Meprin ß knockout reduces brain Aß levels and rescues learning and memory impairments in the APP/lon mouse model for Alzheimer's disease.

ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described ß-secretase to generate Aß peptides in Alzheimer's disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aß peptides generation is the metalloproteinase meprin ß, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin ß expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b-/-). We examined levels of canonical and truncated Aß species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b-/-. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aß1-40 and 1-42 levels are reduced in APP/lon mice when meprin ß is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aß2-x peptide deposition is decreased in APP/lon × Mep1b-/- mice. Importantly, loss of meprin ß improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin ß within the amyloidogenic pathway and Aß production in vivo.

Venetoclax and dexamethasone synergize with inotuzumab ozogamicin-induced DNA damage signaling in B-lineage ALL.

Adult patients with relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a dismal prognosis. To improve pharmacotherapy, we analyzed induction of apoptosis by venetoclax and inotuzumab ozogamicin in terms of cytotoxicity and mode of action. Flow cytometry-based analyses of mitochondrial outer membrane permeabilization (MOMP) and ataxia telangiectasia mutated activation demonstrate rapid induction of MOMP by venetoclax and DNA damage signaling by inotuzumab ozogamicin, respectively. In primary ALL samples and patient-derived xenograft (PDX) models, venetoclax and inotuzumab ozogamicin cooperated and synergized in combination with dexamethasone in vitro in all tested samples of ALL. In murine PDX models, inotuzumab ozogamicin, but not venetoclax, induced complete remission in a dose-dependent manner but constantly failed to achieve relapse-free survival. In contrast, combination therapy with venetoclax, dexamethasone, and inotuzumab ozogamicin induced long-term leukemia-free survival and treatment-free survival in all 3 ALL-PDX models tested. These data demonstrate synergistic and highly efficient pharmacotherapy in preclinical models that qualify for evaluation in clinical trials.

Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase.

The oncogenic fusion protein RUNX1-ETO is a product of the t(8;21) translocation and consists of the hematopoietic transcriptional master regulator RUNX1 and the repressor ETO. RUNX1-ETO is found in 10-15% of acute myeloid leukemia and interferes with the expression of genes that are essential for myeloid differentiation. The neutrophil serine protease Cathepsin G is one of the genes suppressed by RUNX1-ETO, but little is known about its impact on the regulation of other lysosomal proteases. By lentiviral transduction of the t(8;21) positive cell line Kasumi-1 with an RUNX1-ETO specific shRNA, we analyzed long-term effects of stable RUNX1-ETO silencing on cellular phenotypes and target gene expression. Stable anti RUNX1-ETO RNAi reduces both proliferation and apoptosis in Kasumi-1 cells. In addition, long-term knockdown of RUNX1-ETO leads to an upregulation of proteolytic activity in Kasumi-1 cells, which may be released in vitro upon cell lysis leading to massive degradation of cellular proteins. We therefore propose that protein expression data of RUNX1-ETO-silenced Kasumi-1 cells must be analyzed with caution, as cell lysis conditions can heavily influence the results of studies on protein expression. Next, a mass spectrometry-based approach was used to identify protease cleavage patterns in RUNX1-ETO-depleted Kasumi-1 cells and Neutrophil Elastase has been identified as a RUNX1-ETO candidate target. Finally, proteolytic activity of Neutrophil Elastase and Cathepsin G was functionally confirmed by si/shRNA-mediated knockdown in Kasumi-1 cells.

miR-125b regulates chemotaxis and survival of bone marrow derived granulocytes in vitro and in vivo.

The evolutionary conserved miR-125b is highly expressed in hematopoietic stem cells (HSC) enhancing self-renewal and survival. Accordingly, over-expression of miR-125b in HSC may induce myeloproliferative neoplasms and leukemia with long latency. During hematopoietic cell maturation miR-125b expression decreases, and the function of miR-125b in mature granulocytes is not yet known. We here use transplantation of miR-125b over-expressing HSC into syngeneic hosts to generate and analyse miR-125b over-expressing granulocytes. Under steady state conditions, miR-125b over-expression inhibits granulocytic chemotaxis and LPS- but not PMA- and TNFα- induced cell death. Inflammatory signals modulate the effects of miR-125b over-expression as demonstrated in a sterile peritonitis and a polymicrobial sepsis model. In particular, survival of mice with miR-125b over-expressing granulocytes is significantly reduced as compared to controls in the polymicrobial sepsis model. These data demonstrate inflammation dependent effects of miR-125b in granulocytes and may point to therapeutic intervention strategies in the future.

Peptide microarray profiling identifies phospholipase C gamma 1 (PLC-γ1) as a potential target for t(8;21) AML.

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) which has a need for improved therapeutic strategies. We found PLC-γ1 as one of the highest phosphorylated peptides in t(8;21) AML samples compared to NBM or CN-AML in our previous peptide microarray. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in t(8;21) AML. In this study, PLC-γ1 expression was significantly higher in t(8;21) AML compared to other karyotypes. The PLC-γ1 protein expression was suppressed in AML1-ETO knock down cells indicating that it might induce kasumi-1 cell death. ShRNA-mediated PLC-γ1 knockdown in kasumi-1 cells significantly blocked cell growth, induced apoptosis and cell cycle arrest which was explained by the increased activation of apoptotic related and cell cycle regulatory protein expressions. Gene expression array analysis showed the up-regulation of apoptotic and DNA damage response genes together with the downregulation of cell growth, proliferation and differentiation genes in the PLC-γ1 suppressed kasumi-1 cells, consistent with the observed phenotypic effects. Importantly, PLC-γ1 suppressed kasumi-1 cells showed higher chemosensitivity to the chemotherapeutic drug treatments and lower cell proliferation upon hypoxic stress. Taken together, these in vitro finding strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cells and identify PLC-γ1 as a potential therapeutic target for t(8;21) AML treatment.

Long-range massively parallel mate pair sequencing detects distinct mutations and similar patterns of structural mutability in two breast cancer cell lines.

Cancer genomes frequently undergo genomic instability resulting in accumulation of chromosomal rearrangement. To date, one of the main challenges has been to confidently and accurately identify these rearrangements by using short-read massively parallel sequencing. We were able to improve cancer rearrangement detection by combining two distinct massively parallel sequencing strategies: fosmid-sized (36 kb on average) and standard 5 kb mate pair libraries. We applied this combined strategy to map rearrangements in two breast cancer cell lines, MCF7 and HCC1954. We detected and validated a total of 91 somatic rearrangements in MCF7 and 25 in HCC1954, including genomic alterations corresponding to previously reported transcript aberrations in these two cell lines. Each of the genomes contains two types of breakpoints: clustered and dispersed. In both cell lines, the dispersed breakpoints show enrichment for low copy repeats, while the clustered breakpoints associate with high copy number amplifications. Comparing the two genomes, we observed highly similar structural mutational spectra affecting different sets of genes, pointing to similar histories of genomic instability against the background of very different gene network perturbations.

Interleukin-27 acts on hepatic stellate cells and induces signal transducer and activator of transcription 1-dependent responses.

BACKGROUND: Interleukin (IL)-27 is a cytokine belonging to the IL-6/IL-12 cytokine family that is secreted by activated macrophages and dendritic cells and which strongly acts on T-cells and cells of the innate immune system. Not much is known about possible effects of IL-27 on other cell types. It signals via the common IL-6-type-cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. We previously described that IL-27 also stimulates hepatoma cells and primary hepatocytes. The aim of this study was to investigate whether IL-27 would also act on hepatic stellate cells (HSC), the second most abundant hepatic cell type, which would demonstrate a more general role of this cytokine in the liver. RESULTS: Using a human HSC line and primary rat HSC we investigated the signalling characteristics of IL-27 in these cells. We show that IL-27 activates signal transducer and activator of transcription (STAT) 1 and to a minor extent STAT3 in a human HSC cell line and that it leads to the induction of STAT1 target genes such as interferon response factor-1, myxovirus resistance A and STAT1 itself. Similarly we find that IL-27 also elicits STAT1-dependent responses in primary rat HSC. CONCLUSIONS: We provide the first evidence for a function of IL-27 in HSC and show that its responses resemble Interferon-gamma-like functions in these cells. Our data suggests that IL-27 may play an important role in the context of liver inflammation by acting on the different liver cell types.

Interleukin-27 displays interferon-gamma-like functions in human hepatoma cells and hepatocytes.

UNLABELLED: Interleukin-27 (IL-27) is a cytokine belonging to the IL-6/IL-12 cytokine family. It is secreted by antigen-presenting cells, strongly acts on T cells, and also stimulates innate immune cells. In most studies, the effects of IL-27 on T cells were investigated; however, not much is known about possible effects of IL-27 on other cell types. IL-27 signals via the common IL-6-type cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. Given the importance of gp130 in regulating liver responses such as the acute phase response or liver regeneration, we investigated whether IL-27 could also have a function in liver cells. We find that IL-27 stimulates hepatoma cells and hepatocytes by inducing a sustained signal transducer and activator of transcription (STAT)1 and STAT3 activation. Whereas the STAT3 mediated responses to IL-27 (gamma-fibrinogen and hepcidin induction) are not detectable, we observe an interferon-gamma (IFN-gamma)-like STAT1 response leading to the induction of interferon-regulated proteins such as STAT1, STAT2, interferon response factor (IRF)-1, IRF-9, myxovirus resistance A and guanylate binding protein 2. CONCLUSION: Our study provides evidence for a function of IL-27 in hepatoma cells and hepatocytes and shows that IL-27 responses are not restricted to the classical immune cells. Our results suggest that IL-27 exerts IFN-like functions in liver cells and that it can contribute to the antiviral response in these cells.