Daniel W. Fults

The genetic landscape of the childhood cancer medulloblastoma

Genomic analysis of a childhood cancer reveals markedly fewer mutations than what is typically seen in adult cancers. Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Somatic mutations in the neurofibromatosis 1 gene in human tumors

The neurofibromatosis 1 (NF1) gene product, neurofibromin, contains a GTPase-activating protein (GAP)-related domain, or NF1 GRD, that is able to down-regulate p21ras by stimulating its intrinsic GTPase. Since p21ras.GTP is a major regulator of growth and differentiation, mutant neurofibromins resulting from somatic mutations in the NF1 gene might interfere with ras signaling pathways and contribute to the development of tumors. We describe an amino acid substitution in the NF1 GRD, altering Lys-1423, that has occurred in three tumor types: colon adenocarcinoma, myelodysplastic syndrome, and anaplastic astrocytoma, and in one family with neurofibromatosis 1. The GAP activity of the mutant NF1 GRD is 200- to 400-fold lower than that of wild type, whereas binding affinity is unaffected. Thus, germline mutations in NF1 that cause neurofibromatosis 1 can also occur in somatic cells and contribute to the development of sporadic tumors, including tumors not associated with neurofibromatosis 1.

Mutations of PIK3CA in Anaplastic Oligodendrogliomas, High-Grade Astrocytomas, and Medulloblastomas

The phosphatidylinositol 3′-kinase pathway is activated in multiple advanced cancers, including glioblastomas, through inactivation of the PTEN tumor suppressor gene. Recently, mutations in PIK3CA, a member of the family of phosphatidylinositol 3′-kinase catalytic subunits, were identified in a significant fraction (25–30%) of colorectal cancers, gastric cancers, and glioblastomas and in a smaller fraction of breast and lung cancers. These mutations were found to cluster into two major “hot spots” located in the helical and catalytic domains. To determine whether PIK3CA is genetically altered in brain tumors, we performed a large-scale mutational analysis of the helical and catalytic domains. A total of 13 mutations of PIK3CA within these specific domains were identified in anaplastic oligodendrogliomas, anaplastic astrocytomas, glioblastoma multiforme, and medulloblastomas, whereas no mutations were identified in ependymomas or low-grade astrocytomas. These observations implicate PIK3CA as an oncogene in a wider spectrum of adult and pediatric brain tumors and suggest that PIK3CA may be a useful diagnostic marker or a therapeutic target in these cancers.

Clonal selection drives genetic divergence of metastatic medulloblastoma

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Sonic hedgehog and insulin-like growth factor signaling synergize to induce medulloblastoma formation from nestin-expressing neural progenitors in mice

Medulloblastoma (MB) is a malignant brain tumor that arises in the cerebellum of children. Activation of the Sonic hedgehog/Patched (Shh/Ptc) signaling pathway in neural progenitor cells of the cerebellum induces MBs in mice. The incomplete penetrance of tumor formation in mice, coupled with the low frequency of mutations in Shh/Ptc pathway genes in human tumors, suggests that other signaling molecules cooperate with Shh to enhance MB formation. We modeled the ability of insulin-like growth factor (IGF) signaling to induce MB using the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell-type-specific manner. We used RCAS retroviral vectors to target expression of Shh, IGF2, and activated Akt to nestin-expressing neural progenitors in the cerebella of newborn mice. The incidence of Shh-induced tumor formation (15%) was enhanced by coexpression with IGF2 (39%) and Akt (48%). Neither IGF2 nor Akt caused tumors when expressed independently. The induced tumors showed upregulated expression of insulin receptor substrate 1 and phosphorylated forms of IGF1 receptor and Akt, mimicking activated IGF signaling found in human MBs. These results indicate that combined activation of the Shh/Ptc and IGF signaling pathways is an important mechanism in MB pathogenesis.

c-Myc Enhances Sonic Hedgehog-Induced Medulloblastoma Formation from Nestin-Expressing Neural Progenitors in Mice

Medulloblastomas are malignant brain tumors that arise in the cerebella of children. The presumed cells-of-origin are undifferentiated precursors of granule neurons that occupy the external granule layer (EGL) of the developing cerebellum. The overexpression of proteins that normally stimulate proliferation of neural progenitor cells may initiate medulloblastoma formation. Two known mitogens for neural progenitors are the c-Myc oncoprotein and Sonic hedgehog (Shh), a crucial determinant of embryonic pattern formation in the central nervous system. We modeled the ability of c-Myc and Shh to induce medulloblastoma in mice using the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell type-specific manner. We targeted the expression of Shh and c-Myc to nestin-expressing neural progenitor cells by injecting replication-competent ALV splice acceptor (RCAS) vectors into the cerebella of newborn mice. Following injection with RCAS-Shh alone, 3/32 (9%) mice developed medulloblastomas and 5/32 showed multifocal hyperproliferation of the EGL, possibly a precursor stage of medulloblastoma. Following injection with RCAS-Shh plus RCAS-Myc, 9/39 (23%) mice developed medulloblastomas. We conclude that nestin-expressing neural progenitors, present in the cerebellum at birth, can act as the cells-of-origin for medulloblastoma, and that c-Myc cooperates with Shh to enhance tumorigenicity.

Loss of heterozygosity on chromosome 10 in human glioblastoma multiforme

Recessive mutations, revealed by loss of the wild-type allele, have been associated with the development of a variety of cancers in children and adults. Polymorphic chromosome 10 markers were used to screen paired tumor and lymphocyte DNA samples in 13 patients with glioblastoma multiforme. Ten patients showed loss of constitutional heterozygosity in the tumor samples. This finding suggests that a recessive gene involved in the development of glioblastoma multiforme is present on chromosome 10.

Molecular, genetic, and cellular pathogenesis of neurofibromas and surgical implications.

Neurofibromatosis 1 (NF1) is a common autosomal dominant disease characterized by complex and multicellular neurofibroma tumors. Significant advances have been made in the research of the cellular, genetic, and molecular biology of NF1. The NF1 gene was identified by positional cloning. The functions of its protein product, neurofibromin, in RAS signaling and in other signal transduction pathways are being elucidated, and the important roles of loss of heterozygosity and haploinsufficiency in tumorigenesis are better understood. The Schwann cell was discovered to be the cell of origin for neurofibromas, but understanding of a more complicated interplay of multiple cell types in tumorigenesis, specifically recruited heterogenous cell types such as mast cells and fibroblasts, has important implications for surgical therapy of these tumors. This review summarizes the most recent NF1 and neurofibroma literature describing the pathogenesis and treatment of nerve sheath tumors. Understanding the biological underpinnings of tumorigenesis in NF1 has implications for future surgical and medical management of neurofibromas.

N-myc Can Substitute for Insulin-Like Growth Factor Signaling in a Mouse Model of Sonic Hedgehog–Induced Medulloblastoma

Medulloblastoma is a malignant brain tumor that arises in the cerebellum in children, presumably from granule neuron precursors (GNP). Advances in patient treatment have been hindered by a paucity of animal models that accurately reflect the molecular pathogenesis of human tumors. Aberrant activation of the Sonic hedgehog (Shh) and insulin-like growth factor (IGF) pathways is associated with human medulloblastomas. Both pathways are essential regulators of GNP proliferation during cerebellar development. In cultured GNPs, IGF signaling stabilizes the oncogenic transcription factor N-myc by inhibiting glycogen synthase kinase 3beta-dependent phosphorylation and consequent degradation of N-myc. However, determinants of Shh and IGF tumorigenicity in vivo remain unknown. Here we report a high frequency of medulloblastoma formation in mice following postnatal overexpression of Shh in cooperation with N-myc. Overexpression of N-myc, alone or in combination with IGF signaling mediators or with the Shh target Gli1, did not cause tumors. Thus, Shh has transforming functions in addition to induction of N-myc and Gli1. This tumor model will be useful for testing novel medulloblastoma therapies and providing insight into mechanisms of hedgehog-mediated transformation.