Nancy Ratner - US grants

Individuals with neurofibromatosis type 1 (NF1) carry mutations in the NF1 tumor suppressor gene and develop benign peripheral nerve sheath tumors (neurofibromas) that cause significant morbidity, and mortality if they compress vital structures, and which can transform to malignant peripheral nerve sheath tumors (MPNST). Neurofibromas contain normal nerve constituents: axons, Schwann cells, fibroblasts, macrophages and mast cells, and aberrant Schwann cells free of axons. Tumorigenesis results from complete loss of function at NF1, as neurofibroma Schwann cells are characterized by biallelic mutations in NF1, with other cell types recruited secondarily. We developed mouse model systems that are accurate mimics of neurofibroma formation in NF1, and performed large scale gene expression array analyses to identify candidate genes relevant to neurofibroma formation. Our robust Preliminary Data shows that a chemokine receptor, Cxcr3, is crucial for neurofibroma formation. In this application, we propose to use unique mouse models, neurofibroma Schwann cell precursors and Schwann cells to define the chemokine pathways through which Nf1 loss in Schwann cells leads to the presence of Cxcr3+ cells in neurofibroma (Aim 1) and to define CXCr3-expressing cells in neurofibroma and test their function (Aim 2). Finally, we will define molecules that are overexpressed in neurofibroma Schwann cells and are necessary for their survival (Aim 3). Together these studies will identify cellular and molecular underpinnings of tumor formation in the nervous system, and identify therapeutic targets for the treatment of NF1.

[unreadable] DESCRIPTION (provided by applicant): About 10% of all intracranial tumors are schwannomas, benign tumors comprised of Schwann cells with biallelelic NF2 mutations. NF2 mutation or loss is believed to cause schwannoma formation. Several lines of evidence implicate merlin, the NF2 protein, in small G-protein signaling. Biochemical evidence and our analysis of primary schwannoma cells suggest that merlin functions in Rac signaling. While normal Schwann cells are polarized, with one surface attached to axons and the other to basal lamina, schwannoma cells are unassociated with axons. This loss of polarity is likely to arise secondary to altered Rac signaling. Because the relevance of the Rac signaling pathway to Schwann cells is not known, we propose to evaluate a merlin-Rac cascade in Schwann cell growth and tumorigenesis. Our specific hypothesis is that merlin limits the duration of Rac signaling in Schwann cells. We further postulate that deregulated Rac signaling accounts for the failed axon-glial interactions characteristic of schwannomas. We plan to use a combination of gain-of-function and loss-of-function mouse models and primary cells from human schwannomas to test these hypotheses. We believe that analysis of primary schwannoma cells offers a rare opportunity to study a pure population of early stage human cancer cells. The proposed studies are expected to provide insight into regulation of normal axon-glial interactions, and to lead to strategies to understand and ultimately treat human schwannomas. [unreadable] [unreadable] [unreadable]

The administrative core for the Cincinnati Center for Neurofibromatosis Research will function to co-ordinate fiscal management of the grants and integration of the separate research projects into a cohesive whole. The responsibilities of the administrative core are: management of the individual project and core budgets to ensure tight fiscal control over expenditures, preparation of subcontracts, and provision of common administrative services including frequent communication by phone conference and teleconference, as well as face-to-face meetings among the project leaders, and integration of the scientific advisory boards into the projects by meeting organization. The core will also administer a pilot grant program.

[unreadable] DESCRIPTION (provided by applicant): In response to an RFA from the National Institute of Neurological Disorders and Stroke, we propose to establish a Cincinnati Center for Neurofibromatosis Research. Our goal is to identify and therapeutically target signaling pathways that underlie peripheral nerve tumors resulting from NF1 loss of function. To accomplish this goal we have brought together three groups to attack NF1 tumors with genetic, molecular and cellular approaches to preclinical testing. In Project 1, George Thomas & Sara Kozma will investigate the contribution of the RAF, PI3K and PKA signaling pathways in the development of NF1, whether the effects of these pathways are mediated through constitutive activation of S6K1, and the impact of novel pharmacological inhibitors in models of NF1 in cell culture and in the mouse. Working closely with John Perentesis this group will initiate combinatorial studies of drugs first in cell based viability assays, then in xenografts as translational models for human clinical studies. In Project 2, Nancy Ratner uses these same models and new mouse strains to test the novel hypothesis that apparently non-Ras signaling in NF1 can actually be attributed to the Ras-related protein TC21, and use the same drugs tested in Project 1 for their ability to block cell migration. TC21 signaling cascades and migration will be tested as targets for novel therapies. In Project 3, David Largaespada will use an innovative transposon insertional mutagenesis strategy in mice to screen for genes that modify peripheral nerve tumorigenesis. Identified genes will lie in signaling pathways that represent next generation targets for therapeutic strategies. All projects use an Administrative Core (Ratner, PI), a Mouse Xenograft Core (Timothy Cripe, PI) and a Pathology Core (Tilat Rizvi, PI). Our long-term goal is to translate research findings into patient therapies. Cincinnati Children's Hospital has an outstanding pediatric NF1 clinic with over 800 patients in its database, has participated in ongoing NF1 clinical trials, and participates in a new OOD NF Clinical Consortium project. It is also a leading center for pediatric anticancer drug development and a member of the NCI's Pediatric Phase I Consortium. Together these projects bring together a world class group of investigators to identify and stratify drugs for use in NF1 treatment, in an environment that can facilitate clinical trials. [unreadable] [unreadable] [unreadable]

Patients with neurofibromatosis type 1 (NF1) develop benign peripheral nerve sheath tumors called neurotlbromas. Within neurofibromas, biallelic mutations in the NF1 gene are found in tumor Schwann cells, indicating that tumorigenesis requires complete loss of function at NF1 in tumor Schwann cells. Malignant progression of neurofibromas to malignant peripheral nerve sheath tumors occurs in about 10% of NF1 patients. No therapies currently exist for neurofibroma or MPNST. The NF1 protein, neurofibromin, is a GTPase-activating protein (GAP) for Ras proteins, molecular switches that control multiple signaling cascades. Loss of neurofibromin leads to increased levels of Ras-GTP in Schwann cells. We used in vitro model systems for Schwann cell tumorigenesis in NF1 to clarify signaling pathways underlying tumorigenesis. Surprisingly, we found that increased migration and cAMP accumulation in Nf1 mutant Schwann cells was not accounted for by Ha-Ras-GTP. Rather, these phenotypes may require the related and little studied Ras protein TC21/R-Ras2. TC21 uses downstream effectors differently from other Ras proteins. We propose to critically evaluate the relevance of TC21 to formation of neurofibroma and MPNST. Specifically, in Aims 1 &2 we will define cellular and molecular effects of TC21 in Nf1 mutant Schwann cells, and human MPNST xenografts. In Aim 3 we will use mouse models to test whether loss of TC21 delays or blocks neurofibroma or MPNST formation Together these studies are anticipated to identify novel intervention points at which to treat human NF1 disease.

DESCRIPTION (Provided by the applicant): Tumors of the nervous system are the second most common types of neoplasm in the pediatric population. In contrast to the most common cancer (leukemia), strides have been made in improving outcomes for some nervous system tumors but many remain difficult or impossible to treat effectively. There is opportunity to dramatically improve outcome for nervous system cancer in children through development of new therapies. It is our hypothesis that interactive programs based in developmental biology studying individual nervous system tumor types will identify critical differences and similarities in molecular processes and extend the applicability of new therapies. Animal models and neural stem/progenitor cell models systems are now being used to study nervous system cancers and for preclinical development of novel therapies for nervous system tumors. Whole genome approaches are adding insight into developmental regulation and carcinogenesis. The goal of this proposal is to contribute to development of a multi-disciplinary, world-class Neuro-Oncology focus group within the Cincinnati Children's Hospital aimed at attacking the problem of neural tumors. Taking advantage of our institutional strengths in developmental biology and informatics, this will be accomplished by recruitment of 2 new Tenure Track faculty members. One is a developmental biologist with interest in the hippo signaling pathway and the other a neural tumor focused bioinformatician.

DESCRIPTION (provided by applicant): The Neurofibromatosis (NF) Conference has been organized by the Children's Tumor Foundation (CTF) annually since 1985. With its roots in a small workshop-style gathering of a group called the 'NF Consortium'dedicated to cloning the genes underlying the Neurofibromatoses, the NF Conference has grown to a gathering of three hundred NF researchers and clinicians from around the world. This meeting is recognized as the premier annual gathering of international NF researchers and physicians. Major contributors to this growth are the significant advances made in NF research in recent years and, particularly, recent advances into clinical trials and drug therapy development. In addition, state-of-the-art genomic approaches have identified mutations in the NF genes as contributors to numerous types of sporadic cancers, signaling pathways including the Hippo and Ras cascades are implicated in NF, and it is now recognized that mutations in genes along the Ras pathway result in a set of disorders, including NF1, called the "Rasopathies". Traditionally a forum for research information exchange and consensus building, in recent years the NF Conference is also the principal international forum for reporting on the neurofibromatosis preclinical therapeutic pipeline and the expanding arena of neurofibromatosis clinical trials.

DESCRIPTION (provided by applicant): We carried out two large screens to identify genes driving MPNST formation or maintenance: 1) An shRNA screen in human MPNST cell lines, and 2) a Sleeping Beauty (SB) transposon- based insertional mutagenesis screen in a mouse model of MPNST. Both research approaches identified Wnt/?atenin-regulated pathways as critical mediators of MPNST maintenance. Preliminary data confirms that the Wnt/?atenin pathway is required for MPNST maintenance and can be targeted using small molecules. Remarkably, we identified multiple mechanisms of activation of ?atenin that operate in MPNST. Our proposal focuses on these. Aim 1 is based on our data demonstrating that genetic or pharmacological activation of the ?atenin destruction complex reduce ?atenin levels, reduce target gene expression, and inhibit cell survival and proliferation in MPNST. Aim 2 is based on data showing that some MPNSTs ectopically express a Wnt/?atenin activator R-spondin 2 (RSPO2) due to transcript fusion with the upstream EIF3E gene, a mechanism we recently identified in human colorectal cancer. Aim 3 is based on data revealing that the ?atenin target gene PITX2 plays a critical role in MPNST cell survival. Our goals are two-fold: to define the molecular landscape of ?atenin de- regulation in MPNST, and perform a thorough pre-clinical evaluation of critical targets for intervention in the Wnt/?atenin pathway in MPNST, setting the stage for effective clinical testing in human patients. The Co-PIs have established a successful collaborative relationship built on complementary skills and resources. Abundant and orthogonal preliminary data support the basic hypothesis of this proposal.

DESCRIPTION (provided by applicant): Malignant peripheral nerve sheath tumors (MPNSTs) are among the most devastating and intractable of the soft tissue sarcomas. MPNSTs are also the most common neurofibromatosis type 1 (NF1)-associated cancer and a leading cause of death in NF1 patients. Taking advantage of the expertise our team has built over the past five years testing single agent treatments in mouse models of MPNST, we propose a set of combination pre-clinical tests to support a next generation of clinical trials. The NF1 gene product, neurofibromin, is an off signal for Ras proteins, resulting in aberrant Ras-Raf-MEK signaling in MPNST cells. Our recent publication shows that a MEK inhibitor effectively delays MPNST growth. In addition we found that an Aurora kinase A (AURKA) inhibitor stops tumor growth. Preliminary data show that a small molecule in combination with an oncolytic virus actually shrinks tumors and induces some cures. This proposal leverages the complimentary expertise of two PIs. In Aim 1Dr. Cripe will lead the studies exploring small molecules combined with oncolytic viral therapy. In Aim 2 Dr. Ratner will combine small molecules to either target G1/S with G2/M inhibition (horizontal inhibition) or enhance G2/M inhibition (vertical inhibition). Together the studies are anticipated to discover therapeutic combinations that cure MPNST in model systems, and can rapidly be moved into clinical trials.

DESCRIPTION (provided by applicant): Ras proteins are molecular switches that in their GTP-bound state control intracellular signaling cascades. Ras signaling is inactivated by GTPase-activating proteins (GAPs), including the tumor suppressor NF1. NF1 loss of function mutations have recently been implicated in driving neuroblastoma, brain, lung, thyroid and other tumor types and implicated in resistance to therapy. In addition, inherited NF1 mutations result in Neurofibromatosis type 1, a disease in which patients develop incurable benign peripheral nerve sheath Schwann cell tumors called neurofibromas. NF1 patients are also predisposed to aggressive sarcomas, MPNST, which are a leading cause of death in NF1 patients. The goals of this application are two-fold. We shall use NF1 loss of function to delineate Ras-specific pathways in neurofibroma cells. This is because NF1 is a GAP for all Ras proteins: H-, N-, K-, M-, R-Ras and RRas2/TC21, and may have additional, non-Ras, functions. When there is complete loss of NF1, all Ras proteins are activated, so that NF1 models provide a unique opportunity to study the contributions of individual Ras proteins to tumorigenesis in vivo. In Aim 1 we will test whether loss of H-Ras specifically delays or blocks neurofibroma formation in mouse models driven by NF1 loss of function, alone or in combination with RRas2 loss. We will also determine if H-Ras activation is sufficient to promote neurofibroma formation. In Aim 2 we will test if Ras activation is sufficient for neurofibroma formation, using a new Nf1 mutant allele and use specialized Schwann cell systems to define Ras-specific effects in NF1 mutant cells. In Aim 3 we will validate our state-of-the-art exome sequencing to identify Ras-related signaling pathway mutations in Schwann cells that co-operate with NF1 loss of function to promote NF1 tumorigenesis. Together these studies will define Ras isoform specific functions in cancer cells, and clarify molecular signals that drive neurofibroma formation.

? DESCRIPTION (provided by applicant): Neurofibromatosis type 1 (NF1) Rasopathy patients are predisposed to macrocephaly and to enlarged white matter tracts. NF1 patient brains also have abnormal bright spots on imaging, possibly representing abnormal myelin. We hypothesized that white matter alteration contributes to the cognitive, behavior and/or motor/sensory deficits that are common features of NF1 patients. We genetically engineered mice with Nf1 mutant brain oligodendrocytes and found that they have aberrant myelin and enlarged white matter tracts. Remarkably, altered signaling in oligodendrocytes also resulted in non-cell autonomous effects leading to increased permeability of the critical blood brain barrier. The Nf1 protein, neurofibromin, is an off signal for Ras GTPases, and mice with elevated Ras-GTP had the same cellular phenotypes as Nf1 mutants and were hyperactive. Downstream of Ras, loss of Nf1 in oligodendrocytes caused high reactive nitrogen species and most cellular abnormalities were reversed by anti-oxidant or nitric oxide synthase inhibition. These new models provide a unique opportunity to study oligodendrocyte effects on brain function, and regulation of the BBB. We plan a multi-faceted approach based on a collaborative team with specialized skills. In Aim 1, we will use a new flow cytometry (FACS) approach and mouse genetics to delineate signaling pathways that cause high ROS/NO in oligodendrocytes, because identified pathways should be relevant therapeutic targets in Rasopathy patients. In Aim 2, we will test if oligodendrocytes contribute to behavioral changes in NF1, as in the Ras mice we studied, and genetically define the critical NOS enzyme. In Aim 3, we will compare brains in which all brain cells or only oligodendrocytes have mutations in Nf1 using cell-type specific control of activity (optogenetics), advanced imaging (whole-brain functional MRI of awake mice) and electrophysiology. These studies will define aspects of brain structure, impulse conduction and changes at the level of brain cell populations in functional connectivity mediated by oligodendrocytes. They will also begin to identify how oligodendrocyte NO signaling contributes to cognitive and motor/sensory function, and to homeostasis of the blood brain barrier. We aim to provide preclinical support for human Rasopathy therapies that can be tested using imaging endpoints we identify.

DESCRIPTION (provided by applicant): We carried out two large screens to identify genes driving MPNST formation or maintenance: 1) An shRNA screen in human MPNST cell lines, and 2) a Sleeping Beauty (SB) transposon- based insertional mutagenesis screen in a mouse model of MPNST. Both research approaches identified Wnt/?atenin-regulated pathways as critical mediators of MPNST maintenance. Preliminary data confirms that the Wnt/?atenin pathway is required for MPNST maintenance and can be targeted using small molecules. Remarkably, we identified multiple mechanisms of activation of ?atenin that operate in MPNST. Our proposal focuses on these. Aim 1 is based on our data demonstrating that genetic or pharmacological activation of the ?atenin destruction complex reduce ?atenin levels, reduce target gene expression, and inhibit cell survival and proliferation in MPNST. Aim 2 is based on data showing that some MPNSTs ectopically express a Wnt/?atenin activator R-spondin 2 (RSPO2) due to transcript fusion with the upstream EIF3E gene, a mechanism we recently identified in human colorectal cancer. Aim 3 is based on data revealing that the ?atenin target gene PITX2 plays a critical role in MPNST cell survival. Our goals are two-fold: to define the molecular landscape of ?atenin de- regulation in MPNST, and perform a thorough pre-clinical evaluation of critical targets for intervention in the Wnt/?atenin pathway in MPNST, setting the stage for effective clinical testing in human patients. The Co-PIs have established a successful collaborative relationship built on complementary skills and resources. Abundant and orthogonal preliminary data support the basic hypothesis of this proposal.

Project Summary/Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer-predisposing syndrome afflicting ~1 in every 3,500 persons worldwide with the majority of patients developing benign plexiform and/or dermal neurofibromas. Plexiform neurofibromas constitute a lifelong source of disfigurement, morbidity and mortality, and have the potential to transform to a malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In fact, approximately 15% of NF1 patients develop poor prognosis MPNSTs, often in the second or third decade of life. Treatment options for MPNSTs are limited to complicated surgical procedures and classical chemotherapy and, so far, molecular targeted therapies have demonstrated limited efficacy. We desperately need new treatment options for the MPNSTs and methods to prevent them from development. It was recently found that plexiform neurofibromas progress to MPNST via an intermediate, ?atypical? neurofibroma (ANF) that in 70% of tumors shows heterozygous or homozygous loss of CDKN2A the gene encoding p16INK4a and p14ARF (Beert et al., 2011; Pemov et al., 2018). Our proposal will address critical unmet needs in this field, including better in vitro and in vivo models of ANF and identification of critical vulnerabilities of these cells. To provide a model for preclinical testing and prevention of ANF to MPNST development, we combined Desert hedgehog (Dhh)-Cre driven biallelic deletion of Nf1 with heterozygous loss of Cdkn2a, creating a unique model of transplantable ANF developing within pre-existing neurofibroma (Chaney et al., submitted). We also combined Dhh-Cre driven biallelic deletion of Nf1 and Pten, generating rapidly developing perinatal ANF-like lesions (Keng et al., 2012). ANF from Dhh-Cre;Nf1fl/fl;Cdkn2a+/- mice grafted subcutaneously into immunocompromised hosts grew, after a delay, providing a more rapid, tractable, transformation system. We plan a complete transcriptome and exome analysis in these models (Aim 1a), and further investigate the model by identifying and validating cell populations and markers altered in mouse and human PNF, ANF, and MPNST in unperturbed tissue sections using a new image analysis method called CO-Detection by IndEXing (Aim 1b). Modulation of the immune environment is increasing used therapeutically. We will therefore define the influence of the nerve microenvironment and immune system on progression from ANF to MPNST (Aim 1c). To identify ANF vulnerabilities, we have completed drug and CRISPR-based genetic synthetic lethality screens in isogenic immortalized human Schwann cells that are NF1 wildtype or were made homozygous for NF1 loss of function mutations using gene editing. Candidate drugs that inhibit PP2A, and other novel targets from the drug screening effort, will be tested for their effects in ANF- like cells in vitro (Aim 2a) and, when successful, in our unique GEMMs (Aim 2b). Similarly, our genetic screening effort will be used to define additional vulnerabilities tested in vitro (Aim 2c) and in vivo (Aim 2d).