Keith L. Ligon - US grants

[unreadable] DESCRIPTION (provided by applicant): Abnormalities of glia contribute to a wide range of neurologic diseases. However, many important aspects of glial biology and development remain poorly understood. The basic helix-loop-helix transcription factor, Olig2, is essential for motor neuron and oligodendrocyte development in the mouse spinal cord. Yet, fundamental aspects of its function have not been elucidated. The in vivo potential of individual Olig2+ progenitor cells within the microenvironment of the neural tube is unknown; and potential functions for Olig2 during forebrain development and glial tumor formation are untested. The key hypothesis of this grant application is that Olig2+ progenitors give rise to neurons and oligodendrocytes - but not astrocytes - in the developing CNS and are a key cell type in glioma formation. The following studies are proposed to test this hypothesis: Specific Aim 1: Generate a multifunctional transgenic mouse "knock-in" of the tva avian retroviral receptor and cre recombinase into the Olig2 locus (Olig2-tva-cre). This mouse will be used for fate mapping and other studies in the proposal. Specific Aim 2: Determine whether individual Olig2+ progenitors in the ventral neural tube are bipotent and give rise, first to neurons, and then to oligodendrocytes. This will be tested by clonally infecting Olig2-tva-cre derived neural tube, explants, and neural stem cells with RCAS reporter viruses to trace the fate of labeled progeny. The major innovation of this approach is that reporter virus will be targeted solely to Olig2-tva+ progenitors. Specific Aim 3: Determine whether Olig2 function is required for development of telencephalic neuronal subtypes. The hypothesis that Olig2 function is required for forebrain neuronogenesis will be tested by comparative immunohistologic analysis and deconvolution microscopy of Olig2 null and heterozygous littermates. Specific Aim 4: Determine the contributions of Olig2+ progenitors to mouse models of glial tumors and the functional requirements for Olig2 in gliomagenesis. Overall, this proposal should provide insight into regulation of glial development and glial tumors. Such understanding is essential for targeted molecular approaches to the diagnosis and treatment of neurologic diseases such as multiple sclerosis, neurodegeneration, and brain tumors. In addition, this proposal outlines an integrated research training plan and detailed career development plan which should facilitate the transition to full academic independence in the neuroscience community of Harvard Medical School. [unreadable] [unreadable]

Malignant gliomas are considered to be among the deadliest of human cancers. Although we have gained detailed knowledge of a number of genetic pathways mutated in gliomas, recent studies suggest additional mutations play significant role in the phenotype and resistance of this disease. The overall goal of the Neuropathology Core is to provide expert, high throughput, and highly integrated tissue based analysis of human and mouse gliomas the P01. The research of each project is heavily based upon the in vtvo biology of human and mouse gliomas, and as such, the Neuropathology Core services are essential for the success of the P01. In addition, the Core strives to improve the services it delivers through the development of emerging tissue technologies that will reduce the difficulties each project encounters in studying increasingly limited tissue resources. Moreover, the centralized nature of the Core will foster intellectual resource sharing and allow investigators to compare and interpret their findings within the spectrum of research performed across all the projects. The Neuropathology Core has three specific aims: 1) to provide pathologic analysis and interpretation of human and mouse gliomas. 2) to characterize human and mouse gliomas using molecular pathology tools. 3) to develop and implement novel technologies for cellular and molecular characterization of human and mouse gliomas. The Core has the requisite neuropathologic expertise, sophisticated resources, and experienced staff to carry out these aims and ensure that the P01 meets its overall goals of discovery of targets for therapy in malignant glioma.

The overall goal of this SPORE application is to develop more effective targeted molecular therapies and biomarkers for glioblastoma. The four proposed projects focus on 1) Overcoming resistance to VEGF inhibition by targeting Angiopoietin-2; 2) Inhibiting the phosphatidylinositol 3-kinase (PIS kinase) pathway; 3) designing novel strategies for imaging and targeting IDH mutant gliomas; and 4) Increasing the radiosensitivity of glioblastomas by inhibiting the bHLH transcription factor 01ig2. One ofthe innovative themes of this SPORE proposal is that biospecimens collected by the Pathology Core will be comprehensively moiecuiariy profiled using multiplexed technologies so projects may effectively evaluate the interaction of targeted therapies and their targets. The Pathology Core will support the goals of the SPORE by providing expert neuropathologic review, biospecimen banking and clinical trial support. In addition, the Pathology Core will be a centralized resource for clinically and moiecuiariy annotated glioblastoma tissues and primary glioblastoma cell cultures that will be essential to the success of the proposed projects. By centralizing these activities, the Pathology Core will ensure the safe and effective use of finite glioblastoma tissue resources without limiting the scope of the translational research planned in this proposal. The SPORE support, combined with significant institutional support for these goals, will ensure that over the next 5 years the Pathology Gore will provide the critical research infrastructure required for successful, collaborative translational research ofthe SPORE program.

PROJECT SUMMARY ? CORE 2 Oncolytic herpes simplex virus (oHSV) based therapeutics represent a promising and novel treatment approach for glioblastoma (GBM), which still has no effective conventional or targeted therapeutics. However, significant barriers exist to optimizing oHSV therapy and the research of the Core and Program are designed to gain knowledge that will allow improved designed and effectiveness of these agents. To achieve this the Glioblastoma Biorepository Core (Core 2) will provide investigators and all projects centralized access to authenticated resources essential to the planned experimental studies. These include human and mouse glioblastoma samples, associated clinical and genomic data, cell lines, and xenografts. The Core will also offer professional neuropathology and immunopathology expertise useful in interpretation of experimental results and their clinical relevance. These services will greatly aid the program's investigations of mechanisms of resistance to oHSV therapy and discovery of new approaches to improve oHSV treatment as single agent or in combination with other treatments (e.g. checkpoint inhibitors). The Biorepository Core will achieve these goals through three specific aims that support all the Projects. Aim 1 is to generate and maintain a repository of consented glioblastoma samples and data. This will include sample triage by Neuropathologists and distribution from the clinical trial of rQNestin34.5 planned in Project 2 and utilized by Projects 2, 3 and 4 (tissue and blood). Aim 2 is to create and distribute glioblastoma patient derived cell lines (PDCL) and xenografts (PDX). These will be created from the rQNestin34.5 trial patients as well as non-trial GBM patients. The Core will also provide and authenticate mouse syngeneic glioma models (e.g. GL261-N4, CT2A, 4C8) utilized by all Projects. Aim 3 is to provide expert neuropathology, immunopathology, and molecular analysis of glioblastoma tissues and models. This includes genomic sequencing (e.g. EGFRvIII in Project 1, RNA expression to characterize targets relevant to oHSV trafficking and effects (e.g. Nestin Project 2, activated NOTCH Project 3), and multiplex co-localization studies of tissue samples for immune cell markers of lymphocytic, NK-cell (Project 4), and macrophage populations (e.g. PD1, PDL1, CD8) in collaboration with the DFCI Center for Immunooncology. In total the studies planned will facilitate centralized and efficient administration of resources and specialized expertise required to achieve the ambitious goals of the Program and advance the field of oHSV therapeutics for glioma and other cancers.

This is the competing renewal of a SPORE initiative on glioma at Dana-Farber/Harvard Cancer Center. Our objective is to improve the standard of care for children, young adults, and adults with these tumors through the use of targeted therapies. Towards this end, basic scientists from Harvard Medical School and the Broad Institute join with clinical/translational investigators from Boston Children?s Hospital, Brigham and Women?s Hospital, Dana-Farber Cancer Institute and Massachusetts General Hospital. There are four projects: Project One targets pediatric low-grade astrocytomas (PLGAs). Nearly 75% of PLGAs are driven by activating mutations in the BRAF protein kinase. Clinician/scientists Daphne Haas-Kogan, M.D. and Karen Wright, M.D. together with structural biologist Michael Eck, M.D., Ph.D. will develop and test brain-penetrant targeted therapeutics for BRAF-mutant PLGAs. Project Two targets IDH-mutant gliomas which present typically in young adults. IDH-mutant gliomas produce extraordinarily high levels of the ?oncometabolite? R-2- hydroxyglutarate (2-HG). However, therapeutic exploitation of the differential 2-HG content between normal and malignant brain tissue has yet to be realized. Neurosurgeon Daniel Cahill, M.D., Ph.D. and cancer biologist William Kaelin, M.D. will address this therapeutic lacuna. Project Three targets adult gliomas. Recent studies by basic scientist Jean Zhao, Ph.D. show that in addition to suppressing cell cycle progression, CDK4/6 antagonists promote anti-tumor immunity and synergistically enhance the response to checkpoint inhibitors. Going forward, Dr. Zhao together with neuro-oncologist Patrick Wen, M.D. will test the hypothesis that brain penetrant CDK4/6 inhibitors can augment immunotherapeutic approaches to GBM. Project Four targets the neuronal microenvironment of adult and pediatric gliomas. Neuro-oncologist and developmental neurobiologist Michelle Monje, M.D., Ph.D. has shown that neurons promote glioma growth through activity- regulated secretion of neuroligin-3 (NLGN3) into the tumor microenvironment. Basic scientist Mario Suva, M.D, Ph.D. has refined methods for single cell sequencing of the multiple cell types within the microenvironment of freshly resected human gliomas. Working together, Monje and Suva will define the molecular mechanisms whereby microenvironmental NLGN3 modulates formation and progression of gliomas and explore a novel therapeutic opportunity embedded within the NLGN3 requirement. Rigor and reproducibility of work conducted in the four projects will be fostered by cores for Pathology and for Biostatistics and Computational Biology. An Administration core will enable and manage the multiple consortium agreements and collaborative interactions between Harvard Medical School, the four participating Harvard teaching hospitals and facilitate clinical trials and imaging studies. Intellectual vigor within the program is sustained and refreshed by annual Career Enhancement Awards to young investigators and by annual Developmental Project Awards.