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1.
Nature ; 623(7986): 366-374, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37914930

ABSTRACT

The role of the nervous system in the regulation of cancer is increasingly appreciated. In gliomas, neuronal activity drives tumour progression through paracrine signalling factors such as neuroligin-3 and brain-derived neurotrophic factor1-3 (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors4,5. The consequent glioma cell membrane depolarization drives tumour proliferation4,6. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity7,8 and strength9-15. Here we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signalling through the receptor tropomyosin-related kinase B16 (TrkB) to CAMKII, BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. Linking plasticity of glioma synaptic strength to tumour growth, graded optogenetic control of glioma membrane potential demonstrates that greater depolarizing current amplitude promotes increased glioma proliferation. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity17-22 that contributes to memory and learning in the healthy brain23-26. BDNF-TrkB signalling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of glioma TrkB expression robustly inhibits tumour progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of paediatric glioblastoma and diffuse intrinsic pontine glioma. Together, these findings indicate that BDNF-TrkB signalling promotes malignant synaptic plasticity and augments tumour progression.


Subject(s)
Adaptation, Physiological , Glioma , Neuronal Plasticity , Synapses , Animals , Child , Humans , Brain-Derived Neurotrophic Factor/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Proliferation , Disease Progression , Glioma/metabolism , Glioma/pathology , Glutamic Acid/metabolism , Neurons/cytology , Neurons/metabolism , Receptor, trkB/genetics , Receptor, trkB/metabolism , Receptors, AMPA/metabolism , Signal Transduction , Synapses/metabolism , Tumor Microenvironment , Optogenetics
2.
Elife ; 112022 03 21.
Article in English | MEDLINE | ID: mdl-35311648

ABSTRACT

Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. Here, we report TRACC (Transcriptional Readout Activated by Cell-cell Contacts), a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. TRACC is derived from our previous protein-protein interaction recorders, SPARK (Kim et al., 2017) and SPARK2 (Kim et al., 2019), reported in this journal. TRACC incorporates light gating via the light-oxygen-voltage-sensing (LOV) domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC detects cell-cell contacts with high specificity and sensitivity in mammalian cell culture and that it can be used to interrogate interactions between neurons and glioma, a form of brain cancer.


Subject(s)
Light , Signal Transduction , Animals , Mammals
3.
Nature ; 603(7903): 934-941, 2022 03.
Article in English | MEDLINE | ID: mdl-35130560

ABSTRACT

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.


Subject(s)
Astrocytoma , Brain Stem Neoplasms , Gangliosides , Glioma , Histones , Immunotherapy, Adoptive , Mutation , Receptors, Chimeric Antigen , Astrocytoma/genetics , Astrocytoma/immunology , Astrocytoma/pathology , Astrocytoma/therapy , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/immunology , Brain Stem Neoplasms/pathology , Brain Stem Neoplasms/therapy , Child , Gangliosides/immunology , Gene Expression Profiling , Glioma/genetics , Glioma/immunology , Glioma/pathology , Glioma/therapy , Histones/genetics , Humans , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/immunology , Spinal Cord Neoplasms/genetics , Spinal Cord Neoplasms/immunology , Spinal Cord Neoplasms/pathology , Spinal Cord Neoplasms/therapy
4.
Cell ; 181(7): 1445-1449, 2020 06 25.
Article in English | MEDLINE | ID: mdl-32533917

ABSTRACT

The COVID19 crisis has magnified the issues plaguing academic science, but it has also provided the scientific establishment with an unprecedented opportunity to reset. Shoring up the foundation of academic science will require a concerted effort between funding agencies, universities, and the public to rethink how we support scientists, with a special emphasis on early career researchers.


Subject(s)
Career Mobility , Research Personnel/trends , Research/trends , Achievement , Biomedical Research , Humans , Research Personnel/education , Science/education , Science/trends , Universities
5.
Sci Transl Med ; 11(519)2019 11 20.
Article in English | MEDLINE | ID: mdl-31748226

ABSTRACT

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.


Subject(s)
Brain Neoplasms/drug therapy , Drug Evaluation, Preclinical , Glioma/drug therapy , High-Throughput Screening Assays/methods , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Stem Neoplasms/drug therapy , Cell Death , Cell Line, Tumor , Drug Synergism , Female , Glioma/genetics , Glioma/metabolism , Humans , Lactones/pharmacology , Lactones/therapeutic use , Male , Metabolomics , Mice , Panobinostat/pharmacology , Panobinostat/therapeutic use , Pyrroles/pharmacology , Pyrroles/therapeutic use , Reproducibility of Results , Sequence Analysis, RNA , Transcription, Genetic/drug effects , Xenograft Model Antitumor Assays
6.
Cancer Cell ; 36(5): 528-544.e10, 2019 11 11.
Article in English | MEDLINE | ID: mdl-31631026

ABSTRACT

H3K27M mutations resulting in epigenetic dysfunction are frequently observed in diffuse intrinsic pontine glioma (DIPGs), an incurable pediatric cancer. We conduct a CRISPR screen revealing that knockout of KDM1A encoding lysine-specific demethylase 1 (LSD1) sensitizes DIPG cells to histone deacetylase (HDAC) inhibitors. Consistently, Corin, a bifunctional inhibitor of HDACs and LSD1, potently inhibits DIPG growth in vitro and in xenografts. Mechanistically, Corin increases H3K27me3 levels suppressed by H3K27M histones, and simultaneously increases HDAC-targeted H3K27ac and LSD1-targeted H3K4me1 at differentiation-associated genes. Corin treatment induces cell death, cell-cycle arrest, and a cellular differentiation phenotype and drives transcriptional changes correlating with increased survival time in DIPG patients. These data suggest a strategy for treating DIPG by simultaneously inhibiting LSD1 and HDACs.


Subject(s)
Antineoplastic Agents/pharmacology , Brain Stem Neoplasms/drug therapy , Glioma/drug therapy , Histone Deacetylase Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/mortality , Brain Stem Neoplasms/pathology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Chromatin/metabolism , DNA Methylation/drug effects , DNA Methylation/genetics , Epigenesis, Genetic/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Glioma/genetics , Glioma/mortality , Glioma/pathology , Histone Code/drug effects , Histone Deacetylase Inhibitors/therapeutic use , Histone Deacetylases/metabolism , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histones/metabolism , Humans , Mice , Mutation , Pons/pathology , RNA-Seq , Xenograft Model Antitumor Assays
7.
Mol Cell ; 76(6): 965-980.e12, 2019 12 19.
Article in English | MEDLINE | ID: mdl-31588023

ABSTRACT

Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.


Subject(s)
Diffuse Intrinsic Pontine Glioma/genetics , Histones/genetics , Brain/pathology , Brain Neoplasms/genetics , Cellular Reprogramming/genetics , Diffuse Intrinsic Pontine Glioma/metabolism , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic/genetics , Epigenomics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Glioma/genetics , Glioma/metabolism , Humans , Lysine/genetics , Mutation/genetics , Pons/metabolism , Signal Transduction , Transcriptome/physiology
8.
Nature ; 573(7775): 539-545, 2019 09.
Article in English | MEDLINE | ID: mdl-31534222

ABSTRACT

High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth, but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron-glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections, forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation, whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth, human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together, these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression.


Subject(s)
Brain/physiopathology , Electrical Synapses/pathology , Electrophysiological Phenomena , Glioma/physiopathology , Animals , Brain/cytology , Cell Membrane/pathology , Cell Proliferation , Gap Junctions/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Mice , Mice, Inbred NOD , Neurons/pathology , Optogenetics , Potassium/metabolism , Synaptic Transmission , Tumor Cells, Cultured
9.
Nat Commun ; 9(1): 3588, 2018 09 04.
Article in English | MEDLINE | ID: mdl-30181541

ABSTRACT

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by extensive intratumoral heterogeneity. To investigate the underlying biology, we conducted single-cell RNA-sequencing (scRNA-seq) of >1500 cells from six primary TNBC. Here, we show that intercellular heterogeneity of gene expression programs within each tumor is variable and largely correlates with clonality of inferred genomic copy number changes, suggesting that genotype drives the gene expression phenotype of individual subpopulations. Clustering of gene expression profiles identified distinct subgroups of malignant cells shared by multiple tumors, including a single subpopulation associated with multiple signatures of treatment resistance and metastasis, and characterized functionally by activation of glycosphingolipid metabolism and associated innate immunity pathways. A novel signature defining this subpopulation predicts long-term outcomes for TNBC patients in a large cohort. Collectively, this analysis reveals the functional heterogeneity and its association with genomic evolution in TNBC, and uncovers unanticipated biological principles dictating poor outcomes in this disease.


Subject(s)
Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Adult , Female , Gene Expression Regulation, Neoplastic , Humans , Lymph Nodes/pathology , Middle Aged , Prognosis , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Transcriptome , Triple Negative Breast Neoplasms/immunology , Triple Negative Breast Neoplasms/mortality
10.
Neuro Oncol ; 20(10): 1292-1299, 2018 09 03.
Article in English | MEDLINE | ID: mdl-29788372

ABSTRACT

Perineuronal satellitosis, the microanatomical clustering of glioma cells around neurons in the tumor microenvironment, has been recognized as a histopathological hallmark of high-grade gliomas since the seminal observations of Scherer in the 1930s. In this review, we explore the emerging understanding that neuron‒glioma cell interactions regulate malignancy and that neuronal activity is a critical determinant of glioma growth and progression. Elucidation of the interplay between normal and malignant neural circuitry is critical to realizing the promise of effective therapies for these seemingly intractable diseases. Here, we review current knowledge regarding the role of neuronal activity in the glioma microenvironment and highlight critical knowledge gaps in this burgeoning research space.


Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Neurons/pathology , Tumor Microenvironment , Animals , Disease Progression , Humans
11.
Cell ; 171(7): 1611-1624.e24, 2017 Dec 14.
Article in English | MEDLINE | ID: mdl-29198524

ABSTRACT

The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis, and response to therapy. We profiled transcriptomes of ∼6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary tumors and lymph node metastases. Stromal and immune cells had consistent expression programs across patients. Conversely, malignant cells varied within and between tumors in their expression of signatures related to cell cycle, stress, hypoxia, epithelial differentiation, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors. By integrating single-cell transcriptomes with bulk expression profiles for hundreds of tumors, we refined HNSCC subtypes by their malignant and stromal composition and established p-EMT as an independent predictor of nodal metastasis, grade, and adverse pathologic features. Our results provide insight into the HNSCC ecosystem and define stromal interactions and a p-EMT program associated with metastasis.


Subject(s)
Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/pathology , Neoplasm Metastasis/pathology , Carcinoma, Squamous Cell/genetics , Cells, Cultured , Epithelial-Mesenchymal Transition , Gene Expression Profiling , Head and Neck Neoplasms/genetics , Humans , Male , Single-Cell Analysis , Tumor Microenvironment
12.
Cell Rep ; 21(3): 784-797, 2017 Oct 17.
Article in English | MEDLINE | ID: mdl-29045844

ABSTRACT

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.


Subject(s)
B-Lymphocytes/metabolism , Gene Expression Regulation, Neoplastic , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/pathology , Oncogenes , Receptors, Notch/metabolism , Signal Transduction , Animals , Biopsy , Cell Differentiation/genetics , Cell Line, Tumor , Enhancer Elements, Genetic/genetics , Gene Rearrangement , Humans , Lymph Nodes/metabolism , Lymph Nodes/pathology , Mice , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Notch/genetics , Tumor Microenvironment , Xenograft Model Antitumor Assays
13.
Nature ; 549(7673): 533-537, 2017 09 28.
Article in English | MEDLINE | ID: mdl-28959975

ABSTRACT

High-grade gliomas (HGG) are a devastating group of cancers, and represent the leading cause of brain tumour-related death in both children and adults. Therapies aimed at mechanisms intrinsic to glioma cells have translated to only limited success; effective therapeutic strategies will need also to target elements of the tumour microenvironment that promote glioma progression. Neuronal activity promotes the growth of a range of molecularly and clinically distinct HGG types, including adult and paediatric glioblastoma (GBM), anaplastic oligodendroglioma, and diffuse intrinsic pontine glioma (DIPG). An important mechanism that mediates this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic adhesion molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway. However, the necessity of NLGN3 for glioma growth, the proteolytic mechanism of NLGN3 secretion, and the further molecular consequences of NLGN3 secretion in glioma cells remain unknown. Here we show that HGG growth depends on microenvironmental NLGN3, identify signalling cascades downstream of NLGN3 binding in glioma, and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of paediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. NLGN3 stimulates several oncogenic pathways, such as early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes that include upregulation of several synapse-related genes in glioma cells. NLGN3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent the release of NLGN3 into the tumour microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting NLGN3 secretion, which could prove transformative for HGG therapy.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Glioma/metabolism , Glioma/pathology , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , ADAM10 Protein/antagonists & inhibitors , ADAM10 Protein/metabolism , Adult , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Adhesion Molecules, Neuronal/deficiency , Cell Adhesion Molecules, Neuronal/genetics , Cell Proliferation , Child , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Glioma/genetics , Heterografts , Humans , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Neoplasm Transplantation , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Neurons/pathology , Oligodendroglia/cytology , Oligodendroglia/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Tumor Microenvironment
14.
Nature ; 547(7663): 355-359, 2017 07 20.
Article in English | MEDLINE | ID: mdl-28678782

ABSTRACT

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.


Subject(s)
Drug Evaluation, Preclinical/methods , Glioblastoma/drug therapy , Glioblastoma/genetics , Molecular Targeted Therapy/trends , Transcriptional Elongation Factors/antagonists & inhibitors , Transcriptional Elongation Factors/metabolism , Animals , Cell Line, Tumor , Cell Survival , Chromatin/metabolism , Enhancer Elements, Genetic/genetics , Female , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Humans , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Jumonji Domain-Containing Histone Demethylases/metabolism , Male , Mice , RNA Interference , Transcription, Genetic , Tumor Microenvironment , Xenograft Model Antitumor Assays
15.
Science ; 355(6332)2017 03 31.
Article in English | MEDLINE | ID: mdl-28360267

ABSTRACT

Tumor subclasses differ according to the genotypes and phenotypes of malignant cells as well as the composition of the tumor microenvironment (TME). We dissected these influences in isocitrate dehydrogenase (IDH)-mutant gliomas by combining 14,226 single-cell RNA sequencing (RNA-seq) profiles from 16 patient samples with bulk RNA-seq profiles from 165 patient samples. Differences in bulk profiles between IDH-mutant astrocytoma and oligodendroglioma can be primarily explained by distinct TME and signature genetic events, whereas both tumor types share similar developmental hierarchies and lineages of glial differentiation. As tumor grade increases, we find enhanced proliferation of malignant cells, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia expression programs in TME. Our work provides a unifying model for IDH-mutant gliomas and a general framework for dissecting the differences among human tumor subclasses.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , Glioma/genetics , Glioma/pathology , Isocitrate Dehydrogenase/genetics , Tumor Microenvironment , Brain Neoplasms/classification , Cell Lineage , Glioma/classification , Humans , Macrophages , Microglia/metabolism , Microglia/pathology , Neoplasm Grading , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Principal Component Analysis , Sequence Analysis, RNA , Single-Cell Analysis
16.
Nat Genet ; 49(2): 289-295, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27941797

ABSTRACT

SMARCB1 (also known as SNF5, INI1, and BAF47), a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex, is inactivated in nearly all pediatric rhabdoid tumors. These aggressive cancers are among the most genomically stable, suggesting an epigenetic mechanism by which SMARCB1 loss drives transformation. Here we show that, despite having indistinguishable mutational landscapes, human rhabdoid tumors exhibit distinct enhancer H3K27ac signatures, which identify remnants of differentiation programs. We show that SMARCB1 is required for the integrity of SWI/SNF complexes and that its loss alters enhancer targeting-markedly impairing SWI/SNF binding to typical enhancers, particularly those required for differentiation, while maintaining SWI/SNF binding at super-enhancers. We show that these retained super-enhancers are essential for rhabdoid tumor survival, including some that are shared by all subtypes, such as SPRY1, and other lineage-specific super-enhancers, such as SOX2 in brain-derived rhabdoid tumors. Taken together, our findings identify a new chromatin-based epigenetic mechanism underlying the tumor-suppressive activity of SMARCB1.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Enhancer Elements, Genetic/genetics , SMARCB1 Protein/genetics , Cell Line, Tumor , Chromatin/genetics , DNA-Binding Proteins/genetics , Epigenesis, Genetic/genetics , Humans , Mutation/genetics , Nuclear Proteins/genetics , Rhabdoid Tumor/genetics
17.
Cell Stem Cell ; 20(2): 233-246.e7, 2017 02 02.
Article in English | MEDLINE | ID: mdl-27989769

ABSTRACT

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.


Subject(s)
Chromatin Assembly and Disassembly , Drug Resistance, Neoplasm , Glioblastoma/pathology , Neoplastic Stem Cells/pathology , Acetylation/drug effects , Base Sequence , Biomarkers, Tumor/metabolism , Brain/drug effects , Brain/growth & development , Brain/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin Assembly and Disassembly/drug effects , Drug Resistance, Neoplasm/drug effects , Enhancer Elements, Genetic/genetics , Glioblastoma/metabolism , Histone Demethylases/metabolism , Histones/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/metabolism , Lysine/metabolism , Methylation/drug effects , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Nuclear Proteins/metabolism , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Transcription, Genetic/drug effects
18.
Nat Genet ; 48(3): 265-72, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26829750

ABSTRACT

Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.


Subject(s)
Carcinoma, Adenoid Cystic/genetics , Enhancer Elements, Genetic , Oncogene Proteins v-myb/biosynthesis , Translocation, Genetic , Carcinoma, Adenoid Cystic/pathology , Cell Line, Tumor , Cell Lineage/genetics , Chromatin/genetics , Gene Expression Regulation, Neoplastic , Humans , Oncogene Proteins v-myb/genetics , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Transcription Factors/biosynthesis , Tumor Suppressor Proteins/biosynthesis
19.
Nature ; 529(7584): 110-4, 2016 Jan 07.
Article in English | MEDLINE | ID: mdl-26700815

ABSTRACT

Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.


Subject(s)
Gene Expression Regulation, Neoplastic , Glioma/enzymology , Glioma/genetics , Insulator Elements/genetics , Isocitrate Dehydrogenase/genetics , Mutation/genetics , Oncogenes/genetics , Base Sequence , Binding Sites , CCCTC-Binding Factor , CRISPR-Cas Systems/genetics , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Cells, Cultured , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , CpG Islands/genetics , DNA Methylation/drug effects , DNA Methylation/genetics , Down-Regulation/drug effects , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glioma/drug therapy , Glioma/pathology , Glutarates/metabolism , Humans , Insulator Elements/drug effects , Isocitrate Dehydrogenase/chemistry , Isocitrate Dehydrogenase/metabolism , Phenotype , Protein Binding , Receptor, Platelet-Derived Growth Factor alpha/genetics , Repressor Proteins/metabolism , Up-Regulation , Cohesins
20.
Mol Cell ; 61(1): 170-80, 2016 Jan 07.
Article in English | MEDLINE | ID: mdl-26687680

ABSTRACT

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation/methods , Chromatin/metabolism , Hematopoietic Stem Cells/metabolism , High-Throughput Nucleotide Sequencing/methods , Histones/metabolism , Leukemia/metabolism , Multiplex Polymerase Chain Reaction/methods , Chromatin/genetics , Chromatin Assembly and Disassembly/drug effects , DNA Barcoding, Taxonomic , Epigenesis, Genetic/drug effects , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Histones/genetics , Humans , K562 Cells , Leukemia/genetics , Mutation
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