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BACKGROUND Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However,resistance inevitably emerges against these therapies,particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels,p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer,neurosphere,and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics,100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS Daoy,ONS76,UW228,and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth,induced apoptosis,and sensitized cells to SHH agents. Notably,RSK expression is correlated with SHH patients. In mice,BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly,BI-D1870 crosses the BBB,acting as a scaffold for development of more long-lived RSK inhibitors. View Publication

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD,and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs,using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected,cell lines harboring the PSEN2 N141I mutation displayed an increase in the A$\beta$42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit,restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased A$\beta$42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in A$\beta$42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology. View Publication

Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study,we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a 20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures,glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions. View Publication

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs),cancers occurring increasingly in children to young adults,is poorly understood. We present a transplantable tumour line,maintained in immune-compromised mice,and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays,spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model,with parallel studies on 27 primary hFL-HCC tumours,provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple,normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells--newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. View Publication

New therapeutic strategies are needed in the treatment of asthma besides vaccines and pharmacotherapies. For the development of novel therapies,the use of mesenchymal stem cells (MSCs) is a promising approach in regenerative medicine. Delivery of compact bone (CB) derived MSCs to the injured lungs is an alternative treatment strategy for chronic asthma. In this study,we aimed to isolate highly enriched population of MSCs from mouse CB with regenerative capacity,and to investigate the impact of these cells in airway remodeling and inflammation in experimental ovalbumin-induced mouse model of chronic asthma. mCB-MSCs were isolated,characterized,labeled with GFP and then transferred into mice with chronic asthma developed by ovalbumin (OVA) provocation. Histopathological changes including basement membrane,epithelium,subepithelial smooth thickness and goblet cell hyperplasia,and MSCs migration to lung tissues were evaluated. These histopathological alterations were increased in ovalbumin-treated mice compared to PBS group (P<0.001). Intravenous administration of mCB-MSC significantly reduced these histopathological changes in both distal and proximal airways (P<0.001). We showed that GFP-labeled MSCs were located in the lungs of OVA group 2weeks after intravenous induction. mCB-MSCs also significantly promoted Treg response in ovalbumin-treated mice (OVA+MSC group) (P<0.037). Our studies revealed that mCB-MSCs migrated to lung tissue and suppressed histopathological changes in murine model of asthma. The results reported here provided evidence that mCB-MSCs may be an alternative strategy for the treatment of remodeling and inflammation associated with chronic asthma. View Publication

Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1+ neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1+ neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis,we found increased expression of CIRP and higher frequencies and numbers of ICAM-1+ neutrophils in the lungs. Conversely,the CIRP-/- mice showed significant inhibition in the frequencies and numbers of ICAM-1+ neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1+ phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1+ neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared,respectively,with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1+ BMDN were significantly higher than those in ICAM-1- BMDN. Following sepsis the ICAM-1+ neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1- neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP-/- mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus,CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1+ neutrophils in the lungs during sepsis. View Publication

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery,radiation and chemotherapy,posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma,identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet,oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent,highly disparate genomic structural variants,restricted to groups 3 and 4,resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes,GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements,including super-enhancers,instigating oncogenic activity. Our results,supported by evidence from mouse models,identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer. View Publication

The embryonic mouse lung is a widely used substitute for human lung development. For example,attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology,gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover,we identify mouse-human differences,including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated. View Publication

Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics,the immune system,and the environment. Type 1 interferons (T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However,the capacity of IFNα/β to modulate human activated autoreactive CD8+ T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here,we engineer human β-cell-specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4,resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet. View Publication

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus,developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein,we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time,we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally,CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets,supporting the role of FA metabolism for Treg function. Furthermore,we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively,our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor,thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo. View Publication

Glioblastoma multiforme (GBM),the most common and lethal tumor of the adult brain,generally shows chemo- and radioresistance. MicroRNAs (miRs) regulate physiological processes,such as resistance of GBM cells to temozolomide (TMZ). Although miRs are attractive targets for cancer therapeutics,the effectiveness of this approach requires targeted delivery. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers,including GBM. We report on an increase in miR-9 in TMZ-resistant GBM cells. miR-9 was involved in the expression of the drug efflux transporter,P-glycoprotein. To block miR-9,methods were developed with Cy5-tagged anti-miR-9. Dye-transfer studies indicated intracellular communication between GBM cells and MSCs. This occurred by gap junctional intercellular communication and the release of microvesicles. In both cases,anti-miR-9 was transferred from MSCs to GBM cells. However,the major form of transfer occurred with the microvesicles. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to TMZ,as shown by increased cell death and caspase activity. The data showed a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse the chemoresistance of GBM cells.Molecular Therapy-Nucleic Acids (2013) 2,e126; doi:10.1038/mtna.2013.60; published online 1 October 2013. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However,clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy,in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression,we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells,we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs,modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type,expressing pro-angiogenic factors. In contrast,phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data. View Publication