Browsing by Subject "Medulloblastoma"

Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.

Studying the early stages of cancer can provide important insight into the molecular basis of the disease. In many human cancers, such as prostate, pancreatic, and colon cancer, a pre-neoplastic, or intermediate, stage of the disease has been identified. The pre-neoplastic stage is presumed to be a transition during which normal cells undergo malignant transformation. However, the link between the pre-neoplastic cells and end-stage disease has never been formally established. To investigate the fate of such cells, the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma was used. Pre-neoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Genetic fate mapping and orthotopic transplantation provided definitive evidence that PNCs give rise to tumors and showed that the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer.

Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Identifying new treatments will require an understanding of early stages of tumor development - the cell types from which the tumors arise and the signals that regulate their growth - as well as identification of pathways that are critical for the growth and maintenance of established tumors.

In these studies, we first explore the role of WNT signaling in cerebellar progenitors and their potential to serve as cells of origin for WNT-driven tumors. The WNT pathway plays multiple roles in neural development, is crucial for establishment of the embryonic cerebellum, and is highly expressed in a subset of MBs. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. We show that expression of activated β-catenin promotes proliferation of cerebellar neural stem cells (NSCs) but not granule neuron precursors (GNPs). Although β-catenin expressing NSCs proliferate in vivo they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation and that cooperating "second hits" may be required for neoplastic transformation.

In addition to understanding early stages of transformation, identifying vulnerabilities of established tumors will be critical for development of targeted therapies. Our studies in Chapter 3 are focused on the role of Survivin in SHH-driven MB and utility of survivin inhibition as a therapeutic approach for MB. Survivin is an inhibitor of apoptosis protein (IAP) that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB, and when expressed at high levels predicts poor clinical outcome. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Using genetic and pharmacological approaches, we demonstrate that inhibition of Survivin impairs proliferation and survival of both murine and human MB cells. Although Survivin antagonists do not cross the blood-brain barrier, they are capable of impeding growth of MB cells in flank allografts. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.

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The eukaryotic cell cycle is characterized by a series of tightly orchestrated events during which cellular components are replicated and subsequently divided. In mammalian cells, the precise spatiotemporal control of cell cycle progression is achieved through multiple cyclin-dependent-kinases (Cdks), which are pivotal for both the proper timing of the cell cycle and the maintenance of genomic integrity. The cell progresses through a series of different phases before division, including the pre-mitotic G2 phase and the mitotic M phase. As the interval between DNA replication and mitosis, G2 phase is the stage where protein synthesis occurs in preparation for mitosis. On the other hand, mitosis is characterized by the most dramatic events of the cell cycle, featuring chromosome condensation and segregation into two daughter cells. Transition from G2 to M phase is driven by the activation of Cdk1, which forms a heterodimer with its obligate allosteric activator Cyclin B1. This heterodimer phosphorylates downstream effectors, thereby inducing entry into mitosis. Entry into mitosis is halted by activation of the G2/M cell cycle checkpoint, which stimulates the Myt1 and Wee1 kinases; these activated kinases phosphorylate Cdk1 at Thr14 and Tyr15, resulting in Cdk1 inactivation. Failure of G2/M cell cycle arrest leads to premature mitosis and triggers a form of cell death marked by multiple micronuclei and decondensed chromatin known as "mitotic catastrophe". Mitotic catastrophe can be stimulated by the presence of incompletely replicated DNA or DNA damage, which leads to the unscheduled activation of the Cdk1/CyclinB complex.

Recent studies in the brain tumor glioblastoma (GBM) have suggested that abrogation of the G2/M checkpoint with a Wee1 inhibitor enhances the efficacy of chemotherapy and ionizing radiation[1]. Interestingly, I have found that Wee1 protein levels vary dramatically between different types of brain tumors including glioblastoma and medulloblastoma. Also, Wee1 protein abundance determines tumor sensitivity to DNA-damaging chemotherapies. Furthermore, similar to findings in the Xenopus egg extract, in a mammalian cell culture system I have found that Wee1 protein abundance is subject to regulation by the arginine methyltransferase PRMT5. Taken together, these findings suggest that Wee1 or PRMT5 may be used as effective targets for therapy in a subset of glioblastoma and medulloblastoma patients.

1. Mir, S.E., et al., In Silico Analysis of Kinase Expression Identifies WEE1 as a Gatekeeper against Mitotic Catastrophe in Glioblastoma. Cancer Cell, 2010. 18(3): p. 244-257.

Development requires a delicate balance of proliferation and differentiation. Too little proliferation can result in dysfunctional tissues, while prolonged or heightened proliferation can result in tumor formation. This is clearly seen with the granule neuron precursors (GNPs) of the cerebellum. Too little proliferation of these cells during development results in ataxia, whereas too much proliferation results in the cerebellar tumor medulloblastoma. While these cells are known to proliferate in response to Shh, it is not clear what controls the differentiation of these cells in vivo.

Previous work from our lab has identified basic fibroblast growth factor (bFGF) as a candidate differentiation factor for these cells. In this thesis, I characterize some of the cellular and molecular mechanisms involved in FGF-mediated inhibition (FMI) of Shh-induced GNP proliferation. In addition, I employ FGFR knockouts and a bFGF gain-of-function mouse to determine whether FGF signaling is necessary and/or sufficient for differentiation of GNPs during cerebellar development. Finally, the question of whether bFGF can be effective as a therapeutic agent for in vivo tumor treatment is tested in a transplant model.

These experiments indicate that FGF signaling is neither necessary nor sufficient for GNP differentiation during cerebellar development. However, transplanted tumors are potently inhibited by bFGF treatment. Furthermore, FMI is shown to occur around the level of Gli2 processing in the Shh pathway, implying that such a treatment has promise to be widely effective in treatment of Shh-dependent medulloblastomas.

Medulloblastoma is the most common malignant brain tumor in children. The understanding of the genetic alterations in this tumor is emergent, and many such genetic driver events have yet to be functionally-characterized. Our studies have sought to understand the causes and consequences of OTX2 dysregulation in established medulloblastomas and in its putative cellular origins. Using a tumor genetic approach, we have uncovered frequent OTX2 copy number gains driving expression of this oncogene in a subset of medulloblastomas. However, OTX2 is frequently expressed in medulloblastomas independent of genomic copy number gain, and we thus sought to understand the transcriptional regulation of this gene in these tumors. We have found that chromatin accessibility, promoter DNA methylation, and activity of a distal downstream enhancer is distinct between OTX2-expressing and -nonexpressing medulloblastomas. Notably, autoregulation serves to maintain OTX2 expression in some medulloblastomas, whereas DNA methylation actively suppresses OTX2 in tumors not expressing this gene. Finally, we describe the effect of expressing Otx2 (the mouse homolog of OTX2) aberrantly in the developing mouse hindbrain, revealing that Otx2 disrupts spatiotemporal restrictions of neuronal progenitor cell proliferation. The effect of Otx2 in vivo is transient, with ectopically-proliferating cells give way to differentiated neurons. We found that OTX2 expression was not able to give rise to high penetrance medulloblastoma when combined with P53 deletion or double heterozygosity for P53 and PTEN. Thus, although Otx2 alters migration and proliferation dynamics of hindbrain neuronal progenitor cells, further studies are needed to identify the genetic alterations that cooperate with this oncogene to give rise to medulloblastoma.

Cytosolic cytochrome c promotes apoptosis by triggering caspase activation. In healthy cells cytochrome c localizes to mitochondria, where it participates in the electron transport chain. Apoptotic stimuli induce permeabilization of the outer mitochondrial membrane and release of cytochrome c. Once cytosolic, cytochrome c binds Apaf-1, inducing the formation of a protein complex that recruits and activates caspases, which serve to dismantle the dying cell. Although the steps of this signaling pathway have been described, many of the regulatory mechanisms influencing the cellular response to cytosolic cytochrome c remain unclear. Using apoptosis assays and microinjection techniques, we investigated the response of several cell-types to cytosolic cytochrome c.

First, we demonstrate that cytosolic cytochrome c kills brain tumor cells but not normal brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in brain tumors compared with negligible Apaf-1 in brain tissue. These differences in Apaf-1 abundance correlate with differences in E2F1, a previously identified activator of Apaf-1 transcription. Chromatin immunoprecipitation assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to Apaf-1 expression in brain tumors. These results demonstrate an unexpected sensitivity of brain tumors to cytochrome c and raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancers.

Secondly, we develop a method for monitoring caspase activity in Xenopus laevis oocytes and early embryos. The approach, utilizing microinjection of a near-infrared dye that emits fluorescence only after its cleavage by active caspases, has enabled the elucidation of subtleties in the apoptotic program. We demonstrate that brief caspase activation is sufficient to cause death. We illustrate the presence of a cytochrome c dose threshold, which is lowered by neutralization of inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of these oocytes is caspase-independent. Imaging caspase activity in the embryo suggests that apoptosis in early development is not cell-autonomous. Finally, we believe this method presents a useful screening modality for identifying novel apoptotic regulators as well as pro-apoptotic small-molecules that could be useful in treating brain tumors.