Pamelyn Woo

Neuronal Activity Promotes Oligodendrogenesis and Adaptive Myelination in the Mammalian Brain

Introduction Myelin is formed by mature oligodendrocytes to facilitate fast propagation of action potentials in axons. Small changes in myelin thickness can confer substantial changes in conduction speed and may thus alter neural circuit function. The idea that active neurons may modulate myelination is supported by in vitro studies and correlations between experience and myelin microstructure, but direct in vivo evidence demonstrating that neuronal activity regulates oligodendrocyte precursor cell (OPC) proliferation, differentiation, or changes in myelin microstructure has been lacking. We use in vivo optogenetic techniques in awake, behaving mice to provide direct evidence that neuronal activity regulates changes in myelin-forming cells within an active circuit. Neuronal activity promotes OPC proliferation, oligodendrogenesis, and myelin remodeling. Optogenetic stimulation of unilateral premotor cortex layer V projection neurons in awake, behaving Thy1::ChR2 mice promotes OPC proliferation (light green cells, red EdU+ nuclei), oligodendrogenesis (newly generated, EdU-marked oligodendrocyte; dark green cell), and an increase in myelin sheath thickness (viewed in cross section, gray). Together with these adaptive myelin changes, motor performance of the correlate limb is improved during normal gait 4 weeks after premotor cortex stimulation. Rationale Demonstrating the direct effects of behaviorally relevant neuronal activity on oligodendroglial lineage cells in vivo has been a challenge because traditional methods of directly promoting neuronal activity involve placement of an electrode, and the resultant tissue injury and subsequent inflammation affects OPC dynamics. Optogenetic technology allows for in vivo control of neuronal firing with millisecond precision using light delivered at a distance from the target and, thus, avoids extensive electrode-related tissue damage. We used an optogenetic (Thy1::ChR2) mouse model in which 470-nm light delivered near the brain surface stimulates the excitatory opsin channelrhodopsin expressed by cortical layer V projection neurons. Wild-type littermate controls lacking channelrhodopsin were identically manipulated to control for effects of surgery, optical fiber placement, and light exposure. In Thy1::ChR2 mice, light stimulation delivered unilaterally to the premotor cortex elicits complex motor behavior (unidirectional ambulation). The thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) was administered at the time of optogenetic stimulation to mark actively dividing cells. Animals were evaluated at various time points to examine the effects of neuronal activity on myelin-forming cells and myelin microstructure, as well as the functional consequences of neuronal activity–regulated myelin changes. Results Optogenetic stimulation of cortical layer V projection neurons resulted in robust proliferation of OPCs within the premotor circuit, from the deep layers of the premotor cortex to the subcortical projections through the corpus callosum. Four weeks later, an increase in newly generated oligodendrocytes and increased myelin sheath thickness were found within the stimulated premotor circuit. Behavioral testing revealed increased swing speed of the correlate forelimb. Pharmacological blockade of OPC differentiation prevented activity-regulated oligodendrogenesis and myelin changes, as well as the associated behavioral change. Conclusion Neuronal activity regulates OPC proliferation, differentiation, and myelin remodeling in the murine brain with accompanying changes in behavioral function. Taken together, these findings suggest that adaptive changes in myelin-forming cells represent a type of behaviorally relevant neural plasticity, raising numerous conceptual and mechanistic questions. Mechanisms regulating myelin plasticity may be important for adaptive neural function and could be leveraged for interventions in diseases of myelin. Conversely, dysregulated myelin plasticity could conceivably contribute to disease. On-Demand Activity Oligodendroglia ensheath axons in the brain with myelin, which provides the insulation that speeds up transmission of neuronal electrical impulses. The process of myelination in the human brain goes on for decades, concurrent with all manner of brain development and cognitive activity. Gibson et al. (p. 10.1126/science.1252304, published online 10 April; see the Perspective by Bechler and ffrench-Constant) used optogenetics to study myelination in response to neural activity. Electrical activity in the motor cortex of the brain of awake mice led to proliferation and differentiation of oligodendrocytes and consequently increased myelination and alterations in motor response. Optogenetic stimulation of the mouse motor cortex incites proliferation of myelin-producing cells and axonal myelination. [Also see Perspective by Bechler and ffrench-Constant] Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells (OPCs). Electrically active neurons may influence OPC function and selectively instruct myelination of an active neural circuit. In this work, we use optogenetic stimulation of the premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural progenitor cells and OPCs, promotes oligodendrogenesis, and increases myelination within the deep layers of the premotor cortex and subcortical white matter. We further show that this neuronal activity–regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement.

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi–histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

Hedgehog signaling drives oncogenesis in several cancers, and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened (SMO). However, resistance to Smoothened inhibitors occurs by genetic changes of Smoothened or other downstream Hedgehog components. Here we overcome these resistance mechanisms by modulating GLI transcription through inhibition of bromo and extra C-terminal (BET) bromodomain proteins. We show that BRD4 and other BET bromodomain proteins regulate GLI transcription downstream of SMO and suppressor of fused (SUFU), and chromatin immunoprecipitation studies reveal that BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites after treatment with JQ1, a small-molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM (genetically engineered mouse model)-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. Altogether, our results reveal BET proteins as critical regulators of Hedgehog pathway transcriptional output and nominate BET bromodomain inhibitors as a strategy for treating Hedgehog-driven tumors with emerged or a priori resistance to Smoothened antagonists.

Neuronal Activity Promotes Glioma Growth through Neuroligin-3 Secretion

Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). By using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma xenograft model, we demonstrate that active neurons similarly promote HGG proliferation and growth in vivo. Conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures, indicating secretion of activity-regulated mitogen(s). The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen, and soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feedforward expression of NLGN3 in glioma cells. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth.

Erratum: Functionally defined therapeutic targets in diffuse intrinsic pontine glioma(Nature Medicine (2015) 21 (555-559) DOI: 10.1038/nm.3855)

Catherine S Grasso, Yujie Tang, Nathalene Truffaux, Noah E Berlow, Lining Liu, Marie-Anne Debily, Michael J Quist, Lara E Davis, Elaine C Huang, Pamelyn J Woo, Anitha Ponnuswami, Spenser Chen, Tessa B Johung, Wenchao Sun, Mari Kogiso, Yuchen Du, Lin Qi, Yulun Huang, Marianne Hütt-Cabezas, Katherine E Warren, Ludivine Le Dret, Paul S Meltzer, Hua Mao, Martha Quezado, Dannis G van Vuurden, Jinu Abraham, Maryam Fouladi, Matthew N Svalina, Nicholas Wang, Cynthia Hawkins, Javad Nazarian, Marta M Alonso, Eric H Raabe, Esther Hulleman, Paul T Spellman, Xiao-Nan Li, Charles Keller, Ranadip Pal, Jacques Grill & Michelle Monje Nat. Med. 21, 555–559 (2015); doi:10.1038/nm.3855; published online 4 May 2015; corrected after print 15 June 2015

Transcriptional Dependencies in Diffuse Intrinsic Pontine Glioma.

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric cancer with limited therapeutic options. The majority of cases of DIPG exhibit a mutation in histone-3 (H3K27M) that results in oncogenic transcriptional aberrancies. We show here that DIPG is vulnerable to transcriptional disruption using bromodomain inhibition or CDK7 blockade. Targeting oncogenic transcription through either of these methods synergizes with HDAC inhibition, and DIPG cells resistant to HDAC inhibitor therapy retain sensitivity to CDK7 blockade. Identification of super-enhancers in DIPG provides insights toward the cell of origin, highlighting oligodendroglial lineage genes, and reveals unexpected mechanisms mediating tumor viability and invasion, including potassium channel function and EPH receptor signaling. The findings presented demonstrate transcriptional vulnerabilities and elucidate previously unknown mechanisms of DIPG pathobiology.

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

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.

Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M + diffuse midline gliomas

Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1–5 are aggressive and universally fatal pediatric brain cancers6. Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7–10, and recent results suggest benefit in central nervous system malignancies11–13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15–17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.Lethal pediatric tumors bearing a particular histone H3 mutation upregulate the disialoganglioside GD2, thereby making these tumors susceptible to chimeric antigen receptor T cell–based immunotherapy.

Abstract PR04: GD2-directed chimeric antigen receptor T cells mediate potent antitumor effect and cure in xenograft models of diffuse intrinsic pontine glioma

Introduction: Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brainstem tumor with a median survival of less than one year. Despite advances in the understanding of the molecular origins of DIPG, improvement in clinical outcomes has yet to materialize. To date, there has been little target exploration for immunotherapy applications in DIPG. Methods: Patient-derived DIPG cell cultures were screened for expression of more than 350 surface antigens as potential immunotherapeutic targets. The disialoganglioside GD2 was found to have the highest expression across cell cultures and was verified by IHC on post-mortem samples. Chimeric antigen receptor (CAR) T-cell therapy against this target was explored both in vitro and in vivo. Results: We found high levels of the disialoganglioside GD2 expressed on cell cultures derived from post-mortem samples of DIPG. Quantification of the number of GD2 molecules per cell demonstrated higher GD2 expression on DIPG than any other tumors, including neuroblastoma, for which GD2 targeted immunotherapy is part of the standard of care. Most cases of DIPG are caused by a mutation in Histone 3.3 (H3K27M). GD2 is highly and uniformly expressed in patient-derived H3K27M DIPG cultures, whereas H3 wild-type pediatric high-grade gliomas, including those diagnosed as DIPG, do not express significant levels of GD2. The H3K27M mutation is associated with increased levels of enzymes in the ganglioside synthesis pathway, suggesting that expression of the target antigen is driven by H3K27M-induced transcriptional dysregulation. Anti-GD2 CAR T cells with a 4-1BB costimulatory domain demonstrate remarkable preclinical activity against H3K27M DIPG. GD2 CAR T cells specifically kill DIPG cells and produce cytokines IL-2 and IFN- upon coculture with tumor. Systemic administration of anti-GD2 CAR T cells achieves potent and durable cure compared to control T cells in multiple orthotopic xenograft models of DIPG. Using a CAR fluorescent protein fusion construct, we demonstrate significant T-cell trafficking to the brainstem where the antitumor effect is mediated. Universal response was observed across multiple cohorts, and treatment-associated toxicity was transient and tolerated during the period of peak antitumor activity. Conclusion: We have previously demonstrated that antigen density drives CAR efficacy. Extremely high expression of GD2 on DIPG makes this a particularly good disease for CAR T-cell therapy. If these results are predictive of human response, CAR T cells could have a transformative impact upon DIPG outcomes. A clinical trial of second generation anti-GD2 CAR T cells in relapsed and progressive DIPG is planned. Citation Format: Robbie G. Majzner, Christopher Mount, Shree Sundaresh, Evan Arnold, Meena Kadapakkam, Louai Labanieh, Pamelyn Woo, Michelle Monje, Crystal L. Mackall. GD2-directed chimeric antigen receptor T cells mediate potent antitumor effect and cure in xenograft models of diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR04.

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

Neuronal 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.