Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis.

INTRODUCTION: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1ß in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. METHODS: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1ß. RESULTS: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1ß was measured in mice treated with rFc-µTP-L309C. CONCLUSION: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1ß production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Aged hind-limb clasping experimental autoimmune encephalomyelitis models aspects of the neurodegenerative process seen in multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is the most common model of multiple sclerosis (MS). This model has been instrumental in understanding the events that lead to the initiation of central nervous system (CNS) autoimmunity. Though EAE has been an effective screening tool for identifying novel therapies for relapsing-remitting MS, it has proven to be less successful in identifying therapies for progressive forms of this disease. Though axon injury occurs in EAE, it is rapid and acute, making it difficult to intervene for the purpose of evaluating neuroprotective therapies. Here, we describe a variant of spontaneous EAE in the 2D2 T cell receptor transgenic mouse (2D2+ mouse) that presents with hind-limb clasping upon tail suspension and is associated with T cell-mediated inflammation in the posterior spinal cord and spinal nerve roots. Due to the mild nature of clinical signs in this model, we were able to maintain cohorts of mice into middle age. Over 9 mo, these mice exhibited a relapsing-remitting course of hind-limb clasping with the development of progressive motor deficits. Using a combined approach of ex vivo magnetic resonance (MR) imaging and histopathological analysis, we observed neurological progression to associate with spinal cord atrophy, synapse degradation, and neuron loss in the gray matter, as well as ongoing axon injury in the white matter of the spinal cord. These findings suggest that mild EAE coupled with natural aging may be a solution to better modeling the neurodegenerative processes seen in MS.

Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia.

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

ß-Catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

Tibial pseudarthrosis causes substantial morbidity in patients with neurofibromatosis type 1 (NF1). We studied tibial pseudarthrosis tissue from patients with NF1 and found elevated levels of ß-catenin compared to unaffected bone. To elucidate the role of ß-catenin in fracture healing, we used a surgically induced tibial fracture model in conditional knockout (KO) Nfl (Nf1(flox/flox)) mice. When treated with a Cre-expressing adenovirus (Ad-Cre), there was a localized knockdown of Nf1 in the healing fracture and a subsequent development of a fibrous pseudarthrosis. Consistent with human data, elevated ß-catenin levels were found in the murine fracture sites. The increased fibrous tissue at the fracture site was rescued by local treatment with a Wingless-type MMTV integration site (Wnt) antagonist, Dickkopf-1 (Dkk1). The murine pseudarthrosis phenotype was also rescued by conditional ß-catenin gene inactivation. The number of colony-forming unit osteoblasts (CFU-Os), a surrogate marker of undifferentiated mesenchymal cells able to differentiate to osteoblasts, correlated with the capacity to form bone at the fracture site. Our findings indicate that the protein level of ß-catenin must be precisely regulated for normal osteoblast differentiation. An up-regulation of ß-catenin in NF1 causes a shift away from osteoblastic differentiation resulting in a pseudarthrosis in vivo These results support the notion that pharmacological modulation of ß-catenin can be used to treat pseudarthrosis in patients with NF1.-Ghadakzadeh, S., Kannu, P., Whetstone, H., Howard A., Alman, B. A. ß-catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund's adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into naïve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS). Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides feedback on the extent of overall CNS injury in live mice.

Fibronectin and beta-catenin act in a regulatory loop in dermal fibroblasts to modulate cutaneous healing.

ß-Catenin is an important regulator of dermal fibroblasts during cutaneous wound repair. However, the factors that modulate ß-catenin activity in this process are not completely understood. We investigated the role of the extracellular matrix in regulating ß-catenin and found an increase in ß-catenin-mediated Tcf-dependent transcriptional activity in fibroblasts exposed to various extracellular matrix components. This occurs through an integrin-mediated GSK3ß-dependent pathway. The physiologic role of this mechanism was demonstrated during wound repair in extra domain A-fibronectin-deficient mice, which exhibited decreased ß-catenin-mediated signaling during the proliferative phase of healing. Extra domain A-fibronectin-deficient mice have wounds that fail at a lower tensile strength and contain fewer fibroblasts compared with wild type mice. This phenotype was rescued by genetic or pharmacologic activation of ß-catenin signaling. Because fibronectin is a transcriptional target of ß-catenin, this suggests the existence of a feedback loop between these two molecules that regulates dermal fibroblast cell behavior during wound repair.

Pax7 expressing cells contribute to dermal wound repair, regulating scar size through a ß-catenin mediated process.

During skin wound healing, fibroblast-like cells reconstitute the dermal compartment of the repaired skin filling the wound gap. A subset of these cells are transcriptionally active for ß-catenin/T-cell factor (TCF) signaling during the proliferative phase of the repair process, and ß-catenin levels control the size of the scar that ultimately forms by regulating the number of dermal fibroblasts. Here, we performed cell lineage studies to reveal a source of the dermal cells in which ß-catenin signaling is activated during wound repair. Using a reporter mouse, we found that cells in the early wound in which TCF-dependent transcription is activated express genes involved in muscle development. Using mice in which cells express Pax7 (muscle progenitors) or Mck (differentiated myocytes) are permanently labeled, we showed that one quarter of dermal cells in the healing wound are Pax7 expressing progeny, but none are Mck progeny. Removing one allele of ß-catenin in Pax7 expressing progeny resulted in a significantly smaller scar size with fewer Pax7 expressing progeny cell contributing to wound repair. During wound healing, ß-catenin activation causes muscle satellite cells to adopt a fibrotic phenotype and this is a source of dermal cells in the repair process.

Peroxisome Proliferator-Activated Receptor-δ Deficiency in Microglia Results in Exacerbated Axonal Injury and Tissue Loss in Experimental Autoimmune Encephalomyelitis.

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear receptor that functions to maintain metabolic homeostasis, regulate cell growth, and limit the development of excessive inflammation during immune responses. Previously, we reported that PPAR-δ-deficient mice develop a more severe clinical course of experimental autoimmune encephalomyelitis (EAE); however, it was difficult to delineate the role that microglia played in this disease phenotype since PPAR-δ-deficient mice exhibited a number of immune defects that enhanced CNS inflammation upstream of microglia activation. Here, we specifically investigated the role of PPAR-δ in microglia during EAE by using mice where excision of a floxed Ppard allele was driven by expression of a tamoxifen (TAM)-inducible CX3C chemokine receptor 1 promoter-Cre recombinase transgene (Cx3cr1CreERT2: Ppardfl/fl). We observed that by 30 days of TAM treatment, Cx3cr1CreERT2: Ppardfl/fl mice exhibited Cre-mediated deletion primarily in microglia and this was accompanied by efficient knockdown of Ppard expression in these cells. Upon induction of EAE, TAM-treated Cx3cr1CreERT2: Ppardfl/fl mice presented with an exacerbated course of disease compared to TAM-treated Ppardfl/fl controls. Histopathological and magnetic resonance (MR) studies on the spinal cord and brains of EAE mice revealed increased Iba-1 immunoreactivity, axonal injury and CNS tissue loss in the TAM-treated Cx3cr1CreERT2: Ppardfl/fl group compared to controls. In early EAE, a time when clinical scores and the infiltration of CD45+ leukocytes was equivalent between Cx3cr1CreERT2: Ppardfl/fl and Ppardfl/fl mice, Ppard-deficient microglia exhibited a more reactive phenotype as evidenced by a shorter maximum process length and lower expression of genes associated with a homeostatic microglia gene signature. In addition, Ppard-deficient microglia exhibited increased expression of genes associated with reactive oxygen species generation, phagocytosis and lipid clearance, M2-activation, and promotion of inflammation. Our results therefore suggest that PPAR-δ has an important role in microglia in limiting bystander tissue damage during neuroinflammation.

Single-cell chromatin accessibility profiling of glioblastoma identifies an invasive cancer stem cell population associated with lower survival.

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

PRMT5 inhibition disrupts splicing and stemness in glioblastoma.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.

Medulloblastoma Arises from the Persistence of a Rare and Transient Sox2+ Granule Neuron Precursor.

Medulloblastoma (MB) is a neoplasm linked to dysregulated cerebellar development. Previously, we demonstrated that the Sonic Hedgehog (SHH) subgroup grows hierarchically, with Sox2+ cells at the apex of tumor progression and relapse. To test whether this mechanism is rooted in a normal developmental process, we studied the role of Sox2 in cerebellar development. We find that the external germinal layer (EGL) is derived from embryonic Sox2+ precursors and that the EGL maintains a rare fraction of Sox2+ cells during the first postnatal week. Through lineage tracing and single-cell analysis, we demonstrate that these Sox2+ cells are within the Atoh1+ lineage, contribute extensively to adult granule neurons, and resemble Sox2+ tumor cells. Critically, constitutive activation of the SHH pathway leads to their aberrant persistence in the EGL and rapid tumor onset. We propose that failure to eliminate this rare but potent developmental population is the tumor initiation mechanism in SHH-subgroup MB.

Side population cells isolated from mesenchymal neoplasms have tumor initiating potential.

Although many cancers are maintained by tumor-initiating cells, this has not been shown for mesenchymal tumors, in part due to the lack of unique surface markers that identify mesenchymal progenitors. An alternative technique to isolate stem-like cells is to isolate side population (SP) cells based on efflux of Hoechst 33342 dye. We examined 29 mesenchymal tumors ranging from benign to high-grade sarcomas and identified SP cells in all but six samples. There was a positive correlation between the percentage of SP cells and the grade of the tumor. SP cells preferentially formed tumors when grafted into immunodeficient mice, and only cells from tumors that developed from the SP cells had the ability to initiate tumor formation upon serial transplantation. Although SP cells are able to efflux rhodamine dye in addition to Hoechst 33342, we found that the ability to efflux rhodamine dye did not identify a population of cells enriched for tumor-initiating capacity. Here, we identify a subpopulation of cells within a broad range of benign and malignant mesenchymal tumors with tumor-initiating capacity. In addition, our data suggest that the proportion of SP cells could be used as a prognostic factor and that therapeutically targeting this subpopulation of cells could be used to improve patient outcome.

Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of bone repair in mice.

The pace of repair declines with age and, while exposure to a young circulation can rejuvenate fracture repair, the cell types and factors responsible for rejuvenation are unknown. Here we report that young macrophage cells produce factors that promote osteoblast differentiation of old bone marrow stromal cells. Heterochronic parabiosis exploiting young mice in which macrophages can be depleted and fractionated bone marrow transplantation experiments show that young macrophages rejuvenate fracture repair, and old macrophage cells slow healing in young mice. Proteomic analysis of the secretomes identify differential proteins secreted between old and young macrophages, such as low-density lipoprotein receptor-related protein 1 (Lrp1). Lrp1 is produced by young cells, and depleting Lrp1 abrogates the ability to rejuvenate fracture repair, while treating old mice with recombinant Lrp1 improves fracture healing. Macrophages and proteins they secrete orchestrate the fracture repair process, and young cells produce proteins that rejuvenate fracture repair in mice.

A Feedforward Mechanism Mediated by Mechanosensitive Ion Channel PIEZO1 and Tissue Mechanics Promotes Glioma Aggression.

Alteration of tissue mechanical properties is a physical hallmark of solid tumors including gliomas. How tumor cells sense and regulate tissue mechanics is largely unknown. Here, we show that mechanosensitive ion channel Piezo regulates mitosis and tissue stiffness of Drosophila gliomas, but not non-transformed brains. PIEZO1 is overexpressed in aggressive human gliomas and its expression inversely correlates with patient survival. Deleting PIEZO1 suppresses the growth of glioblastoma stem cells, inhibits tumor development, and prolongs mouse survival. Focal mechanical force activates prominent PIEZO1-dependent currents from glioma cell processes, but not soma. PIEZO1 localizes at focal adhesions to activate integrin-FAK signaling, regulate extracellular matrix, and reinforce tissue stiffening. In turn, a stiffer mechanical microenvironment elevates PIEZO1 expression to promote glioma aggression. Therefore, glioma cells are mechanosensory in a PIEZO1-dependent manner, and targeting PIEZO1 represents a strategy to break the reciprocal, disease-aggravating feedforward circuit between tumor cell mechanotransduction and the aberrant tissue mechanics. VIDEO ABSTRACT.

Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing.

BACKGROUND: Delayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The beta-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of beta-catenin signaling in bone repair. METHODS AND FINDINGS: Western blot analysis showed significant up-regulation of beta-catenin during the bone healing process. Using a beta-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that beta-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/beta-catenin pathway) inhibited beta-catenin signaling and the healing process, suggesting that WNT ligands regulate beta-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized beta-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific beta-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of beta-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated beta-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture. CONCLUSIONS: These results demonstrate that beta-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of beta-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, beta-catenin positively regulates osteoblasts. This is a different function for beta-catenin than has previously been reported during development. Activation of beta-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage.

Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing.

After cutaneous injury, a variety of cell types are activated to reconstitute the epithelial and dermal components of the skin. beta-Catenin plays disparate roles in keratinocytes and fibroblasts, inhibiting keratinocyte migration and activating fibroblast proliferation, suggesting that beta-catenin could either inhibit or enhance the healing process. How beta-catenin functions in concert with other signaling pathways important in the healing process is unknown. Wound size was examined in mice expressing conditional null or conditional stabilized alleles of beta-catenin, regulated by an adenovirus expressing cre-recombinase. The size of the wounds in the mice correlated with the protein level of beta-catenin. Using mice expressing these conditional alleles, we found that the wound phenotype imparted by Smad3 deficiency and by the injection of TGFbeta before wounding is mediated in part by beta-catenin. TGFbeta was not able to regulate proliferation in beta-catenin null fibroblasts, whereas keratinocyte proliferation rate was independent of beta-catenin. When mice are treated with lithium, beta-catenin-mediated signaling was activated in cutaneous wounds, which healed with a larger size. These results demonstrate a crucial role for beta-catenin in regulating cutaneous wound size. Furthermore, these data implicate mesenchymal cells as playing a critical role regulating wound size.

ASCL1 Reorganizes Chromatin to Direct Neuronal Fate and Suppress Tumorigenicity of Glioblastoma Stem Cells.

Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1hi GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1lo GSCs do not. Increasing ASCL1 levels in ASCL1lo GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.

Regulation of Cholesterol Homeostasis by Hedgehog Signaling in Osteoarthritic Cartilage.

OBJECTIVE: With no effective therapies to attenuate cartilage degeneration in osteoarthritis (OA), the result is pain and disability. Activation of hedgehog (HH) signaling causes changes related to the progression of OA, with higher levels of Gli-mediated transcriptional activation associated with increased disease severity. To elucidate the mechanism through which this occurs, this study sought to identify genes regulated by HH signaling in human OA chondrocytes. METHODS: Using human OA cartilage samples, microarray analyses were performed to detect changes in gene expression when the HH pathway was modulated. Results were analyzed for differentially expressed genes, grouped into functional networks, and validated in independent samples. To investigate the effects of chondrocyte-specific sterol accumulation, we generated mice lacking Insig1 and Insig2, which are major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements. RESULTS: HH signaling was found to regulate genes that govern cholesterol homeostasis, and this led to alterations in cholesterol accumulation in chondrocytes. A higher level of Gli-mediated transcription resulted in accumulation of intracellular cholesterol. In genetically modified mice, chondrocyte-specific cholesterol accumulation was associated with an OA phenotype. Reducing cholesterol accumulation attenuated the severity of OA in mice in vivo and decreased the expression of proteases in human OA cartilage in vitro. CONCLUSION: HH signaling regulates cholesterol homeostasis in chondrocytes, and intracellular cholesterol accumulation contributes to the severity of OA. Our findings have therapeutic implications, since reduction of HH signaling reversed cholesterol accumulation and statin treatment attenuated cartilage degeneration.