HGG-05. NEURONAL ACTIVITY PROMOTES PEDIATRIC HIGH-GRADE GLIOMA GROWTH THROUGH A NLGN3-CSPG4 SIGNALING AXIS

Abstract High-grade gliomas, including diffuse intrinsic pontine glioma (DIPG), are a lethal group of cancers whose progression is strongly regulated by neuronal activity {Venkatesh 2015}{Venkatesh 2017}{Venkatesh 2019}. One way in which glioma cells sense neuronal activity is via interaction with the ectodomain of post-synaptic adhesion protein neuroligin-3 (NLGN3), which is cleaved and released into the tumor microenvironment (TME) by the sheddase ADAM10. This interaction drives glioma growth, but the relevant binding partner of shed NLGN3 (sNLGN3) on glioma cells is currently unknown. Here, we report that sNLGN3 binds to chondroitin sulfate proteoglycan 4 (CSPG4), in turn inducing regulated intramembrane proteolysis (RIP) of CSPG4, and initiating a signaling cascade within DIPG cells to promote tumor growth. CSPG4 RIP involves activity-regulated ectodomain shedding by ADAM10 and subsequent gamma secretase-mediated release of the intracellular domain in healthy oligodendroglial precursor cells (OPCs), putative cells of origin for several forms of high-grade glioma {Sakry 2014}{Nayak 2018}. Incubation of high-grade glioma cells or healthy OPCs with recombinant NLGN3 is sufficient to augment ADAM10-mediated ectodomain release of CSPG4 and subsequent gamma secretase-mediated cleavage of the CSPG4 intracellular domain (ICD). Pre-treatment of glioma cells or OPCs with an ADAM10 inhibitor entirely blocks NLGN3-induced CSPG4 shedding. Acute depletion of CSPG4 via CRISPR gene editing renders glioma cells insensitive to the growth-promoting effects of NLGN3 application in vitro. We are now performing experiments to better discern how the CSPG4 ICD regulates signaling consequences downstream of sNLGN3 binding. In addition, we are using surface plasmon resonance to investigate whether the shed ectodomains of NLGN3 and CSPG4 remain in complex or only transiently interact. Altogether, our data form a critical missing link in understanding how glioma cells sense, translate and respond to neuronal activity in the TME and identify a new therapeutic target to disrupt neuron-glioma interactions.

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TAMI-28. IDENTIFICATION OF A NEUROLIGIN-3 BINDING PARTNER IN HIGH-GRADE GLIOMAS AND NORMAL PROGENITORS

Neuro-Oncology ◽

10.1093/neuonc/noab196.812 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi204-vi204

Author(s):

Shawn Gillespie ◽

Yoon Seok Kim ◽

Anna Geraghty ◽

Michael Quezada ◽

James Reed ◽

...

Keyword(s):

Neuronal Activity ◽

Glioma Cells ◽

Diffuse Intrinsic Pontine Glioma ◽

Size Exclusion ◽

Dependent Manner ◽

High Grade ◽

Gamma Secretase ◽

Binding Partner ◽

High Grade Gliomas

Abstract High-grade gliomas, including diffuse intrinsic pontine glioma, are lethal cancers whose progression is strongly regulated by neuronal activity. One way in which gliomas detect neuronal activity is via interaction with the ectodomain of post-synaptic adhesion protein neuroligin-3 (NLGN3), which is shed from neurons and oligodendrocyte precursors (OPCs) by the ADAM10 sheddase in an activity dependent manner. NLGN3 signaling drives glioma growth, but the cognate binding partner of shed NLGN3 (sNLGN3) on glioma cells is unknown. Here, we employed a proximity labeling technique to identify chondroitin sulfate proteoglycan 4 (CSPG4) as a putative binding partner of sNLGN3 in gliomas. We then confirmed complexing between recombinant proteins with size exclusion chromatography and are determining kinetics and affinity by surface plasmon resonance. When looking for evidence of binding in cells, we were surprised to find that sNLGN3 triggers regulated intramembrane proteolysis (RIP) of CSPG4, leaving no trace of the interaction at the membrane. sNLGN3 binding first induces ADAM10-mediated cleavage and release of the CSPG4 ectodomain, followed by gamma secretase-mediated release of the intracellular domain and downstream signaling in OPCs and gliomas. Pre-treatment of glioma cells or OPCs with an ADAM10 inhibitor entirely blocks sNLGN3-induced CSPG4 shedding. Acute depletion of CSPG4 via CRISPR gene editing renders glioma cells insensitive to the growth-promoting effects of sNLGN3 in vitro. Furthermore, we find that tamoxifen-induced deletion of NLGN3 from murine OPCs reduces the total number of OPCs, suggesting that this signaling axis promotes maintenance of OPC stemness in an autocrine fashion. Indeed, gamma secretase inhibition accelerates OPC differentiation in vitro, pointing towards a fundamental role for sNLGN3-CSPG4 signaling in OPCs and high-grade gliomas. Altogether, our results form a critical missing link in understanding how glioma cells detect a key neuronal activity-regulated signal, suggest intriguing links to OPC biology and identify a therapeutic target to disrupt neuron-glioma interactions.

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H3.3K27M Mutation Promotes The Migration and Invasion of Glioma Cells By Activating β-Catenin/USP1 Signaling

10.21203/rs.3.rs-1215261/v1 ◽

2022 ◽

Author(s):

Zhiyuan Sun ◽

Yufu Zhu ◽

Xia Feng ◽

Xiaoyun Liu ◽

Kunlin Zhou ◽

...

Keyword(s):

Glioma Cells ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Migration And Invasion ◽

Mrna Levels ◽

High Grade ◽

Protein Levels ◽

Adult Glioma

Abstract H3.3K27M is a newly identified molecular pathology marker in glioma and is especially correlated with the malignancy of diffuse intrinsic pontine glioma (DIPG). In recent years, accumulating research has revealed that other types of glioma also contain the H3.3K27M mutation. However, the role of H3.3K27M in high-grade adult glioma, which is the most malignant glioma, has not been investigated. In this study, we focused on exploring the expression and function of H3.3K27M in high-grade adult glioma patients. We found that H3.3K27M is partly highly expressed in high-grade glioma tissues. Then, we introduced H3.3K27M into H3.3 wild-type glioma cells, U87 cells and LN229 cells. We found that H3.3K27M did not regulate the growth of glioma in vitro and in vivo; however, the survival of mice with transplanted tumors was significantly reduced. Further investigation revealed that H3.3K27M expression mainly promoted the migration and invasion of glioma cells. Moreover, we certified that H3.3K27M overexpression enhanced the protein levels of ꞵ-catenin and p-ꞵ-catenin, the protein and mRNA levels of ubiquitin-specific protease 1 (USP1), and the protein level of enhancer of zeste homolog 2 (EZH2). Importantly, the ꞵ-catenin inhibitor XAV-939 significantly attenuated the upregulation of the aforementioned proteins. Overall, the H3.3K27M mutation is present in a certain proportion of high-grade glioma patients and facilitates a poor prognosis by promoting the metastasis of glioma by regulating the ꞵ-catenin/USP1/EZH2 pathway.

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HG-123AN EPIGENOMIC SCREEN REVEALS THE CHROMATIN REMODELING PROTEIN BPTF AS A DRIVER OF HIGH-GRADE GLIOMA AND DIFFUSE INTRINSIC PONTINE GLIOMA GROWTH IN VITRO AND IN VIVO

Neuro-Oncology ◽

10.1093/neuonc/now073.119 ◽

2016 ◽

Vol 18 (suppl 3) ◽

pp. iii76.3-iii76

Author(s):

Adam Green ◽

Patrick Flannery ◽

John DeSisto ◽

Madeleine Lemieux ◽

Shak Ramkissoon ◽

...

Keyword(s):

Chromatin Remodeling ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

High Grade ◽

Pontine Glioma ◽

Glioma Growth

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HGG-04. TARGETING GABAERGIC NEURON-GLIOMA SYNAPSES IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) THROUGH ANTI-EPILEPTIC DRUG REPURPOSING

Neuro-Oncology ◽

10.1093/neuonc/noab090.070 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i17-i18

Author(s):

Tara Barron ◽

Vilina Mehta ◽

Pamelyn Woo ◽

Michelle Monje

Keyword(s):

Gabaa Receptor ◽

Brain Cancer ◽

Glioma Cells ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Gabaergic Neuron ◽

High Grade ◽

Pontine Glioma ◽

Anti Epileptic Drug ◽

Glioma Growth

Abstract Pediatric high-grade gliomas, including diffuse intrinsic pontine glioma (DIPG), are the leading cause of brain cancer-related death in children. While enormous progress has been made in recent years for many forms of cancer, high-grade gliomas remain seemingly intractable, indicating that fundamental aspects of glioma growth are not yet sufficiently understood. Neuronal activity drives glioma growth both through paracrine signaling and through direct neuron-to-glioma synapses. Recently glutamatergic, AMPA receptor-dependent synapses were discovered between microenvironmental neurons and malignant glioma cells. The depolarizing current that results from synaptic and other forms of electrical neuron-glioma signaling promotes pediatric high-grade glioma proliferation and regulates growth. Neuron-glioma cell synapses mediated by other neurotransmitters remain largely unexplored, though glioma cells express genes encoding neurotransmitter receptors such as GABAA receptor subunits. Using whole-cell patch clamp electrophysiology in patient-derived DIPG xenografts, we have identified functional GABAergic neuron-to-glioma synapses mediated by GABAA receptors. GABAergic input has a depolarizing effect on glioma cells, but the magnitude of depolarization is heterogeneous between high-grade glioma subtypes and between patient-derived DIPG xenograft models. As membrane depolarization increases glioma proliferation, depolarizing GABAergic inputs to glioma cells could promote DIPG progression. Drugs that stimulate GABA signaling, such as benzodiazepines, are often given to pediatric glioma patients to treat nausea, seizures or anxiety. In patient-derived DIPG xenograftn models, lorazepam, a benzodiazepine that increases GABAA receptor conductance, increases glioma growth. Conversely, levetiracetam, an anti-epileptic drug that reduces synaptic transmission including at GABAergic neuron-glioma synapses, reduces glioma proliferation in patient-derived DIPG xenografts. This emerging understanding of brain cancer neurophysiology reveals new therapeutic targets and highlights commonly used drugs about which more study is required in this disease context.

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In Vitro Evaluation of Iodine-125-Labeled Monoclonal Antibody (MAb 425) in Human High-Grade Glioma Cells

American Journal of Clinical Oncology ◽

10.1097/00000421-199612000-00015 ◽

1996 ◽

Vol 19 (6) ◽

pp. 601-608 ◽

Cited By ~ 6

Author(s):

Jacqueline G. Emrich ◽

Hans Bender ◽

Reiner Class ◽

Jeffrey Eshleman ◽

Curtis Miyamoto ◽

...

Keyword(s):

Monoclonal Antibody ◽

Glioma Cells ◽

High Grade Glioma ◽

High Grade ◽

In Vitro Evaluation ◽

Iodine 125

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Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423 and Hey2

10.1101/2020.03.04.976811 ◽

2020 ◽

Author(s):

Cristiana Barone ◽

Mariachiara Buccarelli ◽

Francesco Alessandrini ◽

Miriam Pagin ◽

Laura Rigoldi ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Transcription Factor ◽

Cancer Stem Cells ◽

Regulatory Network ◽

Glioma Cells ◽

High Grade Glioma ◽

High Grade

AbstractCancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain tumors. In gliomas, Sox2 is essential to maintain CSC. In mouse high-grade glioma pHGG, Sox2 deletion causes cell proliferation arrest and inability to reform tumors in vivo; 134 genes are significantly derepressed. To identify genes mediating the effects of Sox2 deletion, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Cdkn2b, Ebf1, Zfp423 and Hey2, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis, or pharmacological inactivation, of each of these genes, individually, showed that their activity is essential for the proliferation arrest caused by Sox2 deletion. These Sox2-inhibited antioncogenes also inhibited clonogenicity in primary human glioblastoma-derived cancer stem-like cell lines. These experiments identify critical anti-oncogenic factors whose inhibition by Sox2 is involved in CSC maintenance, defining new potential therapeutic targets for gliomas.Table of Contents ImageMain PointsSox2 maintains glioma tumorigenicity by repressing the antioncogenic activity of a regulatory network involving the Ebf1, Hey2, Cdkn2b and Zfp423 genes.Mutation of these genes prevents the cell proliferation arrest of Sox2-deleted glioma cells.

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HGG-02. NEUROPHYSIOLOGICAL SMALL MOLECULE SCREEN TO TARGET NEURON-GLIOMA INTERACTIONS IN PEDIATRIC HIGH-GRADE GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noab090.068 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i17-i17

Author(s):

David Rogawski ◽

Sara Mulinyawe ◽

Craig Thomas ◽

Michelle Monje

Keyword(s):

Ion Channels ◽

Small Molecule ◽

Glioma Cells ◽

High Grade Glioma ◽

High Grade ◽

In Vivo Experiments ◽

Target Neuron ◽

Glioma Cell Proliferation ◽

Glioma Growth

Abstract Neurons stimulate glioma growth via synaptic and paracrine signaling mechanisms. We recently demonstrated that neurons form AMPA receptor-dependent synapses with glioma cells, and that neuronal activity also induces potassium-evoked currents that are amplified by gap junctions coupling glioma cells. However, our understanding of the neurotransmitters, receptors, and ion channels participating in neuron-glioma signaling remains incomplete. We have recently developed a high-throughput neuron-glioma co-culture strategy to screen small molecules for agents that may disrupt neuron-glioma signaling. Glioma cell proliferation is increased tenfold when cultured together with neurons; this robust biological effect can be probed in a targeted screen of compounds influencing neurotransmitter receptors and ion channels. The neurophysiological small molecule library used was curated to include approved anti-epileptics, neuroleptics, and antidepressants, as well as a variety of other compounds acting on different neurotransmitter types and ion channels. Hits from the primary screen were run through a counter-screen using glioma cells grown alone without neurons, to identify compounds that specifically affect neuron-glioma interactions. Correlation of the screening results with drug mechanisms of action will allow us to map out the key neurotransmitter pathways regulating glioma growth, which can be further validated using genetic and in vivo experiments. Drugs identified in this glioma neuroscience screen may be readily translated into much-needed therapeutics for children with high-grade glioma.

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HGG-12. HUMAN IPSC-DERIVED H3.3K27M NEUROSPHERES: A NOVEL MODEL FOR INVESTIGATING DIPG PATHOGENESIS AND DRUG RESPONSE

Neuro-Oncology ◽

10.1093/neuonc/noab090.078 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i19-i20

Author(s):

Kasey Skinner ◽

Tomoyuki Koga ◽

Shunichiro Miki ◽

Robert F Gruener ◽

R Stephanie Huang ◽

...

Keyword(s):

Principal Component ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Genetically Engineered ◽

High Grade ◽

Primary Tumors ◽

Secondary Tumor ◽

Immunodeficient Mice

Abstract Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). Existing DIPG models have provided insight into the role of H3.3K27M but have limitations: genetically engineered murine models often rely on overexpression of the mutant histone to form tumors; patient-derived xenografts (PDX) are more genetically faithful but preclude examination of the effect of individual mutations on pathogenesis. To address these shortcomings and better recapitulate the genetics of human tumors, we designed a novel DIPG model based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express heterozygous H3.3K27M. Edited iPSC were chemically differentiated into neural progenitor cells, which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Further, neurospheres cultured from primary tumors formed secondary tumors upon reimplantation with more diffuse invasion, suggesting in vivo evolution. To validate this model’s relevance to DIPG transcriptionally, we performed RNA-sequencing on a cohort of primary and secondary tumor neurospheres (termed primary and secondary iDIPG) and compared them to published RNA-seq data from pediatric PDX and patient tumor samples. Hierarchical clustering and principal component analysis on differentially expressed genes (P<0.05) showed that H3.3K27M iDIPG cluster with H3.3K27M PDX and patient tumors. Further, ssGSEA showed that H3.3K27M iDIPG are enriched for astrocytic and mesenchymal signature genes, a defining feature of H3.3K27M DIPG. Finally, we found that primary H3.3K27M iDIPG neurospheres are sensitive to panobinostat, an HDAC inhibitor shown to be effective against H3.3K27M DIPG cells in vitro. Overall, these data suggest that H3.3K27M iDIPG are a promising tool for investigating DIPG biology and new therapeutic strategies.

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In Vitro Drug Response and Efflux Transporters Associated with Drug Resistance in Pediatric High Grade Glioma and Diffuse Intrinsic Pontine Glioma

PLoS ONE ◽

10.1371/journal.pone.0061512 ◽

2013 ◽

Vol 8 (4) ◽

pp. e61512 ◽

Cited By ~ 67

Author(s):

Susanna J. E. Veringa ◽

Dennis Biesmans ◽

Dannis G. van Vuurden ◽

Marc H. A. Jansen ◽

Laurine E. Wedekind ◽

...

Keyword(s):

Drug Resistance ◽

Drug Response ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Efflux Transporters ◽

High Grade ◽

Pontine Glioma ◽

Pediatric High Grade Glioma

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Targeting neuronal activity-regulated neuroligin-3 dependency for high-grade glioma therapy

10.1101/153122 ◽

2017 ◽

Author(s):

Humsa S. Venkatesh ◽

Lydia T. Tam ◽

Pamelyn J. Woo ◽

Surya Nagaraja ◽

Shawn M. Gillespe ◽

...

Keyword(s):

Neuronal Activity ◽

Therapeutic Potential ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

High Grade ◽

Kinase Activation ◽

Promising Strategy ◽

Oligodendrocyte Precursor ◽

Orthotopic Xenografts ◽

Activity Dependent

AbstractNeuronal activity promotes high-grade glioma (HGG) growth. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule and glioma mitogen neuroligin-3 (Nlgn3), but the therapeutic potential of targeting Nlgn3 in glioma remains to be defined. We demonstrate a striking dependence of HGG growth on microenvironmental Nlgn3 and determine a targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric glioblastoma, diffuse intrinsic pontine glioma and adult glioblastoma fail to grow in Nlgn3 knockout mice. Glioma exposure to Nlgn3 results in numerous signaling consequences, including early focal adhesion kinase activation upstream of PI3K-mTOR. Nlgn3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. Administration of ADAM10 inhibitors robustly blocks HGG xenograft growth. This work defines the therapeutic potential of and a promising strategy for targeting Nlgn3 secretion in the glioma microenvironment, which could prove transformative for treatment of HGG.

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