Charles G. Eberhart, MD PhD - US grants

DESCRIPTION (provided by applicant): Medulloblastomas, embryonal neoplasms arising in the cerebellum, are the most common malignant pediatric brain tumor. We propose several lines of investigation into the roles of c-Myc and N-Myc in medulloblastoma pathobiology. The primary investigator, Dr. Charles Eberhart, is a neuropathologist with a scientific background in Drosophila genetics, who plans a career as an independent clinician scientist researching brain tumors. To accomplish his goals he requires additional training in techniques commonly used to study tumors in the laboratory, including cell culture and mouse transgenic models. Pursuing the specific aims we propose will teach Dr. Eberhart these skills, and enable him to make the transition to an independent career in cancer research. Myc transcription factors are emerging as important modulators of medulloblastoma, biology. In Specific Aim 1 we investigate links between Myc levels in medulloblastoma specimens and clinical outcome. The effects of Myc are varied, and while several Myc targets have been isolated in fibroblasts and hematologic malignancies, it is likely that the profile of genes regulated by Myc will differ between tumor types. We therefore propose in Specific Aim 2 to identify c-Myc: targets in medulloblastoma cell lines by modulating c-Myc levels and evaluating the changes in gene expression profiles using microarrays. In Specific Aim 3 we confirm the clinical importance of these targets by analyzing their expression in medulloblastoma tumor samples with varying c-Myc levels. Finally, in Specific Aim 4 we propose developing a novel medulloblastoma transgenic model by overexpressing c-Myc in the cerebellum of transgenic mice.

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to determine how the Notch signaling pathway regulates the formation and growth of brain tumors, and to develop new brain tumor therapies based on Notch pathway inhibition. Notch receptors control the specification, proliferation and survival of stem and progenitor cells in the brain. Notch signaling is also implicated in tumorigenesis, including the formation and growth of brain tumors such as medulloblastoma and glioma. We have recently shown that the Notch2 gene is amplified and overexpressed in primary medulloblastoma/PNET, and promotes growth of medulloblastoma cell lines. In contrast, NotcM inhibits medulloblastoma cell proliferation. We are the first group to define an in vitro system in which two Notch receptors have opposing effects. We will create chimeras between Notch 1 and Notch2 and exploit this system to determine which domains of Notch differentially regulate proliferation and differentiation (Aim I). This should provide basic insights into the mechanism of Notch signaling, and also help guide our development of new therapies. To effectively treat brain tumors via Notch modulation, it will also be important to understand the mechanisms by which the pathway is turned on, as it must be blocked at or below the level of activation. In Aim II, we will therefore determine the roles of ligand stimulation and receptor mutation in activating the Notch pathway in medulloblastoma. A third aim is to assess the effects of Notch pathway blockade on tumor stems cells and tumor propagation. We have already shown that Notch pathway blockade inhibits proliferation and induces apoptosis of medulloblastoma in vitro. Recently, cells with stem- like properties have been isolated from freshly resected human medulloblastoma/PNET. This subset of neoplastic cells appears to be critical in propagating brain tumors, functioning as "tumor stem cells." Targeted therapies causing tumor stem cells to differentiate or die therefore represent a novel therapeutic avenue with great promise. We hypothesize that the Notch pathway, which controls neural stem cell survival and proliferation during normal fetal development, will also be required for tumor stem cells survival, and will test this in Aim III. Finally, it is not known if persistent activation of Nbtch2 is sufficient to initiate the formation of medulloblastoma. In Aim IV, we will introduce activated Notch2 into cerebellar precursor cells and assess its transforming capacity. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this proposal is to determine if malignant brain tumors are sensitive to the Chinese herbal remedy Zen Sheng Ping (ZSP, also known as antitumor B). ZSP is a combination of six herbs which has been shown to prevent the progression of pre-neoplastic lesions of the esophagus. It also appears to slow or prevent progression of lung cancer in both humans and in animal models. It was recently found that ZSP downregulates the Notch signaling pathway in lung cancer. The Notch pathway plays a critical role in the proliferation of lung tumor cells, suggesting Notch inhibition is an important mechanism by which the anti-tumor effects of ZSP are mediated. This raises the possibility that ZSP might also successfully treat other Notch- dependant tumors. We have shown that Notch signaling plays a critical role in the growth of several types of brain cancer, including the most common malignant brain tumors in children (medulloblastoma) and adults (glioblastoma). Other Notch-dependent neoplasms that might be treated using ZSP include pancreatic, breast and ovarian carcinomas, as well as melanoma. Small-molecule Notch inhibitors appear block the formation and growth of brain tumor xenografts by depleting stem-like cancer cells, but this class of compounds has been associated with significant toxicity. We therefore plan to investigate whether ZSP might represent a more potent and/or less toxic treatment for malignant brain tumors by blocking Notch activity. Project Narrative: The goal of this proposal is to determine if malignant brain tumors can is treated using a Chinese herbal remedy known as Zen Sheng Ping (ZSP). It was recently found that ZSP inhibits an important molecular pathway known as Notch in cancer cells. We have shown that Notch signaling plays a critical role in the growth of several types of brain cancer, including the most common malignant brain tumors in children (medulloblastoma) and adults (glioblastoma)

DESCRIPTION (provided by applicant): No cures exist for patients with glioblastoma (GBM) due to the resistance of tumor cells to standard therapies. Stem-like tumor subpopulations seem especially refractory to most treatments, and it is becoming increasingly clear that specific tumor microenvironments can promote stem cell properties and chemoresistance. However, poor understanding of how emerging targeted therapies interact with other agents and the tumor microenvironment has limited their development. The long-term goal of the project is to develop Notch inhibitors as effective new therapies for glioblastoma and other malignant brain tumors. The objective of this proposal is to elucidate how Notch interacts with the tumor microenvironment and other treatments so pathway inhibitors can be effectively used in the clinic. The Notch pathway, which is required for generation and maintenance of non-neoplastic neural stem cells, also plays a key role in GBM cancer stem cells (CSC). It has been shown that the perivascular microenvironment promotes CSC through activation of Notch signaling, and a number of agent targeting blood vessels are currently in use. As tumor-associated blood vessels are removed, GBM and other tumors shift towards a hypoxic phenotype, and it is less clear how Notch will function in this microenvironment. The central hypothesis to be tested in this proposal is that Notch is a key mediator of GBM differentiation and therapeutic response not just in the perivascular niche, but also in the hypoxic microenvironment. Indeed, emerging data suggest that hypoxic tumor cells can recapitulate many of the molecular features which define the perivascular niche, and that Notch induces a stem-like phenotype and modulates the response to traditional chemotherapy in this context. The first two specific aims focus on understanding how Notch is activated in hypoxic GBM cells, and determining if Notch blockade can reverse the increase in CSC and treatment resistance promoted by hypoxia. The second two specific aims focus on the interaction between Notch inhibition, radiation, and temozolomide chemotherapy, and investigate a novel mechanism by which Notch blockade can sensitize GBM to this commonly used alkylating agent. These studies will determine how Notch activity is regulated in hypoxic glioma cells, and to establish a requirement for Notch in CSC induction and aggressive tumor behavior in the hypoxic microenvironment. They will also examine a novel epigenetic mechanism by which the pathway can modulate MGMT expression and temozolomide sensitivity. These results are all of high clinical relevance, and will have a direct impact on the development of a novel agent targeting CSC in glioblastoma.

Project Summary/Abstract Pathologists are uniquely positioned to contribute to cancer research. Their familiarity with disease classifications and pathogenesis, physical manifestations of tumors in tissue, and clinical challenges in cancer care mean that these investigators can provide important perspectives in developing experimental models that recapitulate key aspects of human disease. The Opportunities for Pathology Trainees In Cancer research (OPTIC) program at the Johns Hopkins University School of Medicine (JHUSOM) supports postdoctoral trainees developing research-oriented careers in cancer pathology and is geared towards preparing them for leadership roles in academic pathology and laboratory-based cancer research. Over the first funding period, the mandate of the OPTIC program has been to attract and recruit the brightest pathology trainees interested in cancer research; to pair them with faculty mentors representing the remarkable breadth and diversity of cancer research at JHUSOM; to protect their time for an immersive experience in the research laboratory; and to involve them in a unique and program-specific educational curriculum. With four postdoctoral trainees each year, the program is overseen by internal and external oversight committees consisting of highly successful academic pathologists in cancer research. Fellows select a research mentor from participating faculty ? a stellar group recruited from the full spectrum of cancer research fields at the institution. All are accomplished investigators with an expertise in experimental cancer research and strong track records of laboratory-based mentorship. In addition to pursuing a rigorous research program, trainees participate in program-specific didactics and workshops to develop skills for their independent careers, and enroll in other course work tailored to their individual interests and chosen to complement their previous experiences. Fellows create an Independent Development Plan with their mentor, each trainee convenes meetings of an Individual Progress Committee, members of which provide feedback on their progress, recommend specific near-term research goals and training activities, and are generally be available as career mentors and advocates. Fellows leave the program poised for transitions to mentored and independent faculty positions in academic pathology departments and fully prepared to conduct high impact cancer research.

Project Summary Retinoblastoma is an aggressive tumor of the retina and the most common intraocular cancer in childhood, causing about 4,000 deaths annually worldwide. While the primary tumor can be successfully treated mostly by systemic and/or local chemotherapy, metastases in the central nervous system (CNS) or in distant organs, such as bone and bone marrow, are more resistant to therapy and still remain the leading cause of death due to retinoblastoma. Since the molecular factors driving metastatic spread are still not understood, there is an urgent and unmet need to elucidate the pathways responsible for retinoblastoma invasion, in order to find new therapeutic targets to block dissemination of this aggressive childhood malignancy. The overall goal of this project is therefore to identify invasion-promoting molecular pathways in retinoblastoma, and to develop targeted therapies which will prevent its spread outside the eye and growth at distant sites. Here we propose to investigate the Nodal/TGF-? signaling as a new strategy to efficiently inhibit retinoblastoma invasion and growth into the CNS, since we found that this pathway was potently upregulated in all cases of invasive retinoblastoma that we analyzed by next generation RNA sequencing, as compared to non-invasive cases. Therefore, our central hypothesis is that targeting Nodal/TGF-? signaling represents a new approach to inhibit retinoblastoma invasion and metastatic growth. To test this hypothesis, we plan to repress this pathway either genetically, by short hairpin RNA (shRNA), at both the ligand and the receptor level, or pharmacologically, using chemical inhibitors of the Nodal/TGF-? signaling. We will investigate in vitro the effects that the pharmacological or genetic inhibition of the Nodal/TGF-? signaling might have on invasion, overall growth, proliferation, clonogenicity, survival, cell death, and differentiation, in multiple patient-derived retinoblastoma cell lines, established from either primary tumors or vitreous seeds. Importantly, we will pursue these aims also in vivo, using an innovative orthotopic model of retinoblastoma invasion in zebrafish, which we have recently established in our lab, for monitoring and quantifying retinoblastoma growth and invasion using longitudinal intravital imaging methods. This model will facilitate our efforts by providing a rapid and economical method to test the effects of various genetic and pharmacological manipulations in an in vivo setting. However, to ensure robust and reproducible data across species, confirmatory testing will be performed using intraocular murine xenografts as well.