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Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse), who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I, killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients, KIR gene(s) for missing self recognition on recipient targets, and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003), better event-free survival in patients transplanted in relapse (34% versus 6%, P = .04) and in remission (67% versus 18%, P = .02), and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor, 0.48; 95% CI, 0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage."

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.

Embryonic stem cells (ESCs) represent an important research tool and a potential resource for regenerative medicine. Generally, ESCs are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts, which maintain the ESCs' capacity for self-renewal and block spontaneous differentiation. These cumbersome conditions, as well as the risk of xenobiotic contamination of human ESCs grown on murine embryonic fibroblasts, make it a priority to develop chemically defined methods that can be safely used for the expansion of ESCs. Using a high-throughput, cell-based assay, we identified the small molecule IQ-1 that allows for the Wnt/beta-catenin-driven long-term expansion of mouse ESCs and prevents spontaneous differentiation. We demonstrate that IQ-1, by targeting the PR72/130 subunit of the serine/threonine phosphatase PP2A, prevents beta-catenin from switching coactivator usage from CBP to p300. The increase in beta-catenin/CBP-mediated transcription at the expense of beta-catenin/p300-mediated transcription is critical for the maintenance of murine stem cell pluripotency.

Wortmannin (Wm), a steroid-like molecule of 428.4 Da, appears to be unstable in biological fluids (apparent chemical instability), yet it exhibits an antiproliferative activity in assays employing a 48 hr incubation period (prolonged bioactivity), a situation we refer to as the wortmannin paradox." Under physiological conditions�

Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells (EPCs), for a variety of cell therapies. Recently, EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition, we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde-High). Moreover, hypoxia-inducible factor proteins are up-regulated and VEGF, CXCR4, and GLUT-1 mRNAs are increased in Alde-Low EPCs under hypoxic conditions, while the response was not significant in Alde-High EPCs. In fact, the introduction of Alde-Low EPCs significantly reduced tissue damage in ischemia in a mouse flap model. Thus, the introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and subsequent regeneration of ischemic tissues.

BACKGROUND AND PURPOSE: Brain ischemia stimulates neurogenesis. However, newborn neurons show a progressive decrease in number over time. Under normal conditions, the cAMP-cAMP responsive element binding protein (CREB) pathway regulates the survival of newborn neurons. Constitutive activation of CREB after brain ischemia also stimulates hippocampal neurogenesis. Thus, activation of cAMP-CREB signaling may provide a promising strategy for enhancing the survival of newborn neurons. We examined whether treatment of mice with the phosphodiesterase-4 inhibitor rolipram enhances hippocampal neurogenesis after ischemia. METHODS: Both common carotid arteries in mice were occluded for 12 minutes. Bromodeoxyuridine (BrdU) was used to label proliferating cells. Mice were perfused transcardially with 4% paraformaldehyde, and immunohistochemistry was performed. To evaluate the role of CREB in the survival of newborn neurons after ischemia, intrahippocampal injection of a CRE-decoy oligonucleotide was delivered for 1 week. We examined whether the activation of cAMP-CREB signaling by rolipram enhanced the proliferation and survival of newborn neurons. RESULTS: Phospho-CREB immunostaining was markedly upregulated in immature neurons, decreasing to low levels in mature neurons. The number of BrdU-positive cells 30 days after ischemia was significantly less in the CRE-decoy treatment group than in the vehicle group. Rolipram enhanced the proliferation of newborn cells under physiologic conditions but not under ischemic conditions. Rolipram significantly increased the survival of nascent BrdU-positive neurons, accompanied by an enhancement of phospho-CREB staining and decreased newborn cell death after ischemia. CONCLUSIONS: CREB phosphorylation regulates the survival of newborn neurons after ischemia. Chronic pharmacological activation of cAMP-CREB signaling may be therapeutically useful for the enhancement of neurogenesis after ischemia.

Abnormal activation of B lymphocytes is a feature commonly seen in human immunodeficiency virus type 1 (HIV-1)-infected persons. However, the mechanism(s) responsible for this dysfunction is still poorly understood. Having recently shown that CD40L, the ligand for CD40, is inserted within emerging HIV-1 particles, we hypothesized that the contact between virus-anchored host CD40L and CD40 on the surface of B lymphocytes might result in the activation of this cell type. We report here that CD40L-bearing viruses, but not isogenic virions lacking host-derived CD40L, can induce immunoglobulin G and interleukin-6 production. Furthermore, such viral entities were found to induce B-cell homotypic adhesion. These effects were paralleled at the intracellular level by the nuclear translocation of the ubiquitous transcription factor NF-kappaB. The presence of host-derived CD40L within virions resulted in an increased virus attachment to B cells and a more-efficient B-cell-mediated transfer of HIV-1 to autologous CD4(+) T lymphocytes. All the above processes were independent of the virus-encoded envelope glycoproteins. Altogether, the data gathered from this series of investigations suggest that the incorporation of host-encoded CD40L in HIV-1 is likely to play a role in the B-cell abnormalities that are seen in infected individuals.

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article.

BACKGROUND The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. RESULTS Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3beta, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1alpha, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)alpha, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. CONCLUSION hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.