the plant system. In abiotic stress, salt, and drought are well known to limit cotton productivity. Transcriptomics meta-analysis has arisen as a robust method to unravel the stress-responsive molecular network in crops. In order to understand drought and salt stress tolerance mechanisms, a meta-analysis of transcriptome studies is crucial. To confront these issues, here, we have given details of genes and networks associated with significant differential expression in response to salt and drought stress. The key regulatory hub genes of drought and salt stress conditions have notable associations with functional drought and salt stress-responsive (DSSR) genes. In the network study, nodulation signaling pathways 2 (NSP2), Dehydration-responsive element1 D (DRE1D), ethylene response factor (ERF61), cycling DOF factor 1 (CDF1), and tubby like protein 3 (TLP3) genes in drought and tubby like protein 1 (TLP1), thaumatin-like proteins (TLP), ethylene-responsive transcription factor ERF109 (EF109), ETS-Related transcription Factor (ELF4), and Arabidopsis thaliana homeodomain leucine-zipper gene (ATHB7) genes in salt showed the significant putative functions and pathways related to providing tolerance against drought and salt stress conditions along with the significant expression values. These outcomes provide potential candidate genes for further in-depth functional studies in cotton, which could be useful for the selection of an improved genotype of Gossypium hirsutum against drought and salt stress conditions.

FLI1 regulates inflammation-associated genes to accelerate leukemogenesis

Inflammation plays a critical role in cancer initiation and progression, and is induced by inflammatory factors that are direct target of oncogenes and tumor suppressors. The ETS related transcription factor Fli-1 is involved in the induction and progression of various cancers; yet its role in inflammation is not well-defined. Using RNAseq analysis, we herein demonstrate that FLI1 induces the inflammatory pathway in erythroleukemia cells. Majority of genes within the TNF signaling pathway including TNF and IL1B were identified as transcriptional targets of FLI1. TNF expression is indirectly regulated by FLI1 through upregulation of another ETS related oncogene, SPI1/PU.1. Pharmacological inhibition of TNF significantly inhibited leukemia cell proliferation in culture. In contrast, IL1B expression is directly regulated by FLI1 through promoter binding and transcriptional activation. The secreted factor IL1B binds its canonical receptors to accelerate cancer progression through changes in the surrounding tumor microenvironment, fostering cell survival, proliferation and migration. Through network analysis, we identified IL1B-interacting genes whose expression is also regulated by FLI1. Among these, IL1B-interacting proteins, FOS, JUN, JUNB and CASP1 are negatively regulated by FLI1. Treatment of leukemia cells with inhibitors of AP1 (TAN IIA) and CASP1 (765VX) significantly accelerated FLI1-dependent leukemia progression. These results emphasize the significance of FLI1 in regulating the inflammatory pathway. Targeting these inflammatory genes downstream of FLI1 offers a novel strategy to treat leukemic progression associated with overexpression of this oncogenic ETS transcription factor.

Mithramycin analogues with reduced toxicity for the treatment of ETS transcription factor driven tumors

IntroductionChromosomal translocations involving the ETS family of transcription factors are common in Ewing sarcoma, prostate cancer, and leukemia. These translocations lead in overexpression of aberrant ETS transcription factors, which drive tumorigenesis. Mithramycin (MTM) was shown to inhibit EWS/FLI1, the most common ETS related transcription factor in Ewing sarcoma, presumably through interference at its DNA binding sites on promoter regions. However, MTM has a short half‐life and a narrow therapeutic window marked by severe liver and hematological toxicities. Considering that MTM has a pKa of 5, we hypothesized that its rapid pharmacokinetic (PK) clearance is attributed to liver uptake by organic anion transporter polypeptides (OATP), which may also contribute to the observed hepatotoxicity. Here, we sought to develop analogues with specificity toward ETS transcription factors and reduced toxicity by attempting to modify the physicochemical properties of MTM.MethodsA series of analogues were obtained by modifying the 3‐side chain of MTM to increase its pKa. Specificity toward cells expressing ETS transcription factors was determined by cytotoxicity studies in ETS expressing cancer cells (n=9) vs. cancer cell lines (n=9) lacking ETS. Luciferase reporter constructs were used to indirectly evaluate if MTM analogues affected interaction of EWS/FLI1 and Sp1 binding to promoters. Gene expression was tested by qRT‐PCR and interaction at specific promoter sites was determined by ChIP‐qRT‐PCR. The PK of MTM was determined in the presence and absence of rifampin pretreatment and in OATP1B1/1B3 transgenic mice. Select analogues were tested in vivo to identify the maximum tolerated dose and determine pharmacokinetics (PK).ResultsSemisynthetic substitutions using aromatic amino acids at the 3‐side chain of MTM lead to a significant increase (0.5–1.5 units) of the pKa. Growth inhibition assays identified several MTM analogues with > 10 fold selectivity toward ETS cell lines and this was corroborated by reporter and expression assays. The clearance of mithramycin was significantly decreased by rifampicin pretreatment and MTM exposure was significantly higher in Oatp1a/1b KO mice expressing OATP1B3 (transgenic) vs KO mice expressing OATP1B1. Select analogues were further tested to determine relative toxicity in mice and were found to be tolerated at 10–30 fold higher doses than MTM. Significantly, the presumed shift in pKa afforded a dramatic difference in pharmacokinetic properties resulting in relative drug exposures (area under the concentration‐time curves) that were 50–400 higher than MTM at their respective maximum tolerated doses.ConclusionThese studies show that substitutions at the 3‐side chain of MTM yield analogues that are selectively more cytotoxic against tumor cell lines expressing aberrant ETS transcription factors. Further, significantly altered pharmacokinetics of these analogues may expand the therapeutic window and lead to the development of a new generation of MTM analogues amenable for targeted treatment of ETS expressing cancers.Support or Funding InformationThis work was partially funded by DoD grant W81XWH‐16‐1‐0477; projects PC150300P1, PC150300P2 and by funds from the DanceBlue Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B043: Mithramycin-SA analogues with reduced toxicity for the treatment of ETS transcription factor-driven tumors

Abstract Introduction: Chromosomal translocations involving the ETS family of transcription factors are common in Ewing sarcoma, prostate cancer, and leukemia. These translocations lead in overexpression of aberrant ETS transcription factors, which drive tumorigenesis. Mithramycin (MTM) inhibits EWS-FLI1, the most common ETS-related transcription factor in Ewing sarcoma, presumably through interference at its DNA binding sites on promoter regions. However, MTM has a narrow therapeutic window marked by severe liver and hematologic toxicities. These are likely the result of interference with the ubiquitously expressed SP1 transcription factor. Here, we sought to develop analogues with specificity toward ETS transcription factors and reduced interaction with SP1. Methods: Using MTM-SA and semisynthetic approaches a series of analogues were obtained. To determine specificity toward cells expressing ETS-related chromosomal translocations, MTM-SA analogues were screened for growth inhibition in a panel of Ewing sarcoma cell lines (n=8), all expressing EWS-ETS translocations, and compared to a panel of non-Ewing cell lines (n=9) that do not express these translocations. Luciferase reporter constructs were developed to evaluate interference with EWS-FLI1 and SP1 regulated genes following MTM-SA analogue treatment. Select analogues were tested in vivo to identify the maximum tolerated dose and determine pharmacokinetics (PK). Results: In growth inhibition assays, several MTM-SA analogues resulted in > 10-fold specificity toward Ewing sarcoma cell lines vs. MTM. Luciferase reporter assays identified two analogues, MTM-SA-Tryptophan-A2 and MTM-SA-Tryptophan-A10, with 10-fold reduced inhibition of SP1 but similar inhibition of EWS-FLI1, as compared to MTM. Additionally, MTM-SA-Phe analogue has lower toxicity, despite longer plasma half-life. Conclusion: These data show that MTM-SA analogues are cytotoxic against tumor cell lines expressing aberrant ETS transcription factors. Further, their reduced interference with the ubiquitously expressed SP1 transcription factor, as well as their improved PK, may result in a wider therapeutic window. Citation Format: Markos Leggas, Joseph Eckenrode, Prithiba Mitra, Amit Jha, Shaimaa Salem, Abhisek Mandal, Jon Thorson, Jurgen Rohr. Mithramycin-SA analogues with reduced toxicity for the treatment of ETS transcription factor-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B043.

An Elf2-like transcription factor acts as repressor of the mouse ecto-5'-nucleotidase gene expression in hepatic myofibroblasts.

Hepatic fibrosis represents a pathological wound healing and tissue repair process triggered in response to chronic liver injury. A heterogeneous population of activated non-parenchymal liver cells, known as liver myofibroblasts, functions as the effector cells in hepatic fibrosis. Upon activation, liver myofibroblasts become fibrogenic, acquiring contractile properties and increasing collagen production capacity, while developing enhanced sensitivity to endogenous molecules and factors released in the local microenvironment. Hepatic extracellular adenosine is a bioactive small molecule, increasingly recognized as an important regulator of liver myofibroblast functions, and an important mediator in the pathogenesis of liver fibrosis overall. Remarkably, ecto-5'-nucleotidase/Nt5e/Cd73 enzyme, which accounts for the dominant adenosine-generating activity in the extracellular medium, is expressed by activated liver myofibroblasts. However, the molecular signals regulating Nt5e gene expression in liver myofibroblasts remain poorly understood. Here, we show that activated mouse liver myofibroblasts express Nt5e gene products and characterize the putative Nt5e minimal promoter in the mouse species. We describe the existence of an enhancer sequence upstream of the mouse Nt5e minimal promoter and establish that the mouse Nt5e minimal promoter transcriptional activity is negatively regulated by an Elf2-like Ets-related transcription factor in activated mouse liver myofibroblasts.

A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis.

The ETS-related transcription factor Fli-1 affects many developmental programs including erythroid and megakaryocytic differentiation, and is frequently de-regulated in cancer. Fli-1 was initially isolated following retrovirus insertional mutagenesis screens for leukemic initiator genes, and accordingly, inhibition of this transcription factor can suppress leukemia through induction of erythroid differentiation. To search for modulators of Fli-1, we hereby performed repurposing drug screens with compounds isolated from Chinese medicinal plants. We identified agents that can transcriptionally activate or inhibit a Fli-1 reporter. Remarkably, agents that increased Fli-1 transcriptional activity conferred a strong anti-cancer activity upon Fli-1-expressing leukemic cells in culture. As opposed to drugs that suppress Fli1 activity and lead to erythroid differentiation, growth suppression by these new Fli-1 transactivating compounds involved erythroid to megakaryocytic conversion (EMC). The identified compounds are structurally related to diterpene family of small molecules, which are known agonists of protein kinase C (PKC). In accordance, these PKC agonists (PKCAs) induced PKC phosphorylation leading to activation of the mitogen-activated protein kinase (MAPK) pathway, increased cell attachment and EMC, whereas pharmacological inhibition of PKC or MAPK diminished the effect of our PKCAs. Moreover, in a mouse model of leukemia initiated by Fli-1 activation, the PKCA compounds exhibited strong anti-cancer activity, which was accompanied by increased presence of CD41/CD61 positive megakaryocytic cells in leukemic spleens. Thus, PKC agonists offer a novel approach to combat Fli-1-induced leukemia, and possibly other cancers,by inducing EMC in part through over-activation of the PKC-MAPK-Fli-1 pathway.

Gain of chromosome 21 or amplification of chromosome arm 21q is one mechanism for increased ERG expression in acute myeloid leukemia.

In acute myeloid leukemia (AML), acquired genomic gains and losses are common and lead to altered expression of genes located within or nearby the affected regions. Increased expression of the ETS-related transcription factor gene ERG has been described in myeloid malignancies with chromosomal rearrangements involving chromosome band 21q22, but also in cytogenetically normal AML, where it is associated with adverse prognosis. In this study, fluorescence in situ hybridization on interphase nuclei disclosed an amplification of the ERG gene (more than six copies) in 33 AML patients with structural rearrangements of 21q22. Array comparative genomic hybridization of these cases disclosed a minimal amplified region at the position 39.6-40.0 Mbp from pter that harbors ERG as the only gene. Analysis by quantitative real-time reverse transcription polymerase chain reaction revealed significantly higher ERG mRNA expression in these patients and in a group of 95 AML patients with complete or partial gain of chromosome 21 (three to six copies) compared with 351 AML patients without gain of chromosome 21. Quantification of ERG DNA copy numbers revealed a strong correlation with ERG mRNA expression. Furthermore, in patients with gain of chromosome 21, higher ERG expression was found to be associated with RUNX1 mutations. Our results suggest that acquired gain of chromosome 21 or amplification of chromosome arm 21q is one mechanism contributing to increased ERG expression in AML.

Finding a fairy in the forest: ELF4, a novel and critical element of type I interferon responses

Type I interferon (IFN) production is a key event during innate immune responses. This early and prompt mechanism is mediated by multiple factors that involve various pattern recognition receptors (PRRs), adaptor proteins, kinases and transcription factors. Stimulator of IFN genes (STING) is an important transmembrane adaptor protein that plays a role in the activation of downstream transcription factors, such as IFN regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6, via TANK-binding kinase 1 (TBK1), which has been considered the main innate immune defense weaponry against viruses and intracellular pathogens. In a current issue of Nature Immunology, You et al. showed that, after viral infection, the ETS-related transcription factor ELF4 (previously known as MEF) interacts with STING and is consequently activated by TBK1. This process leads to the nuclear translocation of ELF4 and its binding to IFN promoters (Figure 1). The group also demonstrated that ELF4 acts as an IFN transcription factor, as it has the potential to increase the binding affinity of IRF3 and IRF7 through cooperative binding to newly identified enhancer elements (EICE) in the IFN promoters.1 These new findings not only extend our knowledge about the details of the fine-tuning of the innate IFN responses, but also represent a new milestone by describing the role of EICE elements in this process.

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures:No relevant conflicts of interest to declare.

14-3-3 Proteins Modulate the ETS Transcription Factor ETV1 in Prostate Cancer

Overexpression of the ETS-related transcription factor ETV1 can initiate neoplastic transformation of the prostate. ETV1 activity is highly regulated by phosphorylation, but the underlying mechanisms are unknown. Here we report that all 14-3-3 proteins, with the exception of the tumor suppressor 14-3-3σ, can bind to ETV1 in a condition manner dictated by its prominent phosphorylation site S216. Non-σ 14-3-3 proteins synergized with ETV1 to activate transcription of its target genes MMP-1 and MMP-7, which regulate extracellular matrix in the prostate tumor microenvironment. S216 mutation or 14-3-3τ downregulation was sufficient to reduce ETV1 protein levels in prostate cancer cells, indicating that non-σ 14-3-3 proteins protect ETV1 from degradation. Notably, S216 mutation also decreased ETV1-dependent migration and invasion in benign prostate cells. Downregulation of 14-3-3τ reduced prostate cancer cell invasion and growth in the same manner as ETV1 attenuation. Finally, we showed that 14-3-3τ and 14-3-3ε were overexpressed in human prostate tumors. Taken together, our results showed that non-σ 14-3-3 proteins are important modulators of ETV1 function that promote prostate tumorigenesis.

PRKD2 Serine-Threonine Kinase, an Essential Effector of Gabp Transcription Factor, Is Required for Development of Chronic Myelogenous Leukemia

AbstractAbstract 1672Chronic Myelogenous Leukemia (CML) is a myeloproliferative neoplasm (MPN) caused by transformation of hematopoietic stem cells (HSCs) by the BCR-ABL oncogene. CML is associated with excessive proliferation of HSCs and massive expansion of the myeloid cell pool. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPα, along with various GABPβ partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. In a mouse model of CML, animals transplanted with BCR-ABL-infected bone marrow developed massive myeloid cell expansion and died with a MPN. Induced deletion of Gabpa prevented development of CML, yet mice continued to produce mature BCR-ABL-expressing granulocytes for months without apparent illness. BCR-ABL+ cells were transplantable into secondary recipients without development of CML, and contributed to all hematopoietic lineages, thereby confirming expression of BCR-ABL by long-term HSCs. We used a bioinformatic approach to analyze GABP-bound genes that are upregulated in both human and mouse LSCs compared to normal HSCs. Among 115 GABP-bound, CML-associated genes, we identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase that has been implicated in cancer, but has not previously been associated with HSC functions or CML. Deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In vitro growth of BCR-ABL+ bone marrow cells was prevented by Gabpa deletion, but growth was partially rescued by forced expression of PRKD2. Knockdown and pharmacologic inhibition of PRKD2 blocked cell cycle entry of BCR-ABL+ cells. We conclude that Gabp is required for HSC cell cycle entry and for development of CML, and that these effects of GABP are mediated, in part, by PRKD2. These findings suggest that PRKD2 kinase may serve as a novel therapeutic target in leukemia. Disclosures:No relevant conflicts of interest to declare.

Implication ofHpEtsin Gene Regulatory Networks Responsible for Specification of Sea Urchin Skeletogenic Primary Mesenchyme Cells

The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.

Heterogeneous Nuclear Ribonucleoprotein R Enhances Transcription from the Naturally Configured c-fos Promoter in Vitro

Transcription of a proto-oncogene c-fos is induced rapidly to high levels by various extracellular stimuli. To explore the molecular mechanism of c-fos gene induction, we established a defined in vitro transcription system for the c-fos promoter that consists of purified activators (SRF, Elk-1, cAMP-responsive element-binding protein, and ATF1), general transcription factors, and RNA polymerase II. In this reconstituted transcription system, activation of c-fos transcription was highly dependent upon coactivators such as PC4 and Mediator, indicating a very weak activation potential of the activators in the context of an unaltered promoter structure. This heightened coactivator dependence, however, allowed us to identify from HeLa nuclear extract a coactivator-like activity termed transcriptional regulator of c-fos (TREF) that enhanced c-fos transcription but not GAL4-VP16-dependent transcription. TREF cooperated with Mediator to enhance c-fos transcription by approximately 60-fold over its basal level and, like Mediator, stimulated activator-independent (basal) transcription as well. Further purification of TREF revealed that it consists of at least three distinct components, one of which was purified to near homogeneity and identified as heterogeneous nuclear ribonucleoprotein R. Recombinant heterogeneous nuclear ribonucleoprotein R enhanced transcription from the c-fos promoter and displayed cooperativity with PC4 and Mediator, thus demonstrating its direct transcriptional activity.

E2A Proteins Promote Development of Lymphoid-Primed Multipotent Progenitors

The first lymphoid-restricted progeny of hematopoietic stem cells (HSCs) are lymphoid-primed multipotent progenitors (LMPPs), which have little erythromyeloid potential but retain lymphoid, granulocyte, and macrophage differentiation capacity. Despite recent advances in the identification of LMPPs, the transcription factors essential for their generation remain to be identified. Here, we demonstrated that the E2A transcription factors were required for proper development of LMPPs. Within HSCs and LMPPs, E2A proteins primed expression of a subset of lymphoid-associated genes and prevented expression of genes that are not normally prevalent in these cells, including HSC-associated and nonlymphoid genes. E2A proteins also restricted proliferation of HSCs, MPPs, and LMPPs and antagonized differentiation of LMPPs toward the myeloid fate. Our results reveal that E2A proteins play a critical role in supporting lymphoid specification from HSCs and that the reduced generation of LMPPs underlies the severe lymphocyte deficiencies observed in E2A-deficient mice.

Real-Time Reverse-Transcription PCR to Quantify a Panel of 19 Genes in Breast Cancer: Relationships with Sentinel Lymph Node Invasion

At the Centre Oscar Lambret, the anticancer centre of the North of France, sentinel lymph node (SLN) procedures are routinely performed for localized (T0-T1, N0, M0) breast carcinoma without any previous treatment, in order to prevent the deleterious effects of axillary lymph node dissection. The present study was undertaken to assess if the expression in the tumor of a panel of 19 genes would allow to predict histological SLN involvement. We looked at cytokeratin 19 (CK19), mucin-1 (MUC1), mammaglobin (MGB1), cyclin D1 (CCND1), the four members of the HER/ErbB growth factor receptor family (EGFR, HER2-4), insulin-like growth factor-1 receptor (IGF-1R), estradiol receptors (ERalpha, ERbeta), progesterone receptor (PR), vascular endothelial growth factors (VEGF, VEGF-C), urokinase-like plasminogen activator (uPA), matrix metalloproteinases 2 and 9 (MMP2, MMP9), ets-related transcription factor ERM, and E-cadherin (CDH1). Their expression was quantified by real-time RT-PCR in 134 breast cancer samples and the relationships with SLN metastases were analyzed. A slight increase (35-40%) in CK19 and HER3 expression was observed in the tumors of patients with SLN metastases compared to those of patients without metastases, even if neither CK19 expression nor HER3 expression allowed to distinguish patients with micrometastases from patients with macrometastases. We conclude that the tumoral expression of biological parameters involved in cell proliferation or playing a critical role in the metastatic process, including tumor invasion and angiogenesis, is not strongly associated with SLN metastases.