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2.
Genome Biol ; 21(1): 83, 2020 03 31.
Article in English | MEDLINE | ID: mdl-32234056

ABSTRACT

BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. RESULTS: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy. CONCLUSIONS: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.


Subject(s)
Brain Neoplasms/therapy , CRISPR-Cas Systems , Glioblastoma/therapy , RNA, Long Noncoding/antagonists & inhibitors , Adult , Astrocytes , Brain , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/radiotherapy , Cell Line, Tumor , Combined Modality Therapy , Glioblastoma/genetics , Glioblastoma/pathology , Glioblastoma/radiotherapy , Humans , Oligonucleotides, Antisense , Organoids , Radiation Tolerance
3.
Cell Stem Cell ; 26(1): 48-63.e6, 2020 01 02.
Article in English | MEDLINE | ID: mdl-31901251

ABSTRACT

Glioblastoma is a devastating form of brain cancer. To identify aspects of tumor heterogeneity that may illuminate drivers of tumor invasion, we created a glioblastoma tumor cell atlas with single-cell transcriptomics of cancer cells mapped onto a reference framework of the developing and adult human brain. We find that multiple GSC subtypes exist within a single tumor. Within these GSCs, we identify an invasive cell population similar to outer radial glia (oRG), a fetal cell type that expands the stem cell niche in normal human cortex. Using live time-lapse imaging of primary resected tumors, we discover that tumor-derived oRG-like cells undergo characteristic mitotic somal translocation behavior previously only observed in human development, suggesting a reactivation of developmental programs. In addition, we show that PTPRZ1 mediates both mitotic somal translocation and glioblastoma tumor invasion. These data suggest that the presence of heterogeneous GSCs may underlie glioblastoma's rapid progression and invasion.


Subject(s)
Brain Neoplasms , Glioblastoma , Adult , Brain Neoplasms/genetics , Cell Line, Tumor , Ependymoglial Cells , Glioblastoma/genetics , Humans , Neoplastic Stem Cells , Receptor-Like Protein Tyrosine Phosphatases, Class 5
4.
Genome Biol ; 18(1): 234, 2017 Dec 20.
Article in English | MEDLINE | ID: mdl-29262845

ABSTRACT

BACKGROUND: Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. RESULTS: We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. CONCLUSIONS: We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.


Subject(s)
Glioma/genetics , Glioma/pathology , Macrophage Activation/genetics , Macrophages/metabolism , Macrophages/pathology , Tumor Microenvironment/genetics , Animals , Computational Biology/methods , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Ontology , Glioma/immunology , Glioma/therapy , High-Throughput Nucleotide Sequencing , Humans , Immunotherapy/methods , Macrophage Activation/immunology , Mice , Prognosis , Single-Cell Analysis , Survival Analysis , Transcriptome , Tumor Microenvironment/immunology
5.
Science ; 355(6320)2017 01 06.
Article in English | MEDLINE | ID: mdl-27980086

ABSTRACT

The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Genome, Human , RNA, Long Noncoding/genetics , Cell Growth Processes/genetics , Cell Line , Gene Knockdown Techniques , Gene Regulatory Networks , Genetic Loci , Genetic Testing , Humans , Induced Pluripotent Stem Cells , Machine Learning , RNA Interference , Transcription, Genetic , Transcriptome
6.
Mol Syst Biol ; 12(11): 889, 2016 Nov 25.
Article in English | MEDLINE | ID: mdl-27888226

ABSTRACT

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.


Subject(s)
ErbB Receptors/genetics , Gene Expression Profiling/methods , Receptors, Platelet-Derived Growth Factor/genetics , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Brain Neoplasms , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Genetic Heterogeneity , Glioblastoma , Humans , Mutation
7.
Cancer Res ; 73(20): 6323-33, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23943798

ABSTRACT

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.


Subject(s)
Glioblastoma/genetics , Hedgehog Proteins/genetics , Medulloblastoma/genetics , Transcription Factors/genetics , p300-CBP Transcription Factors/genetics , Animals , Cell Growth Processes/physiology , Cell Line, Tumor , Glioblastoma/enzymology , Glioblastoma/metabolism , Glioblastoma/pathology , Hedgehog Proteins/biosynthesis , Hedgehog Proteins/metabolism , Humans , Medulloblastoma/enzymology , Medulloblastoma/metabolism , Medulloblastoma/pathology , Mice , NIH 3T3 Cells , Promoter Regions, Genetic , RNA, Small Interfering , Signal Transduction , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Zinc Finger Protein GLI1 , p300-CBP Transcription Factors/metabolism
8.
PLoS Genet ; 9(4): e1003461, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23637629

ABSTRACT

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications.


Subject(s)
Jumonji Domain-Containing Histone Demethylases , Repressor Proteins , Animals , Embryonic Development , Genes, Developmental , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Mice , Nuclear Proteins/genetics , Polycomb-Group Proteins/genetics , Repressor Proteins/genetics
9.
EMBO J ; 30(22): 4586-600, 2011 Nov 16.
Article in English | MEDLINE | ID: mdl-22020125

ABSTRACT

H3K4 methylation is associated with active transcription and in combination with H3K27me3 thought to keep genes regulating development in a poised state. The contribution of enzymes regulating trimethylation of lysine 4 at histone 3 (H3K4me3) levels to embryonic stem cell (ESC) self-renewal and differentiation is just starting to emerge. Here, we show that the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) is dispensable for ESC self-renewal, but essential for ESC differentiation along the neural lineage. By genome-wide location analysis, we demonstrate that Jarid1b localizes predominantly to transcription start sites of genes encoding developmental regulators, of which more than half are also bound by Polycomb group proteins. Virtually all Jarid1b target genes are associated with H3K4me3 and depletion of Jarid1b in ESCs leads to a global increase of H3K4me3 levels. During neural differentiation, Jarid1b-depleted ESCs fail to efficiently silence lineage-inappropriate genes, specifically stem and germ cell genes. Our results delineate an essential role for Jarid1b-mediated transcriptional control during ESC differentiation.


Subject(s)
Embryonic Stem Cells/physiology , Histones/metabolism , Neurogenesis , Neurons/physiology , Transcription, Genetic , Animals , Antibodies, Monoclonal , Cell Line , Central Nervous System/embryology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/cytology , Gene Expression Profiling , Gene Knockout Techniques/methods , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/immunology , Jumonji Domain-Containing Histone Demethylases/metabolism , Methylation , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/cytology , Polycomb-Group Proteins , Promoter Regions, Genetic , RNA Interference , RNA, Small Interfering , Repressor Proteins/metabolism
10.
Nucleic Acids Res ; 38(15): 4958-69, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20385584

ABSTRACT

Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.


Subject(s)
Gene Expression Regulation , Histones/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Acetylation , Animals , Embryonic Stem Cells/metabolism , Gene Knockout Techniques , Histone Acetyltransferases/metabolism , Histones/chemistry , Lysine/metabolism , Methylation , Mice , Polycomb Repressive Complex 2 , Polycomb-Group Proteins , Repressor Proteins/genetics
11.
Biochem Pharmacol ; 76(11): 1515-21, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18718449

ABSTRACT

Itch, an E3 protein ubiquitin ligase (E3), which belongs to the homologous to E6-AP carboxy terminus (HECT)-type subfamily, catalyzes its own ubiquitylation. The precise nature of Itch-mediated self-modification and its biological outcome are not completely understood. Here, we show that Itch auto-ubiquitylation is an intermolecular reaction generating Lys63-linkages, rather than the Lys48-linked polyubiquitin chains that target proteins for proteasomal degradation. As a result, Itch is a relatively high stable protein, whose levels are not significantly affected by treatment by either proteasome or lysosome inhibitors. Furthermore, we demonstrate that the decay rate of a catalytic inactive Itch mutant, which is devoided of self-ubiquitylating activity, is barely indistinguishable from the one of the wild-type protein. These data definitely establish a nondegradative role for Lys63-linked Itch self-ubiquitylation.


Subject(s)
Lysine/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Half-Life , Humans , Immunoprecipitation , Mice , Repressor Proteins/chemistry , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitination
12.
Proc Natl Acad Sci U S A ; 104(27): 11280-5, 2007 Jul 03.
Article in English | MEDLINE | ID: mdl-17592138

ABSTRACT

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.


Subject(s)
Carrier Proteins/metabolism , Carrier Proteins/physiology , Enzyme Inhibitors/metabolism , Membrane Proteins/metabolism , Tumor Suppressor Proteins/physiology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/physiology , Animals , Apoptosis/physiology , Cells, Cultured , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Endosomal Sorting Complexes Required for Transport , Enzyme Inhibitors/chemistry , Intercellular Signaling Peptides and Proteins , Mice , Mice, Knockout , Nedd4 Ubiquitin Protein Ligases , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Substrate Specificity/genetics , Substrate Specificity/physiology , Tumor Protein p73 , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
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