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Precise levels of transcription factor PU.1 are critical for differentiation to distinct blood lineages, and even modest decreases leads to leukemogenesis. However, the mechanism of how target genes sense different PU.1 levels is unknown. To address this, we used myeloid progenitors encoding inducible PU.1 transgene. Intermediate PU.1 activity induced differentiation into granulocyte-like cells, while high PU.1 produced macrophages. Comparative analysis of regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Myeloperoxidase (Mpo) and Gelatinase B (Mmp9) identified 14 Mpo and 16 Mmp9 putative enhancers resembling superenhancers. Interestingly, the PU.1 binding motifs within these regions have an intermediate to high affinity (log ods score). To test their functionality we assembled successively these regulatory sequences into luciferase vectors. Specifically, we identified novel enhancer elements in -3.4 and -15 kb of Mpo which responded to intermediate (but not high) PU.1 levels. Interestingly, activity of -3.4 kb enhancer required presence of promoter while the -15kb element required presence of both PP and the -3.4kb element. Similar phenomenon was observed at multiple Mmp9 enhancers. This model is supported by our Chromosome confirmation capture (3C) data. Native activity of tested enhancers was confirmed by expression of bidirectional enhancer RNAs. The mutation of affinity of PU.1 binding sites within enhancers lead to reprogramming of their activity. In conclusion, our data support the model that PU.1 at intermediate concentrations binds to high affinity binding sites in several enhancers of granulocyte genes, causing their successive interaction that leads to transcription activation. Collectively, this suggests that a cooperative assembly of several cell-type specific enhancers is required for optimal Mpo and Mmp9 activation. more...

The oncogenic cluster miR-17-92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR-17-92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR-17-92 cluster. Using transgenic PU.1−/− myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR-17-92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR-17-92 promoter. Conversely, Egr2 itself is targeted by miR-17-92, indicating existence of mutual regulatory relationship between miR-17-92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR-17-92 and low levels of PU.1 and EGR2 leads to downregulation of miR-17-92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR-17-92 cluster promoter by an Egr-2/Jarid1b-mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states. more...

Abstract 3589 Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In our study, B-CLL patients (N=71, F/M 31/40, median age 65) were subject to molecular analyses that found significant overexpression of MYB and miR-155 in B-CLL and physical association of MYB with the promoter of MIR155 host gene (MIR155HG, also known as B-cell integration cluster). In CD19+ B-cells derived from healthy control individuals (N=13), MYB and miR-155 expression was significantly lower. Next, we found that MYB positively regulates MIR155HG transcription in reporter assays. The endogenous chromatin structure of the MIR155HG promoter in B-CLL cells is characterized by spreading of active chromatin mark, histone H3K9 acetylation. Gene expression arrays on B-CLL patients (N=11) identified a set of predicted miR-155 target genes (N=94) that are downregulated. Their expression pattern displayed significant negative correlation with pri-miR-155 levels. Similarly, a number of MYB target genes were found deregulated (N=99) in B-CLL. Gene annotation of differentially expressed miR-155 and MYB targets in B-CLL revealed their biological functions as regulators of apoptosis (BCL2, API5), proliferation (CCND1, CCND2) and mediators of B-cell function (CD200, F11R). In addition, expression patterns of miR-155, MYB and their targets are currently being compared in a larger patient group with prognostic hallmarks of CLL (ZAP70, CD38, del(17)(p13.1), and IgVH). Our data collectively support novel candidate mechanism in B-CLL that includes MYB directly stimulating MIR155HG promoter coincident with its epigenetic dysregulation. (Grant # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507). Disclosures: Trneny: ROCHE: Honoraria, Research Funding. more...

PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid and lymphoid lineages. Distinct nuclear levels of PU.1 are required for macrophage differentiation (DeKoter 2000). MicroRNAs are a novel class of 22nt long non-coding post-transcriptional regulators, associated with differentiation, development and carcinogenesis. To identify miRNAs involved in PU.1 dependent hematopoietic differentiation we used PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the estrogen receptor ligand-binding domain inducible by tamoxifen (PUER) (Walsh 2002). PU.1 affected expression approximately 100 mature miRNA. Among them, we have observed that PU.1 downregulates members of miR-17~92 cluster and its paralog miR-106b~25 previously associated with macrophage differentiation, apoptosis and hematologic and other malignancies (Ota 2004, Hayashita 2005, Fontana 2008). During macrophage differentiation miR-17~92 becomes downregulated and its sustained expression is associated with the differentiation block (Fontana 2007). In tamoxifen-stimulated PUER cells, PU.1 efficiently downregulates mature miRNAs of the miR-17~92 (-17.5p, -20a, -92) cluster and its paralog miR-106b~25 (but not miR-106a~363) within 96 hours and in dose dependent manner. PU.1 in the same cells activates expression of Egr2, myeloid transcription factor promoting macrophage transcriptional program a putative 8-mer-seed sequence-containing target of miR-17~92 cluster (predicted by TargetScan). We tested whether PU.1 represses expression of miR-17~92 cluster by inducing Egr2. We demonstrate that ectopic expression of Egr2 in unstimulated PUER cells inhibited expression of miR-17~92 cluster, both at the levels of mature miRs and pri-miRNAs, and induced expression of macrophage differentiation markers (Cd14, Csf1r) coupled with cell cycle arrest. Trichostatin-dependent derepression of miR-17~92 indicated that its expression is regulated at the chromatin level. Chromatin immunoprecipitation revealed specific Egr2 occupancy within the regulatory region upstream of the miR-17~92 cluster. Our data indicate that in differentiating cells, a mechanism exists involving direct repression of the miR-17~92 cluster that in turn has the potential to inhibit Egr2 expression. We have tested this mechanisms in peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=9) where levels of PU.1 and Egr2 are significantly lower that in normal controls. 5 out of 9 AML patients significantly overexpressed miR- 17.5p, miR-20a, miR-92 coinciding with downregulation of its established targets p21 and BIM. Rescue of Egr2 in AML cells (N=2) led to downregulation of miR-17~92 cluster and upregulation of BIM mRNA levels coincident with induction of apoptosis. We conclude that PU.1 regulates transcription of the miR-17~92 cluster by inducing Egr2 and demonstrate this mechanism in differentiating macrophages and its disruption in AML cells. more...

PU.1 and GATA1 are hematopoietic lineage-specific transcription factors that play key roles in normal myeloid and erythroid differentiation respectively. Inappropriate expression of PU.1 in proerythroblasts causes its binding to GATA-1 on DNA resulting in a block of erythroid differentiation and development of murine erythroleukemia (MEL) (Rekhtman 1999). Activation of a conditional transgene of GATA-1 (fused with the ligand binding domain of the estrogen receptor, ER) in MEL cells disrupts PU.1-mediated repression in chromatin leading to re-intiation of erythroid differentiation and cell cycle arrest (Choe 2003, Stopka 2005). In this study we show that MEL cells can also be induced to express myeloid differentiation programs upon PU.1-ER activation. Gene expression microarray analysis of GATA-1-ER MEL cells and PU.1-ER MEL cells treated with ER activators allowed us to identify mRNAs that are regulated by both GATA-1 and PU.1 including the set of GATA-1 targets repressed by PU.1, as well as the set of PU.1 targets repressed by GATA-1 in MEL cells. The targets of mutual antagonism of PU.1 and GATA-1 consisted of lineage specific transcription factors, differentiation markers and genes that cause cell cycle arrest and antiapoptotic regulators previously associated with myeloid and erythroid cell differentiation. To determine if PU.1 and GATA-1 directly regulate the lineage specific transcription factor genes, we performed chromatin immunoprecipitation (ChIP) and analyzed the ChIP samples on microarrays and by qPCR. We found that PU.1 and GATA-1 are localized near PU.1 binding sites in the genes for myeloid transcription factors Cebpa and Cbfb and near GATA-1 binding sites in the genes for erythroid transcription factors Fog1 and Nfe2. In addition, further ChIP experiments delineated chromatin architecture near the binding sites for PU.1 and GATA-1 including histone H3 content and acetylation of histone H3K9. We propose that the mutual antagonism of PU.1 and GATA-1 in inhibiting the respective differentiation programs is rendered through specific changes in chromatin structure around lineage specific transcription factors genes. These changes may contribute to the block to differentiation evident in leukemias. more...

Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases. In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells. In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker. Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604. Disclosures No relevant conflicts of interest to declare. more...

Abstract 3791 Use of DNA demethylating agent 5-azacytidine (AZA) in high risk Myelodysplastic syndromes (MDS) represents an important strategy yet not fully understood at molecular levels. Previous work demonstrated that normal progenitors treated by DNA demethylation agent Decitabine upregulated a key hematopoietic transcription factor PU.1. This was potentiated in presence of granulocyte colony stimulating factor (G-CSF) prior the DNA demethylation (Hu et al. 2010). Similarly, Decitabine administered to multiple myeloma cells resulted in DNA demethylation and upregulation of PU.1 gene (Tatetsu et al. 2007). To advance our understanding of molecular events mediated by AZA in MDS we measured effect of AZA on transformed cell lines (OCI-M2, SKM-1, MOLM-13, HNT-34, F-36P) and monitored the hematopoietic transcriptional program. We herein present evidence that AZA, while inducing efficiently cell cycle arrest and apoptosis of MDS tumor cells, stimulates also the expression of PU.1 and its target C/EBPa that is followed by acquisition of the myeloid differentiation program. Gene derepression of PU.1 is marked by epigenetic modifications at the upstream regulatory element (URE) of PU.1. G-CSF potentiated effects of AZA in MOLM-13 and SKM-1 cells. The effects of G-CSF and other cytokines (such as M-CSF or GM-CSF) on monocytic versus granulocytic lineage commitment of MDS cells are currently tested. To advance our study on the patient level we used primary high risk MDS CD34+ cells (N=36). Tumor cells with the lowest PU.1 expression displayed partial methylation at URE and incomplete sensitivity to AZA/G-CSF-mediated in vitro differentiation. We observed that patients with permissive chromatin at URE and higher PU.1 (and C/EBPa) levels responded significantly better to AZA/G-CSF in vitro. Currently we enrolled more patients in order to ask whether permissive PU.1 locus predicts responsiveness to AZA and whether it associates with different clinical patient outcome. (Grants: IGA NT10310-3/2009, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507). Disclosures: No relevant conflicts of interest to declare. more...