TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Arhgef2 regulates mitotic spindle orientation in hematopoietic stem cells and is essential for productive hematopoiesis.

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

ß-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus.

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.

Wnt activation as a therapeutic strategy in medulloblastoma.

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/ß-catenin signaling.

A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

GSK3 Deficiencies in Hematopoietic Stem Cells Initiate Pre-neoplastic State that Is Predictive of Clinical Outcomes of Human Acute Leukemia.

Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3α (GSK-3α) and GSK-3ß dependency leads to aggressive AML. Although GSK-3α deletion alone has no effect, GSK-3ß deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3ß and GSK-3α uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3ß provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3α- and GSK-3ß-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution.

Pyrvinium Targets CD133 in Human Glioblastoma Brain Tumor-Initiating Cells.

PURPOSE: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133(+) treatment-refractory population. EXPERIMENTAL DESIGN: Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved. RESULTS: We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/ß-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133(+) stem cells that drive glioblastoma relapse and mortality. CONCLUSIONS: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers.

Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/ß-Catenin Pathway.

Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3ß(-/-), and GSK-3α/ß double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3ß(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3ß, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of ß-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/ß-catenin axis in antiviral innate immunity.

Ectopic γ-catenin expression partially mimics the effects of stabilized ß-catenin on embryonic stem cell differentiation.

ß-catenin, an adherens junction component and key Wnt pathway effector, regulates numerous developmental processes and supports embryonic stem cell (ESC) pluripotency in specific contexts. The ß-catenin homologue γ-catenin (also known as Plakoglobin) is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. Here, we use ß-catenin((+/+)) and ß-catenin((-/-)) mouse embryonic stem cells (mESCs) to investigate the role of γ-catenin in Wnt signaling and mESC differentiation. Although γ-catenin protein is markedly stabilized upon inhibition or ablation of GSK-3 in wild-type (WT) mESCs, efficient silencing of its expression in these cells does not affect ß-catenin/TCF target gene activation after Wnt pathway stimulation. Nonetheless, knocking down γ-catenin expression in WT mESCs appears to promote their exit from pluripotency in short-term differentiation assays. In ß-catenin((-/-)) mESCs, GSK-3 inhibition does not detectably alter cytosolic γ-catenin levels and does not activate TCF target genes. Intriguingly, ß-catenin/TCF target genes are induced in ß-catenin((-/-)) mESCs overexpressing stabilized γ-catenin and the ability of these genes to be activated upon GSK-3 inhibition is partially restored when wild-type γ-catenin is overexpressed in these cells. This suggests that a critical threshold level of total catenin expression must be attained before there is sufficient signaling-competent γ-catenin available to respond to GSK-3 inhibition and to regulate target genes as a consequence. WT mESCs stably overexpressing γ-catenin exhibit robust Wnt pathway activation and display a block in tri-lineage differentiation that largely mimics that observed upon overexpression of ß-catenin. However, ß-catenin overexpression appears to be more effective than γ-catenin overexpression in sustaining the retention of markers of naïve pluripotency in cells that have been subjected to differentiation-inducing conditions. Collectively, our study reveals a function for γ-catenin in the regulation of mESC differentiation and has implications for human cancers in which γ-catenin is mutated and/or aberrantly expressed.

Medulloblastoma stem cells: modeling tumor heterogeneity.

Brain tumors represent the leading cause of childhood cancer mortality, with medulloblastoma (MB) being the most frequent malignant tumor. In this review we discuss the morphological and molecular heterogeneity of this malignant childhood brain tumor and how this key feature has implicated the presence of a MB stem cell. We focus on evidence from cerebellar development, histopathological and molecular subtypes of MB, the recent identification of brain tumor-initiating cells (BTICs, also referred to as MB stem cells), and the current limitations in studying the interplay between MB stem cells and tumor heterogeneity.

Medulloblastoma stem cells: where development and cancer cross pathways.

Brain tumors are the leading cause of childhood cancer mortality, with medulloblastoma (MB) representing the most frequent malignant tumor. The recent molecular classification of MB has reconceptualized the heterogeneity that exists within pathological subtypes by giving context to the role of key developmental signaling pathways in MB pathogenesis. The identification of cancer stem cell (CSC) populations, termed brain tumor-initiating cells (BTICs), in MB has provided novel cellular targets for the study of these aberrantly activated signaling pathways, namely, Sonic hedgehog (Shh) and Wingless (Wnt), along with the identification of novel BTIC self-renewal pathways. In this review, we discuss recent evidence for the presence of a MB stem cell that drives tumorigenesis in this malignant childhood tumor. We focus on evidence from cerebellar development, the recent identification of BTICs, the presence of activated developmental signaling pathways in MB, the role of epigenetic stem cell regulatory mechanisms, and how these developmental and epigenetic pathways may be targeted for novel therapeutic options.

Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism.

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.

Role of glycogen synthase kinase-3 in cell fate and epithelial-mesenchymal transitions.

Epithelial cells usually exist as sheets of immotile, tightly packed, well-coupled, polarized cells with distinct apical, basal and lateral surfaces. Remarkably, these cells can dramatically alter their morphology to become motile, fibroblast-like mesenchymal cells in a process of epithelial-mesenchymal transition (EMT). This process and the reverse, mesenchymal-epithelial transition, occur repeatedly during normal embryonic development. A phenomenon similar to physiological EMT occurs during the pathophysiological progression of some cancers. Tumours of epithelial origin, as they transform to malignancy, appear to exploit the innate plasticity of epithelial cells, with EMT conferring increased invasiveness and metastatic potential. Key to the maintenance of epithelial cell identity is the expression of E-cadherin, a protein that is required for tight intercellular adhesion along the lateral surfaces of adjacent epithelial cells. Loss of functional E-cadherin is a critical event in EMT. An important regulator of E-cadherin expression is the protein Snail, a zinc-finger transcriptional repressor. Snail contains several consensus sites for the kinase, glycogen synthase kinase-3 (GSK-3), and accumulating evidence indicates that it is a GSK-3 substrate. Phosphorylation of Snail by GSK-3 facilitates its proteasomal degradation. Conversely, inhibition of GSK-3 leads to Snail accumulation, E-cadherin downregulation, and development of EMT in cultured epithelial cells. Several signalling pathways implicated in the progression of EMT, including the Wnt and phosphoinositide 3-kinase pathways, use GSK-3 to mediate their responses. In these pathways, GSK-3's regulation of other transcriptional effectors like beta-catenin works in concert with changes in Snail to orchestrate the EMT process. This review focuses on the emerging role of GSK-3 as a modulator of cell fate and EMT in the contexts of development, in vitro cell culture and cancer.

GSK-3: tricks of the trade for a multi-tasking kinase.

Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine kinase found in all eukaryotes. The enzyme is a key regulator of numerous signalling pathways, including cellular responses to Wnt, receptor tyrosine kinases and G-protein-coupled receptors and is involved in a wide range of cellular processes, ranging from glycogen metabolism to cell cycle regulation and proliferation. GSK-3 is unusual in that it is normally active in cells and is primarily regulated through inhibition of its activity. Another peculiarity compared with other protein kinases is its preference for primed substrates, that is, substrates previously phosphorylated by another kinase. Several recent advances have improved our understanding of GSK-3 regulation in multiple pathways. These include the solution of the crystal structure of GSK-3, which has provided insight into GSK-3's penchant for primed substrates and the regulation of GSK-3 by serine phosphorylation, and findings related to the involvement of GSK-3 in the Wnt/beta-catenin and Hedgehog pathways. Finally, since increased GSK-3 activity may be linked to pathology in diseases such as Alzheimer's disease and non-insulin-dependent diabetes mellitus, several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture experiments, these new inhibitors hold promise as therapeutic agents.