Robert J. Wechsler-Reya, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The cerebellum plays a critical role in motor coordination and learning. Proper development of the cerebellum requires a delicate balance between proliferation of neuronal precursors and differentiation of these cells into neurons. The proliferation of the major cell type in the cerebellum, the granule cell, is regulated by the secreted molecule Sonic hedgehog (Shh). But the signals that cause granule cells to stop proliferating and differentiate are unknown. We have found that basic fibroblast growth factor (bFGF) is a potent inhibitor of Shh-induced proliferation, suggesting that it might be a key regulator of granule cell cycle exit and differentiation. The studies described here are aimed at determining the mechanisms of FGF effects on granule cell precursors and the importance of FGF signaling for normal granule cell development. These studies will not only lend insight into the molecular mechanisms that control granule cell differentiation, but will also have important implications for our understanding of Shh and FGF interactions in other parts of the nervous system. Moreover, by deepening our understanding of cell cycle regulation in normal granule cell precursors, these studies may shed light on the loss of cell cycle control in medulloblastoma, the most common malignant brain tumor in children [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Medulloblastoma is the most common malignant brain tumor in children. Its rapid growth and tendency to spread through the nervous system make it extremely difficult to treat, and more than 40% of the children who develop the disease die from it. Improved treatment of medulloblastoma is likely to come from a deeper understanding of the signals that control normal cerebellar development, and an appreciation of how these signals are dysregulated in tumors. To identify such signals, we have studied an animal model of medulloblastoma - the patched mutant mouse - and identified genes whose expression is altered in tumor cells compared to granule cell precursors (GCPs), the cells from which the tumor is believed to arise. Among the genes whose expression decreased most significantly was Unc5c, which encodes a receptor for the netrin family of signaling molecules. Unc5c was originally described as a regulator of cell migration, but has recently been shown to play an important role in apoptosis as well. Moreover, Unc5c is deleted or mutated in a variety of cancers, and has therefore been suggested to function as a tumor suppressor. We hypothesize that Unc5c controls migration and survival of GCPs during normal cerebellar development, and that its loss contributes to the abnormal migration and increased survival observed in medulloblastoma. If this hypothesis is correct, it will have important implications for our understanding of medulloblastoma, and open up new avenues for treatment of the disease. To test our hypothesis, we propose to: 1) Determine whether Unc5c regulates inward migration of granule cell precursors and tumor cells 2) Test whether Unc5c regulates survival of granule cell precursors and tumor cells 3) Determine whether loss of Unc5c is required for medulloblastoma formation Relevance to Public Health: One of the greatest challenges in medulloblastoma treatment is the ability of tumor cells to migrate into regions where they would not normally go, and to survive once they get there. Our observation of altered Unc5c expression in medulloblastoma is significant because it can contribute to both of these behaviors. By elucidating the role of Unc5c in migration and survival, our studies will shed light on the molecular mechanisms that underlie the aggressive growth and dissemination of medulloblastoma. This, in turn, will pave the way for developing new treatments that can be used to fight this devastating disease. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of our experiments is to image pre-neoplastic lesions in patched mutant mice, a model for human medulloblastoma. Our previous studies indicate that 75-85% of these animals have pre-neoplastic cells on the surface of their cerebellum, although only 15% will go on to develop full-blown tumors. We are interested in determining the size, shape and prevalence of these lesions, and in tracking them to determine what happens to them when they do not become tumors. These studies will involve two phases. Phase I: MR histology to determine the characteristics of pre-neoplastic lesions and the sensitivity and resolution with which they can be detected. This will involve high-resolution imaging of 4-6 mice after perfusion. Phase II. Serial in vivo imaging of 4-6 animals to track the disappearance of pre-neoplastic lesions and/or the development of these lesions into tumors. The feasibility of Phase II will be determined during Phase I. Together these studies will provide important insight into the mechanisms of tumor progression in medulloblastoma.

DESCRIPTION (provided by applicant): The cerebellum is critical for motor coordination and cognitive function. Granule neurons are a key component of cerebellar circuitry and are thought to represent the cell of origin for the pediatric brain tumor medulloblastoma. While the development of granule neurons has been studied in detail, the signals that control their growth and differentiation remain unclear. During postnatal development, proliferation of granule neuron precursors (GNPs) is driven by the mitogen Sonic hedgehog (Shh), which is produced from neighboring Purkinje cells. Interestingly, GNPs also proliferate during embryonic development, at a stage prior to Shh secretion;the mitogen responsible for this proliferation is unknown. Our preliminary studies indicate that a mitogenic activity is present in extracts from the embryonic cerebellum. Here we propose to identify this mitogen and study its role in cerebellar development. Specifically, we aim to (1) Use tissue microdissection and cell sorting to determine the source of the mitogen, and (2) Use expression cloning to identify the mitogen at a molecular level. These studies will not only provide insight into the mechanisms that control cerebellar development, but will also identify novel signaling pathways that may be important in medulloblastoma formation. PUBLIC HEALTH RELEVANCE: Identifying the signals that control production of neurons in the cerebellum may have important implications for understanding diseases in which cerebellar structure and function are impaired, including ataxia and autism. In addition, since signals that control growth and differentiation are often dysregulated in cancer, these studies may shed light on the etiology of cerebellar tumors such as medulloblastoma.

DESCRIPTION (provided by applicant): Medulloblastoma is the most common malignant brain tumor in children. Almost half the children who develop medulloblastoma die from it, and survivors often develop severe side effects as a result of the treatment. Improved approaches to treating medulloblastoma are likely to come from a deeper understanding of the cellular and molecular basis of the disease. One critical question about the etiology of medulloblastoma is the cell from which it originates. The morphology of tumor cells and their location on the surface of the cerebellum have led to speculation that the tumors arise from granule cell precursors (GCPs), restricted neural progenitors that give rise only to granule neurons. On the other hand, recent studies have shown that medulloblastomas express stem cell markers and can differentiate into both neurons and glia, suggesting that some of these tumors may arise from multipotent neural stem cells (NSCs). Cells with an NSC phenotype have also been suggested to represent cancer stem cells, cells that are resistant to conventional therapies and critical for the long-term growth and propagation of tumors in vivo. The cell of origin and the cancer stem cell may or may not be related, but identifying each of them has important implications for understanding and treating medulloblastoma. Our goal is to identify the cell of origin (tumor-initiating cell) and the cancer stem cell (tumor-propagating cell) for medulloblastomas resulting from mutations in the Sonic hedgehog-Patched signaling pathway. Humans with mutations in this pathway have an increased susceptibility to medulloblastoma. Moreover, patched mutant mice develop tumors that resemble human medulloblastoma, and represent a valuable model for the disease. Our preliminary studies suggest that the cell of origin in patched- associated tumors is a granule cell precursor, and that this cell type is required not only for tumor initiation but also for tumor propagation. We now propose to identify subsets of GCPs that are enriched for the ability to initiate tumors, and subsets of tumor cells that are uniquely capable of propagating tumors following transplantation. Identifying these cells will provide critical insight into the mechanisms of transformation, and will help us develop and test novel approaches to targeting medulloblastoma.

DESCRIPTION (provided by applicant): Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit large cell/anaplastic (LCA) histology usually fail therapy and die from their disease. Improved approaches to treating these patients are likely to come from a deeper understanding of LCA tumors, including their aggressive growth properties and their invasive and metastatic behavior. Unfortunately, human LCA MB tissue is difficult to obtain, and existing genetically engineered mouse (GEM) models of MB rarely display anaplasia or metastasis. To address this problem, we have collected >100 human LCA MBs, including paired samples of primary/metastatic tumors. In addition, we have generated GEM and transplant-based models of LCA MB, and mobilized the transposable element Sleeping Beauty (SB) to promote anaplasia and metastasis in these models. Through analysis of our human and murine datasets, we propose to identify the cells and genes that drive progression in LCA medulloblastoma. This application brings together investigators from three institutions, with collective experience in neural development, stem cell biology and genomics of both human and murine MB. Our complementary backgrounds and expertise uniquely position us to investigate the cellular and molecular basis of LCA MB, and will ultimately allow us to develop more effective approaches to targeting these aggressive tumors.

ABSTRACT ? TUMOR INITIATION AND MAINTENANCE PROGRAM The overall goals of the Tumor Initiation and Maintenance (TIM) Program are to identify the cells that give rise to tumors and the signals that allow these cells to expand uncontrollably, and to use this information to develop more effective approaches to treating human cancer. The TIM Program was established in 2013, when the Cancer Center was reorganized, with NCI's approval, to enhance interaction and collaboration among members. Led by Dr. Wechsler-Reya, the Program consists of 17 faculty and 2 adjunct faculty, including many members of the former Tumor Development and Signal Transduction Programs. TIM Program members have a wide range of cancer-relevant interests including self-renewal, cell cycle progression, oncogenic signaling pathways, ubiquitin ligases and regulation of cell fate by non-coding RNAs. Encompassing these interests, the Program is comprised of three overarching themes: Stem Cells and Development (focusing on the molecular and cellular mechanisms that control self-renewal, differentiation, and transformation); Cell Growth Signaling (studying the factors and processes that regulate normal and malignant cell growth); and RNA Biology and Epigenetics (investigating the regulation of gene expression and cell fate by DNA and histone modification and by non-coding RNAs). The program has a strong cancer focus, with particular emphasis on brain tumors, melanoma, breast and prostate cancer. Program members interact closely with one another and other Cancer Center members, resulting in numerous collaborative grants and joint publications. Of the 38 grants awarded to Program members through both NCI and other peer-reviewed cancer-related mechanisms, 39% were collaborative. Members also published 254 cancer relevant papers in the last funding period, of which 22% were collaborative (10% intra-programmatic and 12% inter-programmatic). In the coming years, the TIM Program will seek to recruit new faculty, particularly in the areas of genomics, epigenetics and animal models of cancer. In addition, Program members will focus on translational research to move findings from basic cancer biology toward the clinic.

ABSTRACT ? TUMOR INITIATION AND MAINTENANCE PROGRAM The overall goals of the Tumor Initiation and Maintenance (TIM) Program are to identify the cells that give rise to tumors and the signals that allow these cells to expand uncontrollably, and to use this information to develop more effective approaches to treating human cancer. The TIM Program was established in 2013, when the Cancer Center was reorganized, with NCI's approval, to enhance interaction and collaboration among members. Led by Dr. Wechsler-Reya, the Program consists of 17 faculty and 2 adjunct faculty, including many members of the former Tumor Development and Signal Transduction Programs. TIM Program members have a wide range of cancer-relevant interests including self-renewal, cell cycle progression, oncogenic signaling pathways, ubiquitin ligases and regulation of cell fate by non-coding RNAs. Encompassing these interests, the Program is comprised of three overarching themes: Stem Cells and Development (focusing on the molecular and cellular mechanisms that control self-renewal, differentiation, and transformation); Cell Growth Signaling (studying the factors and processes that regulate normal and malignant cell growth); and RNA Biology and Epigenetics (investigating the regulation of gene expression and cell fate by DNA and histone modification and by non-coding RNAs). The program has a strong cancer focus, with particular emphasis on brain tumors, melanoma, breast and prostate cancer. Program members interact closely with one another and other Cancer Center members, resulting in numerous collaborative grants and joint publications. Of the 38 grants awarded to Program members through both NCI and other peer-reviewed cancer-related mechanisms, 39% were collaborative. Members also published 254 cancer relevant papers in the last funding period, of which 22% were collaborative (10% intra-programmatic and 12% inter-programmatic). In the coming years, the TIM Program will seek to recruit new faculty, particularly in the areas of genomics, epigenetics and animal models of cancer. In addition, Program members will focus on translational research to move findings from basic cancer biology toward the clinic.

? DESCRIPTION (provided by applicant): Medulloblastoma (MB) is the most common malignant brain tumor in children. Even with an intensive regimen of surgery, radiation and chemotherapy, 25-30% of MB patients still die from their disease. While the vast majority of MB studies have focused on primary tumors, relatively few have examined the biology of leptomeningeal metastasis (LM), the spread of tumor cells through the meninges to the brain and spinal cord. LM, most commonly seen in patients with MYC-driven MB, is not amenable to surgical resection and is associated with extremely poor patient outcomes. Thus, there is a critical need to understand the molecular mechanisms driving metastasis so that more effective therapies can be developed. Using a mouse model of MYC-driven MB in which tumor cells exhibit LM, we recently found that metastatic tumor cells are more likely than primary tumor cells to regenerate metastatic lesions when injected into the cerebrospinal fluid. To identify pathways that mediate this difference in metastatic potential, we compared gene expression in primary vs. metastatic tumor cells from our animal model as well as from MB patients, and found that the tetraspan membrane protein Epithelial membrane protein 1 (Emp1) is significantly elevated in both murine and human metastases. Emp1 has been implicated in cell motility, adhesion and proliferation in leukemia, lymphoma and lung cancer, but has never been studied in the context of MB. In preliminary studies we found that a large proportion of metastatic cells express Emp1, and that overexpression of Emp1 in primary MB cells increases their metastatic behavior in vivo. These data have led us to hypothesize that Emp1 may function as a marker and a driver of metastasis. We will test this by determining 1) whether Emp1 marks cells with increased metastatic potential and 2) whether Emp1 is required for metastatic dissemination. These studies will provide critical insight into the mechanisms of metastasis in MB and yield novel approaches for treating metastatic disease.

Project Summary Medulloblastoma (MB) is the most common malignant brain tumor in children. Although aggressive treatments have improved outcomes, many MB patients still die of their disease, and survivors suffer severe long-term side effects from the therapy. Thus, an increased understanding of the disease and improved therapeutic approaches are critical. MB is comprised of four subgroups ? WNT, Sonic hedgehog (SHH), Group 3 and Group 4 ? that differ in terms of genetics, epigenetics, clinical characteristics and outcomes. Although we have some mechanistic understanding of WNT, SHH and Group 3 tumors, the genes and pathways that underlie Group 4, the most prevalent form of the disease, remain a mystery. Identifying oncogenic drivers and using them to create animal models of Group 4 MB is critical for development and testing of new therapies. Our analysis of the genome and epigenome of >400 human Group 4 MBs has identified single nucleotide variants (SNVs) as well as chromosomal copy number alterations (CNAs). While these studies have generated lists of candidate drivers, none of these has been tested or proven necessary or sufficient to initiate tumor growth. We hypothesize that perturbing expression of candidate genes or CNAs in neural progenitors will generate models of Group 4 MB, thereby validating their functional importance. To test this hypothesis, we propose to knock out or overexpress genes in murine and human (iPS-derived) neural progenitors and evaluate tumorigenic potential in vivo. These studies will create robust models of Group 4 MB that can be used to study tumor biology and to identify novel approaches to therapy.

PROJECT SUMMARY Medulloblastoma (MB) is a highly malignant tumor of the cerebellum that occurs most frequently in children. Despite advances in treatment ? including surgery, radiation, and chemotherapy ? approximately one-third of MB patients still die from the disease and survivors suffer severe side effects as a result of treatment. Genomic profiling and bioinformatic analyses of patient samples have identified four subgroups of MB ? WNT, Sonic hedgehog (SHH), Group 3 (MYC-driven) and Group 4. These subgroups differ in terms of mutations, gene expression profiles and patient outcomes, and patients can be stratified using relevant genetic and immunohistochemical markers. Approximately one-quarter of MBs are Group 3 (G3) tumors, which exhibit overexpression or amplification of the c-Myc (MYC) oncogene. Patients with G3 MB are more likely to present with metastatic disease at time of diagnosis, have a higher incidence of recurrence and the poorest survival rate. Thus, more effective therapies for G3 MB are critically needed. To identify MYC inhibitors that would be effective in G3 MB, we developed a phenotypic, target-agnostic assay using disease-relevant cells isolated from G3 MB orthotopic patient-derived xenografts (PDXs). The assay was designed to identify small molecules that reduce endogenous MYC levels ? the signature molecular marker of G3 MB ? in 4 hours, to preferentially modulate targets directly affecting MYC stability and avoid indirect or off- target effects at later time points. We applied this assay to screen a 100,000 compound collection and identified small molecule scaffolds that robustly decrease cellular MYC levels. Hits were validated in a rigorous testing funnel designed to avoid undesired mechanisms of action, and initial SAR was explored for several scaffolds. From these studies the most promising series was prioritized with activities in the 100 nM potency range as well as compound properties indicative of good BBB penetration. We further confirmed that the compounds in this series decrease cell viability after 48 hours exposure and that this effect correlates with potency in the MYC assay. Preliminary data for the most active compound of this series, SBI1242, indicate that this decrease in cell viability of G3 MB patient cells is selective over iPSC-derived neurons, suggesting a possible therapeutic window. The goal of this proposal is to further optimize SBI1242 for preclinical in vivo testing and use the best analog to test our hypothesis that inhibitors of the G3 MB signature biomarker MYC identified in a highly disease-relevant context can safely and effectively arrest or reverse tumor growth in our G3 MB-specific orthotopic PDX mouse model. We anticipate that successful completion of these studies will be a significant step towards our long-term goal of identifying novel, safe, and effective treatments for MB and other MYC-driven cancers.

ABSTRACT ? TUMOR INITIATION AND MAINTENANCE PROGRAM The overall goals of the Tumor Initiation and Maintenance (TIM) Program are to identify the cells that give rise to tumors and the signals that allow these cells to expand uncontrollably, and to use this information to develop more effective approaches to treating human cancer. The TIM Program was established in 2013, when the Cancer Center was reorganized, with NCI's approval, to enhance interaction and collaboration among members. Led by Dr. Wechsler-Reya, the Program consists of 16 faculty and five adjunct faculty. Members of the TIM Program have a wide range of cancer-relevant interests related to the development and growth of tumor cells. Themes include: Self-renewal and Differentiation, Proliferation Signaling, and Genomic and Epigenomic Regulation. Areas of shared interest include control of self-renewal and differentiation in normal development and cancer, regulation of proliferative signals in cancer, and genomic and epigenomic regulation of tumorigenesis. The program has a strong cancer focus, with particular emphasis on brain, breast, pancreatic, and prostate cancers as well as leukemia and melanoma. Program members interact closely with one another and other Cancer Center members, resulting in numerous collaborative grants and joint publications. During the past funding cycle, TIM members were awarded 274 grants, of which 100 (36%) were collaborative. Grant funding for the past year was $4.7M in direct costs, of which $1.7 (35%) was from NCI. Members also published 229 cancer relevant papers in the last funding period, of which 16% were collaborative (5% intra-programmatic and 11% inter-programmatic). In the coming years, the TIM Program will seek to recruit new faculty, particularly in the areas of cancer stem cell biology, functional genomics, and computational biology. In addition, Program members will focus on translational research to move findings from basic cancer biology toward the clinic.

PROJECT SUMMARY Medulloblastoma is the most common malignant brain tumor in children. Although aggressive treatments have improved outcomes, many patients still die of their disease, and survivors suffer severe long-term side effects from the therapy. Thus, safer and more effective approaches to therapy are critical. In our previous studies we have created new animal models of medulloblastoma and used them to define cells of origin, identify key regulators of tumor initiation and maintenance, and uncover agents that may be effective for therapy of the disease. Although we have made important strides over the last few years, there are a number of critical questions that remain unanswered. In the coming years, we will take advantage of the models and approaches we have developed to: (1) Identify drivers and create models for the deadliest forms of medulloblastoma; (2) Uncover the molecular mechanisms of leptomeningeal metastasis, and find novel strategies for targeting metastatic disease; (3) Use primary patient tumors as a platform to discover new therapies; and (4) Identify mechanisms that regulate immune responses to medulloblastoma. The overall goal of our research is to gain a deeper understanding of medulloblastoma biology and use this information to develop more effective approaches to therapy. Although these studies are ambitious, they are essential if we are to make a difference in the lives of children with this devastating disease.