MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNAdependent phenotypes and miRNAregulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colonyformation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, genespecific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNAbinding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/ miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. IntroductionMicroRNAs (miRNAs) represent an emerging class of noncoding single-stranded RNAs of approximately 22 nucleotides 1,2 that play an important role in posttranscriptional regulation of gene expression. miRNAs are processed from primary primiRNA transcripts to pre-miRNAs and mature miRNAs in a multistep process. Mature miRNAs are incorporated into and subsequently recruit a multi-protein effector complex RISC (RNA-induced silencing complex) to complementary miRNAbinding sites located preferentially within the 3ЈUTR of target mRNAs. Sequence-specific binding of RISC results in reduced mRNA translation and/or degradation through RNA interference (RNAi). 3 The interaction between a miRNA and its target mRNAs usually requires complementarity only within so-called seed sequence (miRNA nucleotides 2-8). Hence, a single miRNA has the potential to regulate hundreds of proteins 4-6 but resulting target protein repression is typically relatively mild. 5,6 Thereby, the ratio of regulatory RNAs to target mRNAs may modulate the silencing activity with a negative correlation between target abundance and target down-regulation. 7 To identify miRNA target genes, several prediction programs based on the hybridization energy of complementary miRNA/mRNAs sequences have been described. [8][9][10][11][12] However, the use of these programs is error-prone and identification of miRNA tar...

The double-stranded RNA (dsRNA)-activated serine/threonine kinase R (PKR) is well characterized as an essential component of the innate antiviral response. Recently, PKR has been implicated in Toll-like receptor (TLR) signal transduction in response to bacterial cell wall components. Its contribution to pulmonary immunity, however, has not yet been elucidated. In this report we investigated whether PKR is involved in TLR2/TLR4-mediated immune responses of primary alveolar macrophages (AM). We found that both TLR2 (Pam3CSK4) and TLR4 (LPS) ligands induced rapid phosphorylation of PKR. Moreover, this activation was strictly dependent on the functionality of the respective TLR. Pharmacologic inhibition of PKR activity using 2-aminopurine (2-AP) and PKR gene deletion was found to reduce the TLR2/TLR4-induced activation of the JNK signaling pathway (MKK4/JNK/c-Jun), but did not affect p38 and extracellular signal-regulated kinase 1/2 activation. Moreover, inhibition of PKR phosphorylation severely impaired TNF-alpha and IL-6 production by AM in response to LPS and Pam3CSK4. In addition, we found that PKR phosphorylation plays a major role in LPS- but not Pam3CSK4-induced activation of the p65 subunit of NF-kappaB. Collectively, these results indicate that functional PKR is critically involved in inflammatory responses of primary AM to gram-positive as well as gram-negative bacterial cell wall components.

To integrate available clinical and molecular information for cytogenetically normal acute myeloid leukemia (CN-AML) patients into one risk score, 275 CN-AML patients from multicenter treatment trials AML SHG Hannover 0199 and 0295 and 131 patients from HOVON/SAKK protocols as external controls were evaluated for mutations/polymorphisms in NPM1, FLT3, CEBPA, MLL, NRAS, IDH1/2, and WT1. Transcript levels were quantified for BAALC, ERG, EVI1, ID1, MN1, PRAME, and WT1. Integrative prognostic risk score (IPRS) was modeled in 181 patients based on age, white blood cell count, mutation status of NPM1, FLT3-ITD, CEBPA, single nucleotide polymorphism rs16754, and expression levels of BAALC, ERG, MN1, and WT1 to represent low, intermediate, and high risk of death. Complete remission (P ‫؍‬ .005), relapse-free survival (RFS, P < .001), and overall survival (OS, P < .001) were significantly different for the 3 risk groups. In 2 independent validation cohorts of 94 and 131 patients, the IPRS predicted different OS (P < .001) and RFS (P < .001). High-risk patients with related donors had longer OS (P ‫؍‬ .016) and RFS (P ‫؍‬ .026) compared with patients without related donors. In contrast, intermediate-risk group patients with related donors had shorter OS (P ‫؍‬ .003) and RFS (P ‫؍‬ .05). Donor availability had no impact on outcome of patients in the low-risk group. Thus, the IPRS may improve consolidation treatment stratification in CN-AML patients. Study registered at www.clinicaltrials.gov as #NCT00209833. IntroductionAcute myeloid leukemia (AML) is characterized by uncontrolled proliferation of hematopoietic progenitor cells, blocked maturation resulting from interruption of normal differentiation pathways, and activation of antiapoptotic pathways. In a large proportion of AML patients, standard cytogenetic analyses can identify specific recurrent chromosomal aberrations that are important for disease pathogenesis, response to therapy, and patient survival. 1,2 However, almost half of all AML patients are classified as cytogenetically normal (CN-AML). 2 Many efforts have been made to identify genetic mutations (eg, FLT3,3,4 NPM1, 5,6 CEBPA, 7-10 MLL, 11,12 NRAS, 13 IDH1/ 2, [14][15][16][17][18][19][20] and WT1 [21][22][23] ) that allow further subclassification and possibly risk-directed therapeutic intervention. In addition to mutations, modulated expression of genes important for proliferation, survival, and differentiation have also been shown to be predictive for CN-AML patient outcome (eg, MN1, 24,25 BAALC, 26 ERG, 27 ID1, 28 and WT1 29,30 ). This molecular heterogeneity of CN-AML is not fully reflected in current classification systems. 31,32 Recently, 2 or 3 prognostic markers were combined to define patient subgroups with distinct prognosis. Most importantly, patients with mutated NPM1 and wild-type FLT3 have a favorable prognosis. 5 Patients with low ERG, low EVI1, and high PRAME levels were also shown to have a good prognosis. 33 In addition, WT1 single nucleotide polymorphism (SNP) rs16754, a recently ...

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the ‘classical’ (M1) and ‘alternative’ (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

High MLL5 expression levels are associated with a favorable outcome and may improve risk and treatment stratification in AML.