David H. Gutmann - US grants

Neurofibromatosis type 1 (NF1), one of the most common inherited diseases, represents a challenge to both clinicians and basic scientists. The protean nature of the disorder and its variable expressivity has made presymptomatic clinical diagnosis difficult and treatment unsatisfactory. The neurofibromatosis gene is thought to behave as a tumor suppressor gene and a better understanding of its role in normal cell biology will provide insights not only into tumor biology but also the mechanisms underlying normal growth and development. Progress in appreciating the function of the NF1 gene will require the identification and characterization of the gene product of the NF1 locus. The work outlined in this grant proposal is directed towards characterizing the NF1 protein and determining its normal function in order to better understand how diseases like NF1 results from abnormalities of the NF1 protein. The specific aims of this project are directed at characterizing the NF1 gene product: (1) Antibodies will be generated to identify the NF1 protein (NF1 GAP-related protein, NF1GRP). These antibodies will provide reagents to determine functional and structural domains of NF1GRP as well as to define its subcellular and tissue distribution. Proteins which associate with NF1GRP will be isolated by the combined approaches of coimmunoprecipitation using these antibodies and column chromatography using baculovirus-expressed NF1GRP matrices. (2) Analysis of the complete NF1 cDNA sequence has demonstrated sequence similarity between a 400 amino acid region of NF1GRP (catalytic domain) and a family of GTPase-activating proteins. There is nothing known about the remaining 85% of the protein. Site-directed mutagenesis and domain swapping experiments will be undertaken to investigate the role of the catalytic as well as noncatalytic domain regions in NF1GRP function. (3) Recent studies have demonstrated that NF1GRP may be expressed in all tissues. In order to determine what role NF1GRP has in non-neural crest tissues, the Drosophila NF1 homolog will be cloned and studied. Its pattern of expression in adult tissues as well as during the course of Drosophila nervous system development will be investigated. Drosophila provides a unique model system for studying NF1GRP given the relative simplicity of the fruitfly genome, the wealth of information on nervous system development and the ability to determine the impact of NF1GRP on development by direct mutagenesis and through the study of known developmental mutants.

Neurofibromatosis type 1 (NF1), one of the most common inherited diseases, represents a challenge to both clinicians and basic scientists. The protean nature of the disorder and its variable expressivity has made presymptomatic clinical diagnosis difficult and treatment unsatisfactory. The neurofibromatosis gene is thought to behave as a tumor suppressor gene and a better understanding of its role in normal cell biology will provide insights not only into tumor biology but also the mechanisms underlying normal growth and development. Progress in appreciating the function of the NF1 gene will require the identification and characterization of the gene product of the NF1 locus. The work outlined in this grant proposal is directed towards characterizing the NF1 protein and determining its normal function in order to better understand how diseases like NF1 results from abnormalities of the NF1 protein. The specific aims of this project are directed at characterizing the NF1 gene product: (1) Antibodies will be generated to identify the NF1 protein (NF1 GAP-related protein, NF1GRP). These antibodies will provide reagents to determine functional and structural domains of NF1GRP as well as to define its subcellular and tissue distribution. Proteins which associate with NF1GRP will be isolated by the combined approaches of coimmunoprecipitation using these antibodies and column chromatography using baculovirus-expressed NF1GRP matrices. (2) Analysis of the complete NF1 cDNA sequence has demonstrated sequence similarity between a 400 amino acid region of NF1GRP (catalytic domain) and a family of GTPase-activating proteins. There is nothing known about the remaining 85% of the protein. Site-directed mutagenesis and domain swapping experiments will be undertaken to investigate the role of the catalytic as well as noncatalytic domain regions in NF1GRP function. (3) Recent studies have demonstrated that NF1GRP may be expressed in all tissues. In order to determine what role NF1GRP has in non-neural crest tissues, the Drosophila NF1 homolog will be cloned and studied. Its pattern of expression in adult tissues as well as during the course of Drosophila nervous system development will be investigated. Drosophila provides a unique model system for studying NF1GRP given the relative simplicity of the fruitfly genome, the wealth of information on nervous system development and the ability to determine the impact of NF1GRP on development by direct mutagenesis and through the study of known developmental mutants.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder which manifests clinically with peripheral neurofibromas, cafe-au-lait spots, learning disabilities, and an increased incidence of tumor development. The NF1 gene was identified by positional cloning and its protein product, neurofibromin, determined to be a large cytoplasmic protein involved in the downregulation of p21-ras. In addition, neurofibromin has been shown to be associated with cytoplasmic microtubules and this association may modulate the ability of neurofibromin to downregulate p21-ras. At least three isoforms of NF1, termed type 1 (lacking either the exon 23a or 48a insertions) type 2 (containing the exon 23a insertion) and type 3 (containing the exon 48a insertion) NF1 are generated by the use of alternate exons. Type 2 neurofibromin contains an additional 21 amino acids (exon 23a) inserted into the domain responsible for p2l-ras regulation which has been shown to reduce the ability of this isoform to downregulate p2l-ras in vitro. The expression of type 2 NF1 is induced during in vitro differentiation of Schwann cells and neuroblasts. Type 3 NF1, on the other hand, contains an additional 18 amino acids (exon 48a) inserted into the extreme carboxyl terminus of the protein and is expressed predominantly in muscle. The ability to modulate the function of neurofibromin through changes in isoform expression may represent an additional mechanism for neurofibromin regulation. In order to understand the contributions of these alternatively-spliced isoforms of NF1 to nervous system differentiation. we propose to study (l) the effect of isoform expression during Schwann cell and neuroblast differentiation and (2) the effect of alterations in isoform expression on neurofibromin function. Therefore, to understand the role of neurofibromin in differentiation and development, one must consider the expression of the individual neurofibromin isoforms. Initially, changes in neurofibromin isoform expression incumbent upon in vitro Schwann cell and neuroblast differentiation will be examined using these isoform-specific reagents. Then, these reagents will be used to examine the adult and embryonic expression of neurofibromin isoforms. The effect of isoform expression on Schwann cell and neuroblast proliferation and division shall next be examined by expressing the NF1 isoforms from inducible promoters. In an effort to understand the effect of isoform expression on the known functions of neurofibromin, the ability of these NF1 isoforms to (l) downregulate p2l-ras and (2) interact with cytoplasmic microtubules will be examined. An examination of the differential expression and functional properties of these isoforms will provide additional insights into the role of this important tumor suppressor gene in both health and disease.

Neurofibromatosis type 1 (NF1), one of the most common inherited diseases, represents a challenge to both clinicians and basic scientists. The protean nature of the disorder and its variable expressivity has made presymptomatic clinical diagnosis difficult and treatment unsatisfactory. The neurofibromatosis gene is thought to behave as a tumor suppressor gene and a better understanding of its role in normal cell biology will provide insights not only into tumor biology but also the mechanisms underlying normal growth and development. Progress in appreciating the function of the NF1 gene will require the identification and characterization of the gene product of the NF1 locus. The work outlined in this grant proposal is directed towards characterizing the NF1 protein and determining its normal function in order to better understand how diseases like NF1 results from abnormalities of the NF1 protein. The specific aims of this project are directed at characterizing the NF1 gene product: (1) Antibodies will be generated to identify the NF1 protein (NF1 GAP-related protein, NF1GRP). These antibodies will provide reagents to determine functional and structural domains of NF1GRP as well as to define its subcellular and tissue distribution. Proteins which associate with NF1GRP will be isolated by the combined approaches of coimmunoprecipitation using these antibodies and column chromatography using baculovirus-expressed NF1GRP matrices. (2) Analysis of the complete NF1 cDNA sequence has demonstrated sequence similarity between a 400 amino acid region of NF1GRP (catalytic domain) and a family of GTPase-activating proteins. There is nothing known about the remaining 85% of the protein. Site-directed mutagenesis and domain swapping experiments will be undertaken to investigate the role of the catalytic as well as noncatalytic domain regions in NF1GRP function. (3) Recent studies have demonstrated that NF1GRP may be expressed in all tissues. In order to determine what role NF1GRP has in non-neural crest tissues, the Drosophila NF1 homolog will be cloned and studied. Its pattern of expression in adult tissues as well as during the course of Drosophila nervous system development will be investigated. Drosophila provides a unique model system for studying NF1GRP given the relative simplicity of the fruitfly genome, the wealth of information on nervous system development and the ability to determine the impact of NF1GRP on development by direct mutagenesis and through the study of known developmental mutants.

DESCRIPTION (From the applicant's abstract): Little is known about the molecular pathogenesis of nervous system tumors such as meningiomas and schwannomas. Individuals affected with neurofibromatosis 2 (NF2) develop these tumors at increased frequency. In addition, mutations and loss of NF2 gene expression are associated with the development of sporadic schwannomas and meningiomas, suggesting that the NF2 gene product, merlin is a critical growth regulator for Schwann cells and meningeal cells. The NF2 tumor suppressor gene bears sequence similarity to Protein4.1 proteins that link the actin cytoskeleton to cell surface glycoproteins. Previous work from out laboratory has demonstrated that merlin regulates cell motility and proliferation as well as cell spreading. In this application we propose to test the hypothesis that merlin integrates several different several cellular processes important for tumor formation and progression reflected by its ability to regulate cell spreading (tumor initiation), cell proliferation (tumor growth) and cell motility (tumor spread). The experiments proposed in this application are aimed at determining how the merlin tumor suppressor functions as a negative growth regulator by defining merlin functional domains, critical merlin protein interactions and relevant intracellular signaling pathways. Our ability to design rational therapies for schwannomas and meningiomas is dependent on an improved understanding of the mechanisms by which loss of merlin expression and function promotes tumor formation.

DESCRIPTION (Adapted from applicant's abstract): Neurofibromatosis 1 (NF1) is a comm mon autosomal dominant disorder in which affected individuals develop benign and malignant tumors. This application targets on of the most important clinical issues in NF1. Optic pathway gliomas (astrocytomas) are the second most common tumor in NF1, often leading to blindness and neurologic impairment. Despite significant advances in our understanding of the molecular biology of the NF1 gene, very little is known about these tumors or the role of the NF1 gene in regulating astrocyte growth. The NF1 gene was identified by positional cloning and its protein product, neurofibromin, was shown to function as a GTPase-activating protein (GAP) for p21-ras. Reduced expression of neurofibromin in some tumors results in increased p21-ras activity and cell proliferation. However, it is not known whether the NF1 gene is a growth regulator for astrocytes, whether it functions as a p21-ras regulator in astrocytes, and whether loss of NF1 expression is required for astrocytoma development. Previously, it was demonstrated that mice heterozygous for a targeted NF1 gene mutation have increased numbers of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in their brains. In addition, it was demonstrated that primary astrocytes from NF1 +/- mice proliferate faster in vitro that primary astrocytes derived from NF1 +/+ littermates. These results suggest that neurofibromin might function as a tumor suppressor protein for astrocytes, such that reduced or absent NF1 gene expression might result in increased astrocyte proliferation and tumor formation. In this application it is proposed to test the hypothesis that neurofibromin functions as a negative growth regulator of astrocytes in vitro and in vivo. Specifically, it is proposed to (1) determine the relationship between neurofibromin expression and p21-ras activity during normal astrocyte development and growth arrest, (2) determine whether decreased NF1 expression results in increased astrocyte proliferation, and (3) generate transgenic mice with a targeted disruption of the NF1 gene restricted to astrocytes.

DESCRIPTION (Provided by applicant): Meningiomas are among the most common nervous system tumors and are prevalent in older adults, particularly women. The genetic events important in the molecular pathogenesis and malignant progression of sporadic meningiomas are only partially characterized. To date, the most frequently detected genetic alterations are loss of heterozygosity (LOH) on chromosome 22q and inactivation of the neurofibromatosis 2 (NF2) tumor suppressor gene, occurring in 40-60 percent of sporadic meningiomas. The NF2 gene product, merlin, is a member of the Protein 4.1 family of membrane-associated proteins. Recently, we identified another Protein 4.1 tumor suppressor gene, DAL- 1 (Differentially expressed in Adenocarcinoma of the Lung), that is lost in approximately 60 percent of meningiomas. We propose that the Protein 4.1 tumor suppressors, DAL-1 and merlin, are leptomeningeal cell growth regulators critical to the development and progression of meningiomas. In this grant, we hypothesize that DAL-1 operates as an independent and functionally distinct Protein 4.1 tumor suppressor in meningioma pathogenesis. We plan to test this by (1) determining DAL-l developmental expression and subcellular localization, (2) characterizing DAL- 1 effector protein interactions. and (3) analyzing the ability of DAL-1 to impair cell growth and motility. These studies are collectively designed to define the role of this novel family of growth regulators in meningioma tumorigenesis and progression.

The prognosis for malignant brain tumors (astrocytomas) remains essentially unchanged despite significant advancements in neuro-oncology and radiation therapy. Our ability to design targeted therapies for astrocytomas (gliomas) is heavily dependent upon a more complete understanding of the molecular pathogenesis of these tumors and the availability of appropriate preclinical models to test potential biological therapies. Genetic alterations in human astrocytomas differ between astrocytoma grades and involve gene products important for regulating (1) growth factor signaling pathways and (2) cell cycle progression. Studies from our laboratory have demonstrated that activation of p21-ras is a common feature of low and high- grade astrocytomas and that approximately 60 percent of GBMs harbor alterations in the rap1 signaling pathway. In addition, high-grade gliomas exhibit loss of PTEN/MMAC1 expression or epidermal growth factor receptor (EGF-R) amplification/activation, suggesting a role for these proteins in astrocytoma progression. Over the past year, we have developed transgenic mice with astrocyte-specific expression of EGF-R, EGF- RvIII and p21-ras (G12V). The B8 p2l-ras (G12V) transgenic mouse strain develops astrocytomas with a latency of 3-4 months that are histologically and biologically similar to human astrocytomas. In this proposal, we propose to employ transgenic mouse models to critically evaluate the hypothesis that abnormalities in growth factor signaling and cell cycle control genetically cooperate in the molecular pathogenesis of astrocytomas. Specifically, we wish to determine whether (1) abnormal ras and rap1 signaling in astrocytes is necessary or sufficient for astrocytoma development, (2) loss of PTEN/MMAC1 signaling or EGF-R alterations are associated with astrocytoma progression, and (3) abnormal rap1 and ras signaling in astrocytes combined with defective cell cycle control is associated with astrocytoma progression. The development and characterization of mouse models mimicking the histology and molecular pathogenesis of human astrocytomas would greatly advance our ability to treat human astrocytomas by serving as informative preclinical models to test novel therapeutic agents.

DESCRIPTION (provided by applicant): Funding is requested to support the next meeting of the National Neurofibromatosis Foundation International Consortium for the Molecular and Cell Biology of NF1 and NF2 to be held June 1-4, 2003 at the Hotel Jerome in Aspen, Colorado. Beginning with the first meeting in 1985, these conferences have served as the catalyst for many of the fundamental discoveries that have contributed major insights into these important diseases of the nervous system. These meetings provide a regular forum to bring together molecular and cell biologists, geneticists, clinicians, and others familiar with NF1 and NF2 to share their latest research findings and experiences. Moreover, these meetings have proven to be exceptional opportunities from scientists and clinical researchers to form new collaborative ventures. NNFF Consortia have been instrumental in the rapid advances in the molecular understanding ofNF 1 and NF2, including the cloning of the responsible genes, establishment of diagnostic tests, exploration of protein functions, development of anima models, and creation of a network of patients and clinicians to facilitate clinical trials. Given the rapid scientific progress and subsequent translation to clinical practice, it is necessary to hold these meetings annually to promote synergy, facilitate collaboration, and enable translational research. The 2003 Consortium meeting has been organized as an open meeting that will be advertised to the general scientific community. Each of six platform sessions will be co-chaired by two experts in the field who will guide discussion of the presented work and its relevance to the overall session topic. These sessions will also include invited speakers from the NF research community and other presentations to be selected from submitted abstracts. Participation by junior investigators will be encouraged and the organizers anticipate, on the basis of previous Consortium meetings, that a substantial percentage of the speakers will be young investigators. Speakers will be instructed to allow time for open discussion among all participants. In addition, loner talks by two keynote speakers who are leaders in scientific fields or emerging technologies that are directly relevant to NF research will be incorporated into the scientific sessions. Two poster sessions will also be held which will allow investigators to present abstracts not selected for one of the oral sessions. In addition to updating investigators working on NF 1 and NF2 on the latest research developments, this meeting will help to identify critical gaps in our knowledge as well as strategies and collaborations to address them. An improved understanding of the molecular basis of NF1 and NF2 will play an essential role in developing improved therapies for the complications of these disorders and has broad implications to the fields of developmental neurobiology and cancer research.

[unreadable] DESCRIPTION (provided by applicant): The optic pathway glioma (OPG), a brain tumor composed of neoplastic NF1-deficient astrocytes, is the second most common tumor in individuals affected with the neurofibromatosis 1 (NF1) tumor predisposition syndrome. While these tumors are often regarded as "benign" brain tumors, their continued growth can result in loss of vision and hypothalamic dysfunction (early puberty). Currently, therapy for NF1 OPG is based on the use of compounds that have been successfully employed to treat other low-grade brain tumors, including carboplatin and temozolamide. Unfortunately, tumor progression occurs in one-third of children, necessitating additional therapy. We have recently developed and extensively characterized a mouse model of NF1-associated OPG, in which low-grade optic nerve and chiasm tumors develop by 2 months of age. In an effort to provide an efficient approach for the identification, initial validation, and in vivo preclinical evaluation of new anti-cancer compounds suitable for the treatment of patients with NF1-associated brain tumors, we have initiated a multidisciplinary therapeutic discovery and preclinical chemotherapy testing program. Using this team-based approach, we have identified two novel drug candidates for NF1 -associated brain tumor therapy, rapamycin and AMD3100, which inhibit Nf1-/- astrocyte growth in vitro. In this project, we propose to employ the Nf1 mouse OPG model as a preclinical platform for anti-tumor drug evaluation. First, we plan to characterize the Nf1 mouse OPG model with respect to visual physiology and radiographic features as a function of tumor growth and in response to conventional human NF1 -associated brain tumor therapy. Second, we plan to evaluate rapamycin and AMD3100 as potential therapies for NF1-associated brain tumors. With the unique combination of a multidisciplinary team of scientists and clinicians focused on NF1-associated OPG therapeutics and the availability of a well-characterized mouse model for NF1- associated OPG, we are uniquely positioned to establish such a translational research program. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The cancer stem cell (CSC) hypothesis is predicated on the idea that not all cells have equal proliferative potential and that, in brain tumors, the cells with the greatest ability to proliferate and form new tumors have phenotypic and functional properties similar to normal stem cells (NSCs). Over the past few years, multiple investigators have shown that CSCs isolated from human glial cell tumors (gliomas and ependymomas) undergo self-renewal and multi-lineage cell differentiation, similar to normal NSCs. In addition, CSCs from these glial tumors when implanted into rodent brains generate tumors histologically identical to the parental tumors, suggesting that these stem cells can fully recapitulate the neoplastic phenotype in vivo. While these seminal studies clearly highlight the central role of stem cells in brain tumor formation, they also evoke important questions regarding the nature of the CSC and its relationship to the normal NSC. Recent evidence in our laboratory and others has raised the possibility that low- grade gliomas (pilocytic astrocytoma; PA) and ependymomas may arise from central nervous system (CNS) region-specific progenitors, based on the presence of distinct CNS region-specific molecular signatures. These findings suggest that stem cells may exist within specific CNS regions that each harbor different biological properties relevant to glial cell differentiation and glial tumorigenesis. In this regard, NSCs isolated from different mouse and human embryonic brain regions have been shown to display intrinsic differences in self-renewal, proliferation, and neurogenesis in vitro and in vivo. Moreover, tumor location is an independent prognostic factor for survival in children with astrocytoma. We hypothesize that the biological properties of glial cells and glial neoplasms are defined by unique genetic programs intrinsic to NSCs originating from different brain regions and that these differences dictate the impact of specific cancer-associated genetic changes on NSC self-renewal and differentiation relevant to glial tumor formation and growth. In this proposal, we plan to define the biological properties of NSCs from different brain locations and to determine the impact of one specific glioma-associated genetic change on brain region NSC biological properties. This study will provide new insights into the spectrum of NSC subtypes in the brain and their role in glial cell differentiation and glial tumor formation. The studies outlined in this proposal are focused on the understanding the intrinsic differences of NSCs from different brain locations and the impact of specific glial tumor- causing genetic changes on NSC properties relevant to glial tumor formation. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Neurofibromatosis 1 (NF1) is a common autosomal dominant disorder in which affected individuals are prone to the development of both benign and malignant tumors through loss of the neurofibromin protein. Two of the most clinically challenging tumors in NF1 are the optic glioma (astrocytoma) and the malignant peripheral nerve sheath tumor (MPNST). Recent studies from our laboratories and others have shown that both NF1- associated optic glioma and MPNST exhibit high levels of mammalian target of rapamycin (mTOR) pathway activation and proliferation of Nf1-deficient cells are inhibited by agents that block mTOR activity. Our ability to more effectively treat these tumors is heavily dependent on the identification and preclinical evaluation of new chemotherapeutic compounds that target this important NF1 growth regulatory pathway. The overall objectives of this proposal are to determine how neurofibromin regulates mTOR-pathway-mediated cell growth and to identify new anti-cancer mTOR pathway inhibitory compounds suitable for the treatment of NF1- associated tumors. In this proposal, we plan to determine how neurofibromin regulates mTOR pathway activation in vitro and in vivo. Next, we propose to determine how neurofibromin-mediated mTOR pathway regulation controls cell growth. Lastly, using both a standard luciferase reporter and a recently developed novel mTOR pathway reporter, we propose to employ high-throughput chemical library screening to identify new compounds that inhibit mTOR pathway activation and the growth of Nf1-deficient cells. The availability of genetically-engineered mice and human cell lines affords us a unique opportunity for translational research in which basic science discoveries can be evaluated as potential clinical therapies. Based on the recommendations of the recent Banbury conference on "Barriers and Solutions in the Use of Mouse Models to Develop Therapeutic Strategies for Neurofibromatosis-Associated Tumors", we plan to use Nf1- deficient astrocytes and Nf1-deficient human MPNST cells as "filters" to select mTOR inhibitory agents for future trials that have the greatest likelihood of succeeding in the clinic. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The tumor microenvironment plays an important role in tumor formation and progression by providing both negative and positive signals that influence tumor growth. Similar to normal brain development, brain tumor formation and growth in children may also be dictated in part by the presence of spatially- and developmentally-regulated cues that emanate from the surrounding stroma. Individuals with the inherited tumor predisposition syndrome, neurofibromatosis type 1 (NF1), develop low-grade gliomas (astrocytic neoplasms) along the optic pathway typically during early childhood. Using NF1 as a model system to understand the contribution of the tumor microenvironment to glioma formation and growth, we have shown that Nf1 loss in glial cells is insufficient for glioma formation unless coupled with an Nf1 microenvironment and that Nf1 microglia produce two growth factors, MGEA5 (hyaluronidase) and CXCL12 (stromal cell derived factor-1a;SDF-1a), that stimulate the proliferation and survival of Nf1-/- astrocytes, respectively. Based on these observations, we hypothesize that Nf1 microglia are a critical cellular component of the tumorigenic microenvironment and that MGEA5 and CXCL12 are key stroma-derived paracrine factors which promote Nf1-/- astrocyte and glioma growth in vitro and in vivo. The overall objective of this proposal is to identify the molecular basis for the abnormal cellular phenotypes of Nf1 microglia, to determine whether Nf1 microglia are required for Nf1 glioma formation and continued growth, and to define how these two stroma-derived paracrine factors, MGEA5 and CXCL12, control Nf1-deficient astrocyte growth in vitro and Nf1 glioma formation in vivo. PUBLIC HEALTH RELEVANCE: Similar to normal brain development, brain tumor formation and growth in children may be dictated in part by the presence of spatially- and developmentally-regulated cues that emanate from the surrounding stroma. Using neurofibromatosis type 1 (NF1) as a model system to understand the contribution of the tumor microenvironment to glioma growth, we propose to determine whether Nf1 microglia are required for Nf1 glioma growth and to define how two recently identified stroma-derived paracrine factors control Nf1-deficient astrocyte growth in vitro and Nf1 glioma formation in vivo. These studies provide an excellent opportunity to define the growth regulatory signals produced by the tumor microenvironment, and to develop future therapies that target the trophic relationship between brain tumors and their stroma for pediatric brain tumors.

DESCRIPTION (provided by applicant): Brain tumors (astrocytomas or gliomas) represent the leading cause of cancer-related death in children and the fourth leading cause in adults. While there have been considerable advances in our ability to partially arrest their growth by targeting the genetic and molecular changes within cancer cells, a substantial number of patients with gliomas succumb to their disease, develop secondary brain dysfunction as a result of treatment, or fail to regain normal neurologic function despite treatment. We hypothesize that improved patient outcome from brain tumor treatment requires that therapies consider the bi-directional interactions between neoplastic cells and non-neoplastic cells in the tumor surround. Over the past five years, we have developed and validated several genetically-engineered mouse models (GEMMs) of low-grade glioma that recapitulate the salient features of the human condition. In this application, we will exploit these accurate GEMM systems to understand (1) the role of the tumor microenvironment, including important immune system cells, in gliomagenesis and tumor growth, (2) the effects of glioma growth on normal neuronal function, and (3) the secondary effects of chemotherapy and radiation therapy on non-neoplastic brain cells. Using a cross-disciplinary approach, we aim to unravel the cellular and molecular determinants that underlie the bi-directional interactions between neoplastic cells and non-neoplastic cells in low-grade glioma, and use these insights to identify novel strategies aimed at improving the outcome of patients with these cancers. To this end, we have assembled a new team of investigators with prior involvement in large-scale cooperative research initiatives and expertise in mouse model generation (Dr. David Gutmann), stromal interactions in brain tumors (Drs. David Gutmann, Joshua Rubin), advanced small-animal imaging (Dr. Joel Garbow), genomic influences on tumorigenesis (Dr. Karlyne Reilly), and multi-modality imaging (Dr. Mark Ellisman). Finally, we have individually leveraged the rich research environments at Washington University, University of California-San Diego, and The National Cancer Institute to tackle this complex problem in cancer biology.

DESCRIPTION (provided by applicant): Normal mammalian brain development involves the regulated growth of progenitor cells and their differentiation into specialized cell types. Abnormalities in progenitor cell function during embryogenesis can lead to inappropriate expansion of stem cell populations and abnormal glial maturation, and potentially result in a number of brain abnormalities, including the formation of brain tumors in children. Neurofibromatosis type 1 (NF1) is one of the most common genetic conditions in which affected children develop glial cell tumors (optic pathway gliomas). Using NF1 as a model system to study neural stem/progenitor cell (NSC) function in vitro and in vivo, we have shown that loss of Nf1 protein (neurofibromin) function in embryonic NSCs results in (1) increased NSC proliferation and self-renewal and (2) increased glial lineage expansion. Based on our experimental observations, we hypothesize that neurofibromin is required for NSC maintenance and glial cell maturation in vivo. In this proposal, we have designed complementary in vitro and in vivo experiments to determine how neurofibromin controls NSC maintenance and glial cell differentiation. The overall objective of this proposal is to employ laboratory-generated genetically-engineered Nf1 mutant mice and Nf1-deficient NSCs as tractable experimental platforms to define the critical control mechanisms that govern NSC function in the developing central nervous system. PUBLIC HEALTH RELEVANCE: The most common genetic condition in which children develop brain tumors is neurofibromatosis type 1 (NF1). This proposal employs NF1 as an experimental model system to understand the role of neural stem cells in normal brain development in mice relevant to brain tumor formation. These studies may lead to the development of therapies that specifically target the critical growth and fate control pathways that cause brain tumors in children.

DESCRIPTION (provided by applicant): In summary, this proposal focuses on two of the five themes outlined in the RFA-OD-10-005: "Applying Genomics and Other High Throughput Technologies" and "Translating Basic Science Discoveries into New and Better Treatments" and is ready for immediate implementation. Brain tumors are complex microcosms in which bi-directional interactions between neoplastic cells and non-neoplastic cells drive tumorigenesis and tumor progression. Our limited knowledge of the complex interplay between tumor cells and the tumor surround is a considerable obstacle to fully appreciating brain tumor biology, and most likely represents a significant barrier to successful brain tumor treatment outcomes. Studies from our laboratories using genetically-engineered mice have demonstrated the obligate role of the tumor microenvironment (stroma) in dictating when and where gliomas form and grow. In particular, we have shown that resident non-neoplastic immune system-like cells (microglia) in these tumors elaborate specific growth-promoting factors critical for glioma formation and maintenance. In this proposal, we have assembled a team of investigators with expertise in cross-disciplinary science and prior involvement in large-scale cooperative research initiatives, and focused their efforts on identifying targetable molecules made by microglia present in the tumor microenvironment for preclinical evaluation. Specifically, we plan to extend our ongoing studies with The Genome Center at Washington University School of Medicine to use next-generation sequencing and bioinformatics pipelines to discover new markers and signals shared by mouse and human glioma-associated microglia, and which represent potential targets for stromal-directed therapy (Phase 1). Identified genes will be used to develop immunologic reagents for the subclassification of tumor-associated microglia (Phase 2a) as well as to discover novel glioma-promoting factors ("gliomagens") (Phase 2b). Monoclonal antibodies developed in Phase 2a will next be employed to classify microglia subsets into functionally distinct populations and to determine the relevance of specific microglia populations to clinical tumor behavior (Phase 3a). In addition, lead microglia gliomagens validated by lentiviral manipulation in Nf1 genetically-engineered mouse (GEM) strains (Phase 2b) will be evaluated by high-throughput technology using a stromal co-culture system to identify stromal-directed pharmacologic inhibitors for future preclinical therapeutic studies (Phase 3b). PUBLIC HEALTH RELEVANCE: Brain tumors (gliomas) are the leading cause of cancer-related death in children with few successful treatments available. This proposal applies genomics and novel high throughput technologies to translate basic science discoveries into new and better treatments for children with low-grade glioma.

DESCRIPTION (provided by applicant): As we enter into an era of personalized medicine, it becomes increasingly important to define the factors that confer disease risk and outcome. Since these determinants cannot be easily controlled in human epidemiological studies, genetically-engineered mouse (GEM) strains provide mechanistically-tractable platforms to define the factors underlying disease heterogeneity and translate them to risk assessment tools and treatments. Pediatric low-grade brain tumors (gliomas) represent one such challenging disease with respect to predicting clinical progression, optimizing treatment, and improving neurologic outcome. In the most common inherited cause for pediatric low-grade glioma, neurofibromatosis type 1 (NF1), 15-20% of children develop optic pathway gliomas (OPGs), leading to visual decline in 30-60% of affected individuals. Moreover, conventional chemotherapy results in disease stabilization in only 50-60% of children, and few experience improvement in their visual acuity following treatment. Currently, it is not possible to predict which child with a NF1-OPG will experience visual dysfunction (Barrier 1) and what treatments are most likely to result in tumor response and lead to visual recovery (Barrier 2). Over the past 10 years, as part of the National Cancer Institute Mouse Models of Human Cancers Consortium, we leveraged a collection of novel Nf1 GEM strains with optic glioma to establish that (1) the germline NF1 gene mutation partly determines NF1 protein expression and function, (2) female, but not male, Nf1 mutant mice with optic glioma have reduced visual acuity, (3) non-neoplastic immune system-like cells (microglia) carrying only a germline NF1 gene mutation are required for murine optic glioma formation and growth, and (4) murine optic gliomas contain glioma stem cells (optic GSCs) with unique, and potentially targetable, molecular and cellular properties. Based on these findings, we propose a systematic, team-based dissection of the factors responsible for NF1-optic glioma progression, vision loss, and therapeutic success. For this initiative, we have assembled a cross- disciplinary collaborative team to (a) define the molecular etiology for female susceptibility to OPG-induced visual decline (Aim 1), (b) assess the impact of the germline NF1 gene mutation on OPG-induced visual decline (Aim 2) as well as (c) microglia function relevant to potential stroma-directed glioma treatments (Aim 3), and (d) exploit a series of distinct GSCs from Nf1 optic glioma GEMs for developing new treatments that uniquely target the cells most responsible for maintaining the tumor (Aim 4). Collectively, these studies have immediate translatability to the human condition, given the wide clinical availability of NF1 genetic testing, recent advances in rapid cellular reprograming, and the existence of two large international consortia with proven expertise in NF1-OPG clinical assessment and therapeutic evaluation.

Project Summary As we enter into an era of personalized medicine, it becomes increasingly important to define the factors that confer disease risk and outcome. Since these determinants cannot be easily controlled in human epidemiological studies, genetically-engineered mouse (GEM) strains provide mechanistically-tractable platforms to define the factors underlying disease heterogeneity and translate them to risk assessment tools and treatments. Pediatric low-grade brain tumors (gliomas) represent one such challenging disease with respect to predicting clinical progression, optimizing treatment, and improving neurologic outcome. In the most common inherited cause for pediatric low-grade glioma, neurofibromatosis type 1 (NF1), 15-20% of children develop optic pathway gliomas (OPGs), leading to visual decline in 30-60% of affected individuals. However, it is not currently possible to predict which child with NF1 will develop an OPG or who will experience visual decline or blindness from their tumor. Our ability to identify those children at greatest risk for OPG development and vision loss would provide important clinically-meaningful prognostic information to guide clinical care for a pre-verbal patient population in which accurate visual assessments can be challenging. Recent observations suggest that the specific germline NF1 gene mutation may be one risk factor for OPG development, whereas patient sex influences OPG-associated visual decline. In this regard, children with NF1-associated OPGs are more likely to harbor specific types of germline NF1 gene mutations (5' end frameshift mutations). In addition, we have recently shown that female children and mice with NF1 more frequently experience visual loss from their OPGs. Based on these provocative findings, we hypothesize that the particular germline NF1 gene mutation and gonadal sex hormones are independent risk factors for OPG development and progression, respectively. In this proposal, we aim to critically determine how the specific NF1 gene mutation dictates OPG formation and define the molecular basis for the observed sexually-dimorphic OPG-associated vision loss using a novel series of Nf1 GEM strains and approaches. The resulting outcomes will be leveraged to preclinically evaluate new approaches to identifying children with NF1 at risk for OPG development and vision loss as well as potential alternative therapeutic approaches for attenuating or preventing NF1-OPG-related visual decline.

Project Summary As we enter into an era of personalized (precision) medicine, it becomes increasingly important to define the factors that confer disease risk and outcome. Since these determinants cannot be easily controlled in human epidemiological studies, genetically-engineered mouse (GEM) strains and human induced pluripotent stem cell (iPSC) lines provide mechanistically-tractable platforms to define the factors underlying disease heterogeneity and translate them to risk assessment tools and treatments. This challenge is nicely illustrated by Neurofibromatosis type 1 (NF1), a rare neurogenetic condition caused by a germline mutation in the NF1 gene. Individuals with NF1 are prone to the development of a wide variety of neurological problems, including cognitive and behavioral problems (60-70% of children) and low-grade brain tumors (~20% of children). While establishing the diagnosis of NF1 in an infant is straightforward, it is currently not possible to predict which child will develop future medical problems, determine whether there will be clinical progression requiring treatment, or institute effective therapies that specifically target the subtype of clinical manifestation in that individual. The pressing challenge for clinicians and researchers alike is to dissect the genetic, cellular, molecular, and systems-level etiologies for these common neurologic problems with the ultimate goal of developing prognostic and precision neurology approaches for children affected with NF1. In this proposal, we plan to leverage human NF1-patient iPSCs, mice engineered with patient germline NF1 gene mutations, bioinformatics and systems biology approaches, and novel modeling approaches to mechanistically define the factors that underlie the heterogeneity that characterizes NF1. The overall mission of this project is to establish the etiologic bases for NF1 clinical variability and to create a blueprint for future clinical application necessary to transform the care of individuals with NF1 from a reactive to a more proactive approach.