Gene Expressional Profile of Neonatal Neutrophils in Response to Bacterial Lipopolysaccharide (LPS) and Possible Mechanism of Transcription Factor NR4A2 on the Immune Signal Cascade

Abstract Gram-negative bacterial infection is a serious condition in neonates which could lead to septicemic shock, disseminated intravascular coagulation and death. LPS, the cell wall component of Gram-negative bacteria is a potent stimulator of the host immune response system, mediated by binding to toll-like receptor (TLR)-4 and CD14 receptors. Neutrophils are the first line of innate defense which possess antimicrobial and acute inflammatory activities. Neonatal neutrophils, though incompletely characterized, are suggested to be functionally immature and compromised in their bactericidal capacity compared with adults. We performed a comparative genome-wide expression array analysis on purified (>95%) human cord blood (term delivery) neutrophils upon challenge by LPS for 4 hours at 100 ng/mL. With the criteria of 2-fold differences and statistical significance (P <0.01), 1236 transcripts were increased and 1633 transcripts were decreased in LPS-treated neutrophils (n=3). A panel of 20 target genes was selected by their magnitude of regulation, novelty and relevant immune functions and validated by qPCR. Eleven of these genes could be linked upstream or downstream of the novel transcription factor, nuclear receptor subfamily-4, group-A, member-2 (NR4A2) by pathway analysis (Metacore). We thus proposed a regulatory network mediated by binding of LPS to TLR4/CD14 receptors, leading to NR4A2 upregulation, thereby promoting a cascade of downstream effector genes associated with antibacterial and/or inflammatory activities in neutrophils. To validate the upstream signal cascade of LPS-induced regulation of NR4A2, neonatal neutrophils were pretreated with specific TLR-4, p38MAPK or NF-κB inhibitors, and stimulated with LPS, followed by quantification of target gene expression by qPCR (n=6 in each group). Our results showed that LPS-induced NR4A2 expression was significantly decreased (all P <0.05) after pretreatment with TLR-4 antibody (0.47 fold), p38MAPK inhibitor SB203580 (0.27 fold), and NF-κB inhibitors BAY-117082 (0.14 fold) and pyrrolidine dithiocarbamate (0.07 fold). These results suggested that NR4A2 could be upregulated by LPS-receptor-mediated p38MAPK and NF-κB signals. To compare the capacity and developmental regulation of LPS-induced responses of NR4A2-associated signals, purified cord blood neutrophils from preterm (gestational age 33-36 weeks) and term (37-40 weeks) neonates, and peripheral blood from healthy male adult (age 25-30 years old) were evaluated (n=10 in each group). LPS-induced expression of NR4A2 and associated molecular signals (EGR1, PPARG, NF-κB, ATOH8, G0S2, GPR84, IL-8, PTGS2, TNF-α, CAMK2G and GCH1) were measured by qPCR. Our result showed that expressions of these target genes were significantly up- or downregulated in LPS-activated neutrophils of neonates and adults. However, at the basal level, expressions of EGR1, GOS2, IL-8 and PTSG2 were significantly lower (P< 0.05) in preterm or term neonatal neutrophils compare with those in adults, showing that there might be compromised levels in neonates at the unstimulated status. Upon stimulation with LPS, expressions of GOS2 and IL-8 were significantly lower (P< 0.05) in preterm or term neonates than in adult, indicating that there could be developmental regulation of NR4A2 downstream genes in neutrophils secondary to LPS stimulations. Correlation analysis showed that the expressions of EGR1 and PTGS2 were positively correlated with NR4A2 in LPS-induced neutrophils from both term (EGR1: R=0.805, P< 0.05; PTGS2: R=0.912, P< 0.05) and preterm neonates (EGR1: R=0.636, P< 0.05; PTGS2: R=0.830, P< 0.05), but not in adult peripheral neutrophils. These results indicated that there could be developmental differences in the regulation of the NR4A2 immune cascade involving apoptosis (EGR1, GOS2)/inflammatory cytokine (IL-8) and prostaglandin synthesis (PTGS2) between adult and neonatal neutrophils at basal and/or LPS-activation states. In summary, we reported the expressional profile and deregulated target genes in neonatal neutrophils upon stimulation by LPS and identified the downstream NR4A2 immune cascade that could be regulated by LPS, p38MAPK and NF-κB signals, as well as the developmental status. Disclosures No relevant conflicts of interest to declare.

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C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽

10.1182/blood.v120.21.650.650 ◽

2012 ◽

Vol 120 (21) ◽

pp. 650-650

Author(s):

Cailin Collins ◽

Jingya Wang ◽

Joel Bronstein ◽

Jay L. Hess

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Target Genes ◽

De Novo ◽

Conflicts Of Interest ◽

Epigenetic Modifications ◽

Conditional Knockout ◽

Motif Analysis ◽

Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Gfi1 Upregulates c-Myc Expression and Promotes c-Myc-Driven Cell Proliferation

Blood ◽

10.1182/blood-2019-124833 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3769-3769

Author(s):

Yangyang Zhang ◽

Fan Dong

Keyword(s):

Cell Proliferation ◽

Transcription Factor ◽

Transcriptional Repression ◽

Cell Cycle Progression ◽

Target Genes ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Protein Levels ◽

Enhanced Expression ◽

Ubiquitin Proteasome

Gfi1 is a zinc-finger transcriptional repressor that plays an important role in hematopoiesis. When aberrantly activated, Gfi1 may function as a weak oncoprotein in the lymphoid system, but collaborate strongly with c-Myc in lymphomagenesis. c-Myc is a transcription factor that is frequently activated in human cancers including leukemia and lymphoma mainly due to its overexpression as a result of gene amplifications and chromosomal translocations. c-Myc overexpression may also result from stabilization of c-Myc protein, which is highly unstable and rapidly degraded through the ubiquitin-proteasome pathway. The mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis is incompletely understood. c-Myc activates gene expression by forming a heterodimeric complex with the partner protein Max, but may also repress target genes through interaction with transcription factor Miz-1. We previously showed that Gfi1 indirectly interacts with c-Myc through Miz-1 and collaborates with c-Myc to repress CDK inhibitors p21Cip1 and p15Ink4B. In this study, we show that Gfi1 augmented the level of c-Myc protein transiently expressed in Hela cells and the levels of MycER fusion protein stably expressed in the mouse pro-B Ba/F3 and myeloid 32D cells. The C-terminal ZF domains of Gfi1, but not its transcriptional repression and DNA binding activities, were required for c-Myc upregulation. Notably, although Miz-1 has been shown to stabilize c-Myc protein, the expression of c-Myc V394D mutant, which is defective in Miz-1 interaction, was still upregulated by Gfi1, suggesting that Gfi1-mediated c-Myc upregulation was independent of Miz-1 interaction. We further show that Gfi1 overexpression led to reduced polyubiquitination and increased stability of c-Myc protein. Interestingly, the levels of endogenous c-Myc mRNA and protein were augmented upon induction of Gfi1 expression in Ba/F3 and Burkitt lymphoma Ramos cells transduced with the doxycycline-inducible Gfi1 lentiviral construct, but reduced in Gfi1-knocked down human leukemic HL60 and U937 cells. Additionally, targeted deletion of Gfi1 resulted in reduced c-Myc expression in mouse lineage negative bone marrow cells, which was associated with a decline in the expression of c-Myc-activated target genes. The oncogenic potential of Myc derives from its ability to stimulate cell proliferation. Our results demonstrate that inducible expression of Gfi1 in Ba/F3 cells expressing MycER promoted Myc-driven cell cycle progression and proliferation. Thus, in addition to its role in c-Myc-mediated transcriptional repression, Gfi1 upregulates c-Myc expression at both mRNA and protein levels, leading to enhanced expression of c-Myc-activated genes and augmented cell proliferation driven by c-Myc. Together, these data may reveal a novel mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Blood ◽

10.1182/blood.v128.22.4082.4082 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4082-4082

Author(s):

Daniel Schaefer ◽

Marianne Olsen ◽

Ulrik Lausten-Thomsen ◽

Cyrill Schipp ◽

Martin Stanulla ◽

...

Keyword(s):

Transcription Factor ◽

Cord Blood ◽

B Cell ◽

Childhood Leukemia ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Inverse Pcr ◽

Blood Samples ◽

Chimeric Transcription Factor

Abstract Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns. The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected. Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened. Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented. Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. Table Table. Disclosures No relevant conflicts of interest to declare.

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Large-Scale Integration of Gene Profiling Identifies TCF7L2/TCF4 as the Most Frequently Dysregulated Wnt Signaling Component In AML

Blood ◽

10.1182/blood.v116.21.2480.2480 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2480-2480

Author(s):

Siti Sarah Daud ◽

Alan K Burnett ◽

Richard L Darley ◽

Alex Tonks

Keyword(s):

Transcription Factor ◽

Wnt Signaling ◽

Large Scale ◽

Target Genes ◽

Conflicts Of Interest ◽

Function Analysis ◽

Genetic Models ◽

Nuclear Accumulation ◽

Functional Relationships ◽

Linear Contrasts

Abstract Abstract 2480 Acute myeloid leukemia (AML) represents one of the most genetically heterogeneous malignancies; however, some processes are commonly dysregulated. One of the most frequently dysregulated processes in AML is Wnt signaling. In solid cancers, aberrant Wnt signaling has been shown to promote cancer by increasing nuclear accumulation of β-catenin and with consequent activation of target genes. In AML, overexpression of β-catenin is also common; in addition however, patient studies and genetic models indicate that other components of the Wnt pathway are also commonly dysregulated and may mediate transcriptional changes independently of β-catenin. The aim of this study was to identify aberrantly regulated Wnt components and target genes in AML by interactome analysis of the AML Affymetrix GeneChip® 3` expression microarray datasets; a network building algorithm used to understand relationships between genes. Analysis and interpretation of microarray data is still both biologically and computationally challenging. To address this, we performed batch adjustment to the large scale AML dataset by merging gene expression profile (GEP) data derived from different database sources (including different array platforms). GEPs data were generated from our AML patients enrolled in two different AML NCRI-MRC UK clinical trials using two different Affymetrix platforms, HG-U133A (n=216) and HG-U133Plus2.0 (n=139). GEPs from normal CD34+ bone marrow samples were downloaded from ArrayExpress (n=26). In order to compare AML vs. normal haematopoietic GEP, all data were merged into a single dataset. Individual. CEL files were imported into Partek® Genomics Suite™ and GC-RMA normalization was applied. Linear contrasts, mixed model analysis of variance with false discovery rate correction (P<0.05) and threshold analysis (>1.5 or <1.5 fold-change) were applied to the adjusted data followed by gene enrichment analysis using MetaCore™ (GeneGo Inc). Batch adjustment was performed using Distance Weight Discrimination (DWD) method to the merged GEPs. Prior to further inferential and gene ontology testing, the DWD merged datasets showed significant reduction in the source of data bias with GEP clustered according to their biological variation rather than technical variation. As a result, we present a final list of 58 significant changes in the expression of Wnt related genes in AML. Enrichment by protein function analysis highlighted 8 Wnt transcription factors to be dysregulated (TCF7L2/TCF4, MYC, NANOG, WT1, RUNX2, p300, TCF7, SMAD2), along with 5 receptors (CD44, FZD3, FZD4, FZD5, LDLR), 3 types of phosphatases (B56G, PR61-β, PPP2R5A) and other categories of Wnt related objects (n=33). Consistent findings were seen with previously established Wnt-associated genes specific to AML (CD44, WT1, MYC) showing that data sources from DWD adjustment was effective. We sought to evaluate the significant biological and functional relationships within the genes in the final dataset by transcription factor target modeling using MetaCore™ Interactome tools. Direct network interaction uncovered TCF7L2/TCF4 as the most significantly upregulated Wnt transcription factor with concurrent high expression of its downstream Wnt responsive genes CD44, AXIN1, ID2 that were also present in the final list. Importantly, β-catenin is unlikely to contribute to this transcriptional activation due to the fact that our data showed increased transcription of β-catenin degradation complexes (or negative regulation of Wnt signalling). Specifically, RUVBL1, that directly increases β-catenin activity was significantly downregulated, whereas the other significantly overexpressed upstream genes (APC, CSNK1E, AXIN1, WT1) are known to have inhibitory effect on β-catenin-mediated transcription. In summary, by using multiple GEP data from a large AML cohort in conjunction with robust statistical adjustments, we have identified TCF7L2/TCF4 mediated transcription as the most significant Wnt-regulated process to be altered in AML compared with normal blasts. We also predict that transcription of TCF7L2/TCF4 regulated genes is likely to be independent of β-catenin, supporting observations in genetic models which indicate that β-catenin (and γ-catenin) are redundant for normal haematopoiesis and are not required for TCF-mediated transcription. Disclosures: No relevant conflicts of interest to declare.

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The Hbo1-Brd1/Brpf2 HAT Complex Is Required for Erythropoiesis In Fetal Liver

Blood ◽

10.1182/blood.v116.21.3872.3872 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3872-3872

Author(s):

Yuta Mishima ◽

Satoru Miyagi ◽

Atsunori Saraya ◽

Masamitsu Negishi ◽

Mitsuhiro Endoh ◽

...

Keyword(s):

Transcription Factor ◽

Target Genes ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

K562 Cells ◽

Erythroid Differentiation ◽

Pwwp Domain ◽

Transcription Factor Genes ◽

Chip Analysis

Abstract Abstract 3872 Bromodomain-containing protein 1 (Brd1, initially designated as BR140-LIKE; BRL) contains a bromodomain, two plant homology domain (PHD) zinc fingers, and a proline-tryptophan-tryptophan-proline (PWWP) domain, three types of modules characteristic of chromatin regulators. Recently, BRD1 appeared to belong to the BRPF family which includes BRPF1, BRD1/BRPF2, and BRPF3. Among them, BRPF1 is known to be a subunit of the MOZ H3 histone acetyltransferase (HAT) complex. BRD1 has been proposed to be additional subunit of the MOZ H3 HAT complex on the analogy of BRPF1. However, its molecular function remains elusive. To elucidate the biological functions of BRD1, we generated Brd1-null mice and found that they die in utero. Brd1-/- embryos were alive and recovered at nearly the expected Mendelian ratio at 12.5 days postcoitum (dpc) but died by 15.5 dpc. Brd1-/- embryos at 12.5 dpc were pale and the cell number of fetal livers, in which fetal hematopoiesis occurs, was decreased to about 20% of the control. Cytological analysis revealed that Brd1-/- fetal livers had profoundly fewer erythroblasts at maturation stages beyond proerythroblasts compared to wild-type fetal livers. Flow cytometric analysis of Brd1-/- fetal livers revealed a significant accumulation of CD71+Ter119- proerythroblasts and a reduction in CD71+Ter119+ and CD71-Ter119+ maturating erythroblasts. A drastic increase in AnnexinV+ apoptotic cells was detected in the CD71+Ter119+ and CD71-Ter119- cell fractions in Brd1-/- fetal livers. These findings suggested that severe anemia caused by compromised differentiation and/or survival of erythroblasts accounts for embryonic lethality of Brd1-/- embryos. To understand the mechanism underlying defective erythropoiesis in Brd1-null embryos, we performed biochemical analyses and found that Brd1 bridges the HAT, HBO1 but not MOZ, and its activator protein, ING4, to form an enzymatically active HAT complex. Forced expression of Brd1 promoted erythroid differentiation of K562 cells, while Brpf1, which preferentially binds to MOZ, had no significant effect. Correspondingly, depletion of Hbo1 by Hbo1 knockdown perturbed erythroid differentiation of mouse fetal liver progenitors. Of note, the level of global acetylation of histone H3 at lysine 14 (H3K14) was specifically decreased in Brd1-deficient erythroblasts. These results collectively implied that acetylation of H3K14 catalyzed by the Hbo1-Brd1 complex has a crucial role in fetal liver erythropoiesis. To identify the downstream targets for the HBO1-BRD1 complex, we performed the ChIP-on-chip analysis in K562 cells and found that BRD1 and HBO1 largely co-localize on the genome, especially on the promoters of erythroid transcription factor genes. ChIP analysis revealed that acetylation of H3K14 at the promoters of erythroid transcription factor genes, including Gata1, Gata2, Tal1, Stat5a, and ETO2, were profoundly diminished in the Brd1-deficient erythroblasts. Among these target genes, we focused on Gata1, which plays a central role in erythropoiesis, and carried out complementation experiments with Gata1 using a Gata1 retrovirus. Exogenous Gata1, but not Bcl-xL, efficiently improved proliferative capacity and survival of Brd1-deficient erythroid progenitors and also restored, at least partially, their impaired differentiation. These results clearly showed that the Hbo1-Brd1 complex is required for the acetylation of H3K14 at the promoters of erythroid transcription factor genes, thereby is crucial for erythropoiesis in fetal liver. Disclosures: No relevant conflicts of interest to declare.

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GATA2 Transcription Factor Mediates Myeloid Expansion Coupled with Lymphoid Block by Transcriptional Regulation of Key Target Genes.

Blood ◽

10.1182/blood.v120.21.2317.2317 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2317-2317

Author(s):

Satish Kumar Nandakumar ◽

Geoffrey A. Neale ◽

Derek A. Persons

Keyword(s):

Transcription Factor ◽

Transcriptional Regulation ◽

Cell Lines ◽

Liquid Culture ◽

Insertional Mutagenesis ◽

Target Genes ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Control Group ◽

Mouse Bone Marrow

Abstract Abstract 2317 Functions of the transcription factor GATA2 in the hematopoietic system were difficult to explore due to the embryonic lethal phenotype on deletion (Tsai et al., Nature 1994) and HSC quiescence on overexpression (Persons et al., Blood 1999). To overcome this problem we developed a Tamoxifen inducible system by fusing the human GATA2 cDNA to the ligand binding domain of the estrogen receptor (GATA2-ERT). Previously we found that when competitive transplant experiments were performed with mouse bone marrow cells, GATA2-ERT transduced cells greatly outcompeted untransduced myeloid cells while at the same time not contributing to the lymphoid lineage. However, no leukemia was observed. Since these effects were observed in the absence of Tamoxifen, we speculated that GATA2-ERT protein leaked into the nucleus to provide low level tonic activity (known to occur in other ERT systems). Immunoblot analysis of BM cells transduced with the GATA2-ERT vector confirmed the presence of the protein in both nuclear and cytoplasmic fractions. In addition, GATA2-ERT transduced cells also out-competed mock transduced cells in liquid culture and could be serially replated in colony forming assays. GATA2-ERT and control GFP transduced cells were diluted with mock cells to achieve around 25% GFP+ cells. After 9 days in culture the percentage of GFP + cells in the GATA2-ERT group was 91%±6 compared to GFP group which remained at 38%±14. GATA2-ERT transduced cells could be cultured indefinitely while after 7 weeks the control GFP transduced cells differentiated to mast cells. These GATA2-ERT cell lines were dependent only on IL-3 and expressed myeloid cell surface markers (53%±1 Gr+, 87%±4 Mac1+ cells n=3). DNA binding mutants of GATA2-ERT completely abolished both serial colony replating ability and growth advantage in liquid culture. This suggested that GATA2-ERT mediates these effects through transcriptional regulation of target genes. To identify the targets of GATA2 responsible for myeloid expansion, gene expression profiling was done on three independently generated GATA2-ERT cell lines and compared to an immortalized myeloid cell line (due to insertional mutagenesis) which does not express GATA2. Genes involved in myeloid neoplasms like Nmyc, HoxA9 and Bcl11a were significantly elevated (HoxA9 –77-fold, Nmyc 60-fold, Bcl11a 36-fold) in the GATA2-ERT lines. To test if GATA2 mediates myeloid expansion through Nmyc, we knocked down Nmyc in GATA2-ERT cells and tested for growth defects. GATA2-ERT cells were transduced with lentiviral vectors harboring shRNA targeting GATA2-ERT (73%±10.2), Nmyc (77%±9.5) and scrambled (75%±7) and cultured for 18 days. As expected, transduced cell numbers dropped to 5%±1 for GATA2-ERT group, confirming GATA2 dependence, while transduced cells decreased to 40%±8 for the Nmyc group with the control group unchanged. This suggests that Nmyc could be one of the key targets of GATA2 responsible for myeloid expansion. Current studies are aimed at determining whether GATA2 directly regulates the Nmyc promoter. Disclosures: No relevant conflicts of interest to declare.

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AML1-ETO Represses Multiple Transcription Factor Induced CTSG Activation by Reducing H3 Acetylation

Blood ◽

10.1182/blood.v122.21.3750.3750 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3750-3750

Author(s):

Yun Tan ◽

Wen Jin ◽

Kang Wu ◽

Kankan Wang

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Conflicts Of Interest ◽

Critical Role ◽

Negative Regulator ◽

Cathepsin G ◽

Luciferase Reporter ◽

Aberrant Expression ◽

H3 Acetylation

Abstract Acute myeloid leukemia (AML) is often accompanied with the aberrant expression of transcription factors. In t(8;21) AML, the AML1-ETO fusion protein executes its critical role in leukemogenesis through the interference with hematopoietic transcription factors (TFs) including AML1, C/EBPα, PU.1 and c-Myb. These transcription factors cooperate to modulate hematopoiesis by regulating their differentiation-related target genes. In our previous work, we have identified that AML1-ETO suppresses the AML1-dependent transactivation of the gene encoding the neutrophil granule protease, cathepsin G (CTSG). However, the detailed mechanisms of AML1-ETO mediated transrepression, especially coordinated regulation of hematopoietic transcription factors, have not been characterized yet. To investigate the regulatory pattern of CTSG by hematopoietic specific transcription factors, we constructed a luciferase reporter containing the CTSG promoter and co-transfect it with AML1, c-Myb, C/EBPα or PU.1 to 293T cells. The results of luciferase assays showed that these TFs individually activated the CTSG promoter, and synergistic transactivation occurred between AML1 and c-Myb, C/EBPα and PU.1, and PU.1 and c-Myb on the CTSG promoter. Furthermore, AML1/ETO effectively suppressed the transcription factor-dependent transactivation and synergistic transactivation of the CTSG promoter. Chromatin immunoprecipitation assays further demonstrated that AML1-ETO coexisted with these TFs on the CTSG promoter in AML1/ETO-positive Kasumi-1 cell line, indicating AML1-ETO was tethered to the chromatin bound by these TFs. The data suggested that AML1-ETO might act as a negative regulator by interfering the normal function of hematopoietic TFs instead of competing for their binding. In addition, to reveal the underlying mechanism of AML1/ETO-mediated transcription repression at the epigenetic level, we examined the epigenetic status of the CTSG promoter in AML1-ETO negative and positive cells, and found the level of histone H3 Lys9 acetylation on the CTSG promoter was obviously lower in AML1-ETO positive cells than that in AML1-ETO negative cells. The data suggested that AML1-ETO might repress the gene transcription by changing the H3 acetylation status of its target gene. Collectively, our findings demonstrate that AML1-ETO represses the transactivation of the CTSG promoter mediated by multiple hematopoietic transcription factors through a decrease of H3 acetylation. Disclosures: No relevant conflicts of interest to declare.

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Integrated Nuclear Proteomics and Transcriptomics Identifies New High Frequency Dysregulated Transcription Factors in AML

Blood ◽

10.1182/blood.v124.21.3554.3554 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3554-3554

Author(s):

Chinmay R Munje ◽

Andrew J. K. Williamson ◽

Samuel Taylor ◽

Robert K. Hills ◽

Steven Knapper ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Experimental Design ◽

Protein Identification ◽

Target Genes ◽

Conflicts Of Interest ◽

Mrna Level ◽

Transcriptional Level ◽

Cd34 Cells ◽

Nuclear Proteome

Abstract Acute myeloid leukemia (AML) is characterized by developmental arrest which is thought to arise from transcriptional dysregulation of myeloid development programs. Transcription factor (TF) dysregulation has been identified at both the genomic and transcriptomic levels; however the actual transcriptional environment in AML patients characterized by the relative abundance of TF protein expression compared with normal blasts has not yet been described. Here, we have analysed the nuclear proteome of AML blasts in comparison with normal CD34+ cells and carried out a parallel analysis of the transcriptome using Affymetrix arrays. In our experimental design we restricted our analysis to the minimally differentiated FAB M1 since this subtype has little developmental heterogeneity and would also be developmentally matched to normal controls. The final experimental design comprised nuclear protein extracts from 5 normal CD34+ controls and 15 FAB M1 patients (>80% viable blasts; <10% CD14+/CD15+ cells). Purity of nuclear fractions was assessed by western blotting for histone and GAPDH (Figure 1). Nuclear tryptic peptides were generated and in each experiment labelled with 8 channel isobaric tagging to allow relative quantification coupled with peptide/protein identification using tandem mass spectrometry. In parallel mRNA from these samples were analysed using Human Transcriptome Array 2.0 (Affymetrix, USA). In total 6164 proteins were found and quantified. After applying appropriate quality control criteria 437 proteins were found to be significantly dysregulated between normal CD34+ cells and AML blasts. To identify frequently dysregulated proteins we selected those that consistently changed in at least 5 AML patients (± >1.2 fold). This yielded 188 proteins of which 112 (60%) were nuclear proteins. We identified 7 upregulated transcription factors in leukaemic cells compared to normal CD34+ cells; 3 of which had been previously associated with AML (CEBPA, STAT6 and WT1). Comparative analysis of mRNA of these changes showed that these increases were also significantly observed at the transcriptional level (Table 1). The remainder (DRAP1, NFIC, HMGB1 and HMGB2) had not been previously reported in AML, and none of the changes were seen at the transcriptional level indicating increased expression arose at post-transcriptionally or were due to an increased level of nuclear localization. We also identified 7 down-regulated TF, one of which had been previously associated with AML (DAZAP1) with the remaining being newly described abnormalities (MYEF2, NFIX, FUBP1, TARDBP, ILF2, ILF3). Again most of these changes (4 of 6) were not seen at the transcriptional level. We also observed changes in 15 heterogeneous nuclear ribonucleoproteins (hnRNP) affecting mRNA processing (including: A0, A1, A2B1, A3, AB, C, D, DL, PF, H1, H3, K, L, R and UL2) and we are currently examining whether their expression correlates with increased alternative splicing that we have observed in these patients from analysis of the exon arrays. These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression that would not have been seen at the mRNA level. We are performing in silico analysis to determine whether dysregulation of these TF give rise to corresponding changes in known target genes. Disclosures No relevant conflicts of interest to declare.

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Gipfel – a Novel Method for Unbiased Molecular ETV6-RUNX1 Screening of Healthy Newborns

Blood ◽

10.1182/blood.v124.21.5340.5340 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5340-5340

Author(s):

Ute Fischer ◽

Daniel Schäfer ◽

Elisa Füller ◽

Katrine Ask ◽

Pina Fanny Ida Krell ◽

...

Keyword(s):

Transcription Factor ◽

Cord Blood ◽

Output Signal ◽

Childhood Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Population Based ◽

Inverse Pcr ◽

Blood Samples ◽

Chimeric Transcription Factor

Abstract Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns. The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented. In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. Disclosures No relevant conflicts of interest to declare.

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Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL

Blood ◽

10.1182/blood.v126.23.2445.2445 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2445-2445

Author(s):

Marshall S Horwitz ◽

Matthew R Hart

Keyword(s):

Transcription Factor ◽

B Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

B Cell Differentiation ◽

Loss Of Function ◽

Genetic Redundancy ◽

Factor Expression ◽

Pax Family

Abstract Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies. Disclosures No relevant conflicts of interest to declare.

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