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In sarcoma, the activity of NF-κB (nuclear factor κB) reduces the abundance of the microRNA (miRNA) miR-29. The tumor suppressor A20 [also known as TNFAIP3 (tumor necrosis factor–α–induced protein 3)] inhibits an upstream activator of NF-κB and is often mutated in lymphomas. In a panel of human sarcoma cell lines, we found that the activation of NF-κB was increased and, although the abundance of A20 protein and mRNA was decreased, the gene encoding A20 was rarely mutated. The 3′ untranslated region (UTR) of A20 mRNA has conserved binding sites for both of the miRNAs miR-29 and miR-125. Whereas the expression of miR-125 was increased in human sarcoma tissue, that of miR-29 was decreased in most samples. Overexpression of miR-125 decreased the abundance of A20 mRNA, whereas reconstituting miR-29 in sarcoma cell lines increased the abundance of A20 mRNA and protein. By interacting directly with the RNA binding protein HuR (human antigen R; also known as ELAVL1), miR-29 prevented HuR from binding to the A20 3′UTR and recruiting the RNA degradation complex RISC (RNA-induced silencing complex), suggesting that miR-29 can act as a decoy for HuR, thus protecting A20 transcripts. Decreased miR-29 and A20 abundance in sarcomas correlated with increased activity of NF-κB and decreased expression of genes associated with differentiation. Together, the findings reveal a unique role of miR-29 and suggest that its absence may contribute to sarcoma tumorigenesis.

SummaryT cells infiltrate affected organs in chronic infections and malignancy, but they may fail to eradicate virus-infected cells or tumor because of exhaustion. This report describes a Yin Yang-1 (YY1)-centered mechanism for diverse components that have been correlated with exhaustion. Utilizing an in vitro reconstruction of chronic T cell activation, YY1 is shown to positively regulate the checkpoint receptors PD1, Lag3, and Tim3 and to negatively regulate the type I cytokines interleukin-2 (IL-2) (in collaboration with Ezh2 histone methyltransferase) and interferon gamma (IFN-γ). Other tests suggest that IL-2 failure drives a large component of cytotoxic functional decline rather than solely checkpoint receptor-ligand interactions that have been the focus of current anti-exhaustion therapies. Clinical evaluations confirm elevated YY1 and Ezh2 in melanoma tumor-infiltrating lymphocytes and in PD1+ T cells in patients with HIV. Exhaustion is revealed to be an active process as the culmination of repetitive two-signal stimulation in a feedback loop via CD3/CD28→p38MAPK/JNK→YY1→ exhaustion.

CCAAT/enhancer-binding protein ␣ (C/ EBP␣) is a critical regulator for early myeloid differentiation. Mutations in C/EBP␣ occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominantnegative isoform (C/EBP␣p30). In the present study, using a global proteomics approach to identify the target proteins of C/EBP␣p30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBP␣p30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBP␣p30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBP␣p42 decreases the transactivation capacity on a minimal C/EBP␣ promoter. Importantly, overexpression of C/EBP␣p30 in granulocyte colony-stimulating factor (G-CSF)-stimulated human CD34 ؉ cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBP␣p30 target through which C/EBP␣p30 enhances the sumoylation of C/EBP␣p42 to inhibit granulocytic differentiation. IntroductionThe transcription factor CCAAT/enhancer-binding protein ␣ (C/ EBP␣) is crucial for granulocytic differentiation. [1][2][3] Alterations of the function of C/EBP␣ are a common feature of leukemic cells. 4,5 It was discovered in 10% of patients with acute myeloid leukemia (AML) that the CEBPA gene is mutated. 6,7 These mutations are found in AMLs with a myeloblast phenotype (French-AmericanBritish [FAB]-M1 and -M2 subtypes). The mutated gene results in the predominant expression of a 30-kDa protein initiated at an internal AUG start codon. This mutated isoform lacks the Nterminal transactivation domain 1 (TAD1). However, it possesses the intact bZIP protein-protein interaction domain and can interact with activators and repressors that affect its biological roles. The mutated 30-kDa isoform has been shown to act in a dominantnegative manner over the wild-type isoform. 5 The ratio of p30/p42 is critical for a physiologic granulopoiesis. 5,8 In contrast to C/EBP␣p42, C/EBP␣p30 fails to induce myeloid cell differentiation. It inhibits the expression of the endogenous granulocyte colony-stimulating factor (G-CSF) receptor and leads to an enhanced proliferation. 9,10 Recently, it was reported that C/EBP␣p30 directly interacts with the BCL2 promotor to fulfill this role. 11 Relatively little is understood about how C/EBP␣p30 exerts its dominant-negative effect over C/EBP␣p42 and how it inhibits C/EBP␣p42 during normal myeloid lineage development. We applied high-throughput proteomics to identify the target proteins of C/EBP␣p30. In our screen, we identified the ubiquitinconjugating enzyme (Ubc9) as a novel target of C/EBP␣p30.Ubc9 is an essential E2 enzyme required for small ubiquitinrelated modifier (SUMO) conjugation, or sumoylation. 12,13 Ubc9 is known to play a central role in su...

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Acute myeloid leukaemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogenetic abnormalities differs considerably, we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and post-translational modifications (PTM). We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MS) and MSMS tandem MS. We could identify significant differences in the proteome and PTM of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals major regulating networks: MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53-MYC-PRKAC for 11q23 and JUN and MYC for Inv(16). Further, we analysed 42 MS spectra representative of hnRNPH1, calreticulin and hnRNPA2/B1 in a peak explorer, which reveals a cytogenetic-specific PTM of b-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about AML pathogenesis, as differences at PTM level could be used to distinguish different subtypes of AML.

BackgroundWe recently reported that bacterial lipopolysaccharide (LPS)-induced inflammation decreases the expression of the primary thyroid hormone transporters at the blood–brain barrier, organic anion-transporting polypeptide 1c1 (OATP1c1) and monocarboxylate transporter 8 (MCT8). l-type amino acid transporters 1 and 2 (LAT1 & LAT2) are regarded as secondary thyroid hormone transporters, and are expressed in cells of the blood–brain or blood-cerebrospinal fluid barrier and by neurons. The purpose of this study was to examine the effect of LPS-induced inflammation on the expression of LAT1 and LAT2, as these may compensate for the downregulation of OATP1c1 and MCT8.MethodsLPS (2.5 mg/kg body weight) was injected intraperitoneally to adult, male, Sprague–Dawley rats and C57Bl/6 mice, which were euthanized 2, 4, 9, 24 or 48 h later. LAT1 and LAT2 mRNA expression were studied on forebrain sections using semiquantitative radioactive in situ hybridization. LAT1 protein levels in brain vessels were studied using LAT1 immunofluorescence. Statistical comparisons were made by the non-parametric Kruskal–Wallis and Dunn’s tests.ResultsIn both species, LAT1 mRNA decreased in brain blood vessels as soon as 2 h after LPS injection and was virtually undetectable at 4 h and 9 h. During recovery from endotoxemia, 48 h after LPS injection, LAT1 mRNA in brain vessels increased above control levels. A modest but significant decrease in LAT1 protein levels was detected in the brain vessels of mice at 24 h following LPS injection. LPS did not affect LAT1 and LAT2 mRNA expression in neurons and choroid plexus epithelial cells.ConclusionsThe results demonstrate that LPS-induced inflammation rapidly decreases LAT1 mRNA expression at the blood–brain barrier in a very similar manner to primary thyroid hormone transporters, while changes in LAT1 protein level follow a slower kinetics. The data raise the possibility that inflammation may similarly down-regulate other blood–brain barrier transport systems at the transcriptional level. Future studies are required to examine this possibility and the potential pathophysiological consequences of inflammation-induced changes in blood–brain barrier transport functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-015-0016-8) contains supplementary material, which is available to authorized users.