Ryan J. Emnett

Distinct Genetic Signatures among Pilocytic Astrocytomas Relate to Their Brain Region Origin

Pilocytic astrocytomas (PAs) are the most common glioma in children. Whereas many PAs are slow-growing or clinically indolent, others exhibit more aggressive features with tumor recurrence and death. To identify genetic signatures that might predict PA clinical behavior, we did gene expression profiling on 41 primary PAs arising sporadically and in patients with neurofibromatosis type 1 (NF1). Whereas no expression signature was found that could discriminate clinically aggressive or recurrent tumors from more indolent cases, PAs arising in patients with NF1 did exhibit a unique gene expression pattern. In addition, we identified a gene expression signature that stratified PAs by location (supratentorial versus infratentorial). Lastly, we also identified a gene expression pattern common to PAs and normal mouse astrocytes and neural stem cells from these distinct brain regions as well as a gene expression pattern shared between PAs and another human glial tumor (ependymoma) arising supratentorially compared with those originating in the posterior fossa. These results suggest that glial tumors share an intrinsic, lineage-specific molecular signature that reflects the brain region in which their nonmalignant predecessors originated.

Neurofibromatosis-1 Regulates Neuronal and Glial Cell Differentiation from Neuroglial Progenitors In Vivo by Both cAMP- and Ras-Dependent Mechanisms

Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

Alterations of BRAF and HIPK2 loci predominate in sporadic pilocytic astrocytoma.

Objective: Independent studies have previously demonstrated that both the HIPK2 and BRAF genes are amplified and rearranged, respectively, in pilocytic astrocytomas (PAs). The purpose of this study was to further investigate the frequency of BRAF and HIPK2 alterations in PAs, the concordance of these events, and their relationship to clinical phenotype. Methods: We performed extensive characterization by array-based copy number assessment (aCGH), HIPK2 copy number analysis, and BRAF rearrangement and mutation analysis in a set of 79 PAs, including 9 tumors from patients with neurofibromatosis type 1 (NF1). Results: We identified 1 of 3 previously identified BRAF rearrangements in 42/70 sporadic PAs. An additional 2 tumors with no rearrangement also exhibited BRAF mutation, including a novel 3-base insertion. As predicted from the genomic organization at this locus, 22/36 tumors with BRAF rearrangement also exhibited corresponding HIPK2 amplification. However, 14/36 tumors with BRAF rearrangement had no detectable HIPK2 gene amplification and 6/20 tumors demonstrated HIPK2 amplification without apparent BRAF rearrangement or mutation. Only 12/70 PAs lacked detectable BRAF or HIPK2 alterations. Importantly, none of the 9 PA tumors from NF1 patients exhibited BRAF rearrangement or mutation. Conclusions: BRAF rearrangement represents the most common genetic alteration in sporadic, but not neurofibromatosis type 1-associated, pilocytic astrocytomas (PAs). These findings implicate BRAF in the pathogenesis of these common low-grade astrocytomas in children, and suggest that PAs arise either from NF1 inactivation or BRAF gain of function.

Neurofibromatosis-1 regulates neuroglial progenitor proliferation and glial differentiation in a brain region-specific manner

Recent studies have shown that neuroglial progenitor/stem cells (NSCs) from different brain regions exhibit varying capacities for self-renewal and differentiation. In this study, we used neurofibromatosis-1 (NF1) as a model system to elucidate a novel molecular mechanism underlying brain region-specific NSC functional heterogeneity. We demonstrate that Nf1 loss leads to increased NSC proliferation and gliogenesis in the brainstem, but not in the cortex. Using Nf1 genetically engineered mice and derivative NSC neurosphere cultures, we show that this brain region-specific increase in NSC proliferation and gliogenesis results from selective Akt hyperactivation. The molecular basis for the increased brainstem-specific Akt activation in brainstem NSCs is the consequence of differential rictor expression, leading to region-specific mammalian target of rapamycin (mTOR)/rictor-mediated Akt phosphorylation and Akt-regulated p27 phosphorylation. Collectively, these findings establish mTOR/rictor-mediated Akt activation as a key driver of NSC proliferation and gliogenesis, and identify a unique mechanism for conferring brain region-specific responses to cancer-causing genetic changes.

Reduced striatal dopamine underlies the attention system dysfunction in neurofibromatosis-1 mutant mice

Learning and behavioral abnormalities are among the most common clinical problems in children with the neurofibromatosis-1 (NF1) inherited cancer syndrome. Recent studies using Nf1 genetically engineered mice (GEM) have been instructive for partly elucidating the cellular and molecular defects underlying these cognitive deficits; however, no current model has shed light on the more frequently encountered attention system abnormalities seen in children with NF1. Using an Nf1 optic glioma (OPG) GEM model, we report novel defects in non-selective and selective attention without an accompanying hyperactivity phenotype. Specifically, Nf1 OPG mice exhibit reduced rearing in response to novel objects and environmental stimuli. Similar to children with NF1, the attention system dysfunction in these mice is reversed by treatment with methylphenidate (MPH), suggesting a defect in brain catecholamine homeostasis. We further demonstrate that this attention system abnormality is the consequence of reduced dopamine (DA) levels in the striatum, which is normalized following either MPH or l-dopa administration. The reduction in striatal DA levels in Nf1 OPG mice is associated with reduced striatal expression of tyrosine hydroxylase, the rate-limited enzyme in DA synthesis, without any associated dopaminergic cell loss in the substantia nigra. Moreover, we demonstrate a cell-autonomous defect in Nf1+/- dopaminergic neuron growth cone areas and neurite extension in vitro, which results in decreased dopaminergic cell projections to the striatum in Nf1 OPG mice in vivo. Collectively, these data establish abnormal DA homeostasis as the primary biochemical defect underlying the attention system dysfunction in Nf1 GEM relevant to children with NF1.

Neurofibromatosis-1 regulates mTOR-mediated astrocyte growth and glioma formation in a TSC/Rheb-independent manner

Converging evidence from the analysis of human brain tumors and genetically engineered mice has revealed that the mammalian target of rapamycin (mTOR) pathway is a central regulator of glial and glioma cell growth. In this regard, mutational inactivation of neurofibromatosis-1 (NF1), tuberous sclerosis complex (TSC), and PTEN genes is associated with glioma formation, such that pharmacologic inhibition of mTOR signaling results in attenuated tumor growth. This shared dependence on mTOR suggests that PTEN and NF1 (neurofibromin) glial growth regulation requires TSC/Rheb (Ras homolog enriched in brain) control of mTOR function. In this report, we use a combination of genetic silencing in vitro and conditional mouse transgenesis approaches in vivo to demonstrate that neurofibromin regulates astrocyte cell growth and glioma formation in a TSC/Rheb-independent fashion. First, we show that Nf1 or Pten inactivation, but not Tsc1 loss or Rheb overexpression, increases astrocyte cell growth in vitro. Second, Nf1-deficient increased mTOR signaling and astrocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in glial progenitors of Nf1+/− mice does not lead to glioma formation. Collectively, these findings establish TSC/Rheb-independent mechanisms for mTOR-dependent glial cell growth control and gliomagenesis relevant to the design of therapies for individuals with glioma.

Neurofibromatosis-1 Heterozygosity Increases Microglia in a Spatially- and Temporally-Restricted Pattern Relevant to Mouse Optic Glioma Formation and Growth

Whereas carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression are heavily influenced by the local microenvironment. Previous studies from our laboratory have used Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/− microglia in the tumor microenvironment are critical cellulardeterminants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we used CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times after ganciclovir administration. Ganciclovir-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/− optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/− optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similarincrease in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance ofNf1 optic glioma.

Array-based comparative genomic hybridization identifies CDK4 and FOXM1 alterations as independent predictors of survival in malignant peripheral nerve sheath tumor

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with variable patient survival and few known prognostically relevant genomic biomarkers. To identify survival-associated genomic biomarkers, we performed high-resolution array-based comparative genomic hybridization (aCGH) on a large set of MPNSTs. Experimental Design: Candidate gene alterations identified by aCGH in 38 MPNSTs were validated at the DNA, RNA, and protein levels on these same tumors and an independent set of 87 MPNST specimens. Results: aCGH revealed highly complex copy number alterations, including both previously reported and completely novel loci. Four regions of copy number gain were associated with poor patient survival. Candidate genes in these regions include SOX5 (12p12.1), NOL1 and MLF2 (12p13.31), FOXM1 and FKBP1 (12p13.33), and CDK4 and TSPAN31 (12q14.1). Alterations of these candidate genes and several others of interest (ERBB2, MYC and TP53) were confirmed by at least 1 complementary methodology, including DNA and mRNA quantitative real-time PCR, mRNA expression profiling, and tissue microarray-based fluorescence in situ hybridization and immunohistochemistry. Multivariate analysis showed that CDK4 gain/amplification and increased FOXM1 protein expression were the most significant independent predictors for poor survival in MPNST patients (P < 0.05). Conclusions: Our study provides new and independently confirmed candidate genes that could serve as genomic biomarkers for overall survival in MPNST patients. Clin Cancer Res; 17(7); 1924–34. ©2011 AACR.

Pediatric glioma-associated KIAA1549:BRAF expression regulates neuroglial cell growth in a cell type-specific and mTOR-dependent manner

Tandem duplications involving the BRAF kinase gene have recently been identified as the most frequent genetic alteration in sporadic pediatric glioma, creating a novel fusion protein (f-BRAF) with increased BRAF activity. To define the role of f-BRAF in gliomagenesis, we demonstrate that f-BRAF regulates neural stem cell (NSC), but not astrocyte, proliferation and is sufficient to induce glioma-like lesions in mice. Moreover, f-BRAF-driven NSC proliferation results from tuberin/Rheb-mediated mammalian target of rapamycin (mTOR) hyperactivation, leading to S6-kinase-dependent degradation of p27. Collectively, these results establish mTOR pathway activation as a key growth regulatory mechanism common to both sporadic and familial low-grade gliomas in children.

The neurofibromatosis 2 protein, merlin, regulates glial cell growth in an ErbB2- and Src-dependent manner.

ABSTRACT Individuals with the inherited cancer predisposition syndrome neurofibromatosis 2 (NF2) develop several central nervous system (CNS) malignancies, including glial cell neoplasms (ependymomas). Recent studies have suggested that the NF2 protein, merlin (or schwannomin), may regulate receptor tyrosine kinase signaling, intracellular mitogenic growth control pathways, or adherens junction organization in non-nervous-system cell types. For this report, we used glial fibrillary acidic protein conditional knockout mice and derivative glia to determine how merlin regulates CNS glial cell proliferation. We show that the loss of merlin in glial cells results in increased proliferation in vitro and in vivo. Merlin regulation of glial cell growth reflects deregulated Src activity, such that pharmacologic or genetic inhibition of Src activation reduces Nf2−/− glial cell growth to wild-type levels. We further show that Src regulates Nf2−/− glial cell growth by sequentially regulating FAK and paxillin phosphorylation/activity. Next, we demonstrate that Src activation results from merlin regulation of ErbB2 activation and that genetic or pharmacologic ErbB2 inhibition reduces Nf2−/− glial cell Src/Src effector activation and proliferation to wild-type levels. Lastly, we show that merlin competes with Src for direct binding to ErbB2 and present a novel molecular mechanism for merlin regulation of ErbB2-dependent Src signaling and growth control.