Vit Pospisil

PU.1 is a transcription factor absolutely required for normal hematopoiesis. Cumulating evidence indicates that precise levels of PU.1 expression are critical for differentiation to distinct blood lineages, and if perturbed, even modest decreases in PU.1 can lead to leukemogenesis. In contrast to extensive knowledge of regulation of PU.1 gene itself, the mechanism of how target genes senses different PU.1 levels remain largely unknown. To address this, we used PU.1-/- mouse myeloid progenitors encoding inducible PU.1 transgene (PU.1ER, PUER, Walsh 2002) that allows tight control of PU.1 activity. Interestingly, intermediate PU.1 activity induced differentiation of PUER progenitors into granulocyte like cells, while high PU.1 produced macrophages, supporting the model that different PU.1 expression is not a consequence but a driver of cell fate choice. Global expression analysis using 4 different levels of PU.1 at 8 time points (2-96 hrs) revealed that granulocyte specific genes were activated exclusively by intermediate PU.1 levels in 3 distinct modes: 1. not expressed in progenitors while strongly induced at intermediate PU.1 (e.g. Gelatinase B (Mmp9) and Neutrophil collagenase (NC) 2. moderately expressed in progenitors while strongly activated at intermediate PU.1 and repressed at high PU.1 (e.g. Myeloperaxidase (Mpo) 3. highly expressed in unstimulated progenitors with expression maintained at intermediate PU.1 but strongly repressed at high PU.1 (e.g. Neutrophil elastase (NE), Proteinase 3 (primary granule proteins), Cebpe and Gfi1 (Growth factor independent1) Majority of macrophage genes (incl. CD14, Csf1R, Egr2) were regulated as early PU.1 target genes; being gradually activated by high PU.1 activity within 8hrs. However, most granulocyte genes (NE, Mmp9, Mpo, NC but not Cebpe and GFI1) were late activated PU.1 targets (48 and 96hrs) indicating that these genes are coregulated by additional factor(s), likely an early PU.1 target. Next we analyzed the regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Mpo and Mmp9, using own and public ChIP(seq) data of transcription factors (TFs) (PU.1, GFI.1), DNAseI hypersensitive sites, histone modifications (H3K4Me, H3K27Ac, H3K9Ac) and expression of enhancer specific bidirectional ncRNAs (eRNA) (CAGE). 14 Mpo and 16 Mmp9 putative enhancers, selected by above mentioned criteria, were cloned into luciferase vector containing their proximal promoter (PP) and were tested for functional activity in response to PU.1 levels. Interestingly, the PU.1 binding motifs within these regions have a low to intermediate affinity (log of score, Jaspar) and are often present in multiples and/or enriched for binding sites of other lineage determining transcription factors. Although PU.1 bound to all of these DNA regions resembling superenhancer, just a small fraction of PU.1 binding was functionally responsive. Specifically, we identified novel enhancer elements at -3.4 kb and...

MicroRNAs (miRs) represent a distinct class of posttranscriptional modulators of gene expression with remarkable stability in sera. Several miRs are oncogenic (oncomiRs) and are deregulated in the pathogenesis of breast cancer and function to inhibit tumor suppressors. Routine blood monitoring of these circulating tumor-derived products could be of significant benefit to the diagnosis and relapse detection of early-stage breast cancer (EBC) patients.

MicroRNAs (miRNAs) are single-stranded non-coding RNAs (21-25nt) that negatively regulate gene expression at post-transcriptional level by blocking translation and mRNA destabilization. Diffuse large B cell lymphomas (DLBCL) overexpress oncogenic microRNAs (including miR-17-92 and miR-155) that regulate tumor growth and spreading. Unlike longer species of RNA, microRNAs are stable and detectable in body fluids including serum/plasma and cerebrospinal fluid (CSF), however their kinetics in body fluids upon course the therapy of DLBCL has not been fully explored. Notably DLBCLs with primary (PCNSL) or secondary CNS involvement display poor prognosis, short overall survival and their current diagnosis and disease monitoring is based on conventiontional methods (magnetic resonance imaging, histopathology, CSF cytology and flow cytometry) with unsatisfactory sensitivity. We investigated abundance of microRNAs in the CSF and sera of DLBCL patients with (N=18) or without (N=30) CNS involvement and compared their levels at diagnosis and during treatment-response phase. Extensive time-points to collect CSF and sera (5-20) were used during and after administration of therapy. Twenty candidate miRs from DLBCL patients were quantitated by TaqMan RT-PCR and their levels adjusted to those of control miRs and normal donors. Analysis of CSF at diagnosis revealed that most prominently deregulated miRs (members of oncogenic miR-17-92 cluster and miR-21) were specificaly elevated in both PCNSL (N=11) and systemic lymphomas with CNS involvement (SCNSL, N=7) to similar extent while in systemic lymphomas without CNS involvement (N=30) their levels were significantly lower. Analysis of sera at diagnosis revealed that all SCNSL patients and ∼50% of systemic lymphomas without CNS involvement expressed high levels of the oncomiRs. Surprisingly, also a subset of PCNSL patients had elevated these miRs in the sera and in turn, a subset of systemic lymphomas without CNS involvement showed moderately elevated oncomiRs in the CSF. These data indicate that 1) lymphoma patients with CNS involvement (PCNSL or SCNSL) are characterized by increase of oncogenic microRNAs in CSF, 2) blood brain barrier is likely permissive for miRs in both directions. Next, we investigated the dynamics of abundance of the oncomiRs in CSF upon course of the treatment in 8 patients with PCNSL or SCNSL. In therapy-responders (N=4) the oncomiRs gradually decreased in CSF. However, their levels were not completely normalized as compared to normal individuals. In contrast, refractory lymphomas (N=2) displayed gradual increase of these miRs in CSF regardless the treatment. Interestingly, clinical relapse within CNS as confirmed by MRI and cytology (N=2) was preceded (>3 months) by significant and gradual elevation of miRs in CSF. In systemic lymphoma (N=2) increased levels of studied oncomiRs in CSF preceded the CNS progression. In all cases high levels of miRs in CSF preceded and positively correlated with data from flow cytometry and cytology. The measurement of oncomiRs in CSF and sera represents sensitive tool for detection of CNS lymphoma, for monitoring and estimation of therapy efficacy and prediction of the CNS relapse. Furthermore, our data indicate that elevated expression of oncomiRs in CSF and sera in systemic lymphomas may become helpful to predict CNS lymphoma involvement. Grants: GACR305 13-12449P, UNCE204021, PRVOUK: P24/LF1/3, P27/LF1/1, P 27/2012 LF3 Trneny: Roche: Honoraria, Research Funding.

PU.1 downregulation within hematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to their aggressiveness; however, its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1(ure/ure) mice (PU.1(ure/ure)p53(-/-)) results in more aggressive AML with shortened overall survival. PU.1(ure/ure)p53(-/-) progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1 levels in PU.1(ure/ure)p53(-/-) mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved in the pathogenesis of AML and its aggressiveness characterized by p53 mutation.

Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.