Inhibition of Erb-B2 Receptor Tyrosine Kinase 3 and Associated Regulatory Pathways Potently Impairs Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, currently untreatable Schwann cell-derived neoplasms with hyperactive mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. To identify potential therapeutic targets, previous studies used genome-scale shRNA screens that implicated the neuregulin-1 receptor erb-B2 receptor tyrosine kinase 3 (erbB3) in MPNST proliferation and/or survival. The current study shows that erbB3 is commonly expressed in MPNSTs and MPNST cell lines and that erbB3 knockdown inhibits MPNST proliferation and survival. Kinomic and microarray analyses of Schwann and MPNST cells implicate Src- and erbB3-mediated calmodulin-regulated signaling as key pathways. Consistent with this, inhibition of upstream (canertinib, sapitinib, saracatinib, and calmodulin) and parallel (AZD1208) signaling pathways involving mitogen-activated protein kinase and mammalian target of rapamycin reduced MPNST proliferation and survival. ErbB inhibitors (canertinib and sapitinib) or erbB3 knockdown in combination with Src (saracatinib), calmodulin [trifluoperazine (TFP)], or proviral integration site of Moloney murine leukemia kinase (AZD1208) inhibition even more effectively reduces proliferation and survival. Drug inhibition enhances an unstudied calmodulin-dependent protein kinase IIα phosphorylation site in an Src-dependent manner. The Src family kinase inhibitor saracatinib reduces both basal and TFP-induced erbB3 and calmodulin-dependent protein kinase IIα phosphorylation. Src inhibition (saracatinib), like erbB3 knockdown, prevents these phosphorylation events; and when combined with TFP, it even more effectively reduces proliferation and survival compared with monotherapy. These findings implicate erbB3, calmodulin, proviral integration site of Moloney murine leukemia kinases, and Src family members as important therapeutic targets in MPNSTs and demonstrate that combinatorial therapies targeting critical MPNST signaling pathways are more effective.

Down Syndrome Biobank Consortium: A perspective.

Individuals with Down syndrome (DS) have a partial or complete trisomy of chromosome 21, resulting in an increased risk for early-onset Alzheimer's disease (AD)-type dementia by early midlife. Despite ongoing clinical trials to treat late-onset AD, individuals with DS are often excluded. Furthermore, timely diagnosis or management is often not available. Of the genetic causes of AD, people with DS represent the largest cohort. Currently, there is a knowledge gap regarding the underlying neurobiological mechanisms of DS-related AD (DS-AD), partly due to limited access to well-characterized brain tissue and biomaterials for research. To address this challenge, we created an international consortium of brain banks focused on collecting and disseminating brain tissue from persons with DS throughout their lifespan, named the Down Syndrome Biobank Consortium (DSBC) consisting of 11 biobanking sites located in Europe, India, and the USA. This perspective describes the DSBC harmonized protocols and tissue dissemination goals.

LPAR1 and aberrantly expressed LPAR3 differentially promote the migration and proliferation of malignant peripheral nerve sheath tumor cells.

Schwann cell-derived neoplasms known as malignant peripheral nerve sheath tumors (MPNSTs) are the most common malignancy and the leading cause of death in individuals with neurofibromatosis Type 1. Using genome-scale shRNA screens, we have previously found evidence suggesting that lysophosphatidic acid receptors (LPARs) are essential for MPNST proliferation and/or survival. Here, we examine the expression and mutational status of all six LPA receptors in MPNSTs, assess the role that individual LPA receptors play in MPNST physiology and examine their ability to activate key neurofibromin-regulated signaling cascades. We found that human Schwann cells express LPAR1 and LPAR6, while MPNST cells express predominantly LPAR1 and LPAR3. Whole exome sequencing of 16 MPNST cell lines showed no evidence of mutations in any LPAR genes or ENPP2, a gene encoding a major LPA biosynthetic enzyme. Oleoyl-LPA, an LPA variant with an unsaturated side chain, promoted MPNST cell proliferation and migration. LPAR1 knockdown ablated the promigratory effect of LPA, while LPAR3 knockdown decreased proliferation. Inhibition of R-Ras signaling with a doxycycline-inducible dominant negative (DN) R-Ras mutant, which inhibits both R-Ras and R-Ras2, blocked LPA's promigratory effect. In contrast, DN R-Ras did not affect migration induced by neuregulin-1ß (NRG1ß), suggesting that LPA and NRG1ß promote MPNST migration via distinct pathways. LPA-induced migration was also inhibited by Y27632, an inhibitor of the ROCK1/2 kinases that mediate R-Ras effects in MPNSTs. Thus, LPAR1 and aberrantly expressed LPAR3 mediate distinct effects in MPNSTs. These receptors and the signaling pathways that they regulate are potentially useful therapeutic targets in MPNSTs.

Suppression of Fli-1 protects against pericyte loss and cognitive deficits in Alzheimer's disease.

Brain pericytes regulate cerebral blood flow, maintain the integrity of the blood-brain barrier (BBB), and facilitate the removal of amyloid ß (Aß), which is critical to healthy brain activity. Pericyte loss has been observed in brains from patients with Alzheimer's disease (AD) and animal models. Our previous data demonstrated that friend leukemia virus integration 1 (Fli-1), an erythroblast transformation-specific (ETS) transcription factor, governs pericyte viability in murine sepsis; however, the role of Fli-1 and its impact on pericyte loss in AD remain unknown. Here, we demonstrated that Fli-1 expression was up-regulated in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aß accumulation compared with cognitively normal individuals and wild-type (WT) mice. Antisense oligonucleotide Fli-1 Gapmer administered via intrahippocampal injection decelerated pericyte loss, decreased inflammatory response, ameliorated cognitive deficits, improved BBB dysfunction, and reduced Aß deposition in 5xFAD mice. Fli-1 Gapmer-mediated inhibition of Fli-1 protected against Aß accumulation-induced human brain pericyte apoptosis in vitro. Overall, these studies indicate that Fli-1 contributes to pericyte loss, inflammatory response, Aß deposition, vascular dysfunction, and cognitive decline, and suggest that inhibition of Fli-1 may represent novel therapeutic strategies for AD.

Developmental, neurochemical, and behavioral analyses of ErbB4 Cyt-1 knockout mice.

Neuregulins (NRGs) and their cognate neuronal receptor ERBB4, which is expressed in GABAergic and dopaminergic neurons, regulate numerous behaviors in rodents and have been identified as schizophrenia at-risk genes. ErbB4 transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26, which encodes a cytoplasmic domain that imparts ErbB4 receptors the ability to signal via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although ErbB4 Cyt-1/2 isoforms have been studied in transfected cultured cells, their functions in vivo remain unknown. Here, we generated ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice to investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon. Overall receptor mRNA levels remain unchanged in germline ErbB4 Cyt-1 knockouts (Cyt-1 KOs), with all transcripts encoding Cyt-2 variants. In contrast to mice lacking all ErbB4 receptor function, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. However, basal extracellular dopamine (DA) levels in the medial prefrontal cortex are increased in Cyt-1 heterozygotes. Despite these neurochemical changes, Cyt-1 heterozygous and homozygous mice do not manifest behavioral abnormalities previously reported to be altered in ErbB4 null mice. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the DA-rich ventral tegmental area of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Taken together, our observations indicate that ErbB4 Cyt-1 function in vivo is important for DA balance and behaviors in adults.

The Role of R-Ras Proteins in Normal and Pathologic Migration and Morphologic Change.

The contributions that the R-Ras subfamily [R-Ras, R-Ras2/teratocarcinoma 21 (TC21), and M-Ras] of small GTP-binding proteins make to normal and aberrant cellular functions have historically been poorly understood. However, this has begun to change with the realization that all three R-Ras subfamily members are occasionally mutated in Noonan syndrome (NS), a RASopathy characterized by the development of hematopoietic neoplasms and abnormalities affecting the immune, cardiovascular, and nervous systems. Consistent with the abnormalities seen in NS, a host of new studies have implicated R-Ras proteins in physiological and pathologic changes in cellular morphology, adhesion, and migration in the cardiovascular, immune, and nervous systems. These changes include regulating the migration and homing of mature and immature immune cells, vascular stabilization, clotting, and axonal and dendritic outgrowth during nervous system development. Dysregulated R-Ras signaling has also been linked to the pathogenesis of cardiovascular disease, intellectual disabilities, and human cancers. This review discusses the structure and regulation of R-Ras proteins and our current understanding of the signaling pathways that they regulate. It explores the phenotype of NS patients and their implications for the R-Ras subfamily functions. Next, it covers recent discoveries regarding physiological and pathologic R-Ras functions in key organ systems. Finally, it discusses how R-Ras signaling is dysregulated in cancers and mechanisms by which this may promote neoplasia.

The Proteome of the Dentate Terminal Zone of the Perforant Path Indicates Presynaptic Impairment in Alzheimer Disease.

Synaptic dysfunction is an early pathogenic event in Alzheimer disease (AD) that contributes to network disturbances and cognitive decline. Some synapses are more vulnerable than others, including the synapses of the perforant path, which provides the main excitatory input to the hippocampus. To elucidate the molecular mechanisms underlying the dysfunction of these synapses, we performed an explorative proteomic study of the dentate terminal zone of the perforant path. The outer two-thirds of the molecular layer of the dentate gyrus, where the perforant path synapses are located, was microdissected from five subjects with AD and five controls. The microdissected tissues were dissolved and digested by trypsin. Peptides from each sample were labeled with different isobaric tags, pooled together and pre-fractionated into 72 fractions by high-resolution isoelectric focusing. Each fraction was then analyzed by liquid chromatography-mass spectrometry. We quantified the relative expression levels of 7322 proteins, whereof 724 showed significantly altered levels in AD. Our comprehensive data analysis using enrichment and pathway analyses strongly indicated that presynaptic signaling, such as exocytosis and synaptic vesicle cycle processes, is severely disturbed in this area in AD, whereas postsynaptic proteins remained unchanged. Among the significantly altered proteins, we selected three of the most downregulated synaptic proteins; complexin-1, complexin-2 and synaptogyrin-1, for further validation, using a new cohort consisting of six AD and eight control cases. Semi-quantitative analysis of immunohistochemical staining confirmed decreased levels of complexin-1, complexin-2 and synaptogyrin-1 in the outer two-thirds of the molecular layer of the dentate gyrus in AD. Our in-depth proteomic analysis provides extensive knowledge on the potential molecular mechanism underlying synaptic dysfunction related to AD and supports that presynaptic alterations are more important than postsynaptic changes in early stages of the disease. The specific synaptic proteins identified could potentially be targeted to halt synaptic dysfunction in AD.

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.

BACKGROUND: Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome. METHODS: NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches. RESULTS: R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles. CONCLUSIONS: R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. Video Abstract.

The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.

Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.

Mechanisms of Receptor Tyrosine-Protein Kinase ErbB-3 (ERBB3) Action in Human Neoplasia.

It is well established that the epidermal growth factor (EGF) receptor, receptor tyrosine-protein kinase erbB-2 (ERBB2)/human EGF receptor 2 (HER2), and, to a lesser extent, ERBB4/HER4, promote the pathogenesis of many types of human cancers. In contrast, the role that ERBB3/HER3, the fourth member of the ERBB family of receptor tyrosine kinases, plays in these diseases is poorly understood and, until recently, underappreciated. In large part, this was because early structural and functional studies suggested that ERBB3 had little, if any, intrinsic tyrosine kinase activity and, thus, was unlikely to be an important therapeutic target. Since then, however, numerous publications have demonstrated an important role for ERBB3 in carcinogenesis, metastasis, and acquired drug resistance. Furthermore, somatic ERBB3 mutations are frequently encountered in many types of human cancers. Dysregulation of ERBB3 trafficking as well as cooperation with other receptor tyrosine kinases further enhance ERBB3's role in tumorigenesis and drug resistance. As a result of these advances in our understanding of the structure and biochemistry of ERBB3, and a growing focus on the development of precision and combinatorial therapeutic regimens, ERBB3 is increasingly considered to be an important therapeutic target in human cancers. In this review, we discuss the unique structural and functional features of ERBB3 and how this information is being used to develop effective new therapeutic agents that target ERBB3 in human cancers.

ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms.

BACKGROUND: We have found that erbB receptor tyrosine kinases drive Ras hyperactivation and growth in NF1-null malignant peripheral nerve sheath tumors (MPNSTs). However, MPNSTs variably express multiple erbB receptors with distinct functional characteristics and it is not clear which of these receptors drive MPNST pathogenesis. Here, we test the hypothesis that altered erbB4 expression promotes MPNST pathogenesis by uniquely activating key cytoplasmic signaling cascades. METHODS: ErbB4 expression was assessed using immunohistochemistry, immunocytochemistry, immunoblotting and real-time PCR. To define erbB4 functions, we generated mice that develop MPNSTs with floxed Erbb4 alleles (P0-GGFß3;Trp53+/-;Erbb4flox/flox mice) and ablated Erbb4 in these tumors. MPNST cell proliferation and survival was assessed using 3H-thymidine incorporation, MTT assays, Real-Time Glo and cell count assays. Control and Erbb4-null MPNST cells were orthotopically xenografted in immunodeficient mice and the growth, proliferation (Ki67 labeling), apoptosis (TUNEL labeling) and angiogenesis of these grafts was analyzed. Antibody arrays querying cytoplasmic kinases were used to identify erbB4-responsive kinases. Pharmacologic or genetic inhibition was used to identify erbB4-responsive kinases that drive proliferation. RESULTS: Aberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P0-GGFß3 and P0-GGFß3;Trp53+/- mice); multiple erbB4 splice variants that differ in their ability to activate PI3 kinase and nuclear signaling were present in MPNST-derived cell lines. Erbb4-null MPNST cells demonstrated decreased proliferation and survival and altered morphology relative to non-ablated controls. Orthotopic allografts of Erbb4-null cells were significantly smaller than controls, with reduced proliferation, survival and vascularization. ERBB4 knockdown in human MPNST cells similarly inhibited DNA synthesis and viability. Although we have previously shown that broad-spectrum erbB inhibitors inhibit Ras activation, Erbb4 ablation did not affect Ras activation, suggesting that erbB4 drives neoplasia via non-Ras dependent pathways. An analysis of 43 candidate kinases identified multiple NRG1ß-responsive and erbB4-dependent signaling cascades including the PI3K, WNK1, STAT3, STAT5 and phospholipase-Cγ pathways. Although WNK1 inhibition did not alter proliferation, inhibition of STAT3, STAT5 and phospholipase-Cγ markedly reduced proliferation. CONCLUSIONS: ErbB4 promotes MPNST growth by activating key non-Ras dependent signaling cascades including the STAT3, STAT5 and phospholipase-Cγ pathways. ErbB4 and its effector pathways are thus potentially useful therapeutic targets in MPNSTs.

Recent Advances in the Diagnosis and Pathogenesis of Neurofibromatosis Type 1 (NF1)-associated Peripheral Nervous System Neoplasms.

The diagnosis of a neurofibroma or a malignant peripheral nerve sheath tumor (MPNST) often raises the question of whether the patient has the genetic disorder neurofibromatosis type 1 (NF1) as well as how this will impact the patient's outcome, what their risk is for developing additional neoplasms and whether treatment options differ for NF1-associated and sporadic peripheral nerve sheath tumors. Establishing a diagnosis of NF1 is challenging as this disorder has numerous neoplastic and non-neoplastic manifestations which are variably present in individual patients. Further, other genetic diseases affecting the Ras signaling cascade (RASopathies) mimic many of the clinical features of NF1. Here, we review the clinical manifestations of NF1 and compare and contrast them with those of the RASopathies. We also consider current approaches to genetic testing for germline NF1 mutations. We then focus on NF1-associated neurofibromas, considering first the complicated clinical behavior and pathology of these neoplasms and then discussing our current understanding of the genomic abnormalities that drive their pathogenesis, including the mutations encountered in atypical neurofibromas. As several neurofibroma subtypes are capable of undergoing malignant transformation to become MPNSTs, we compare and contrast patient outcomes in sporadic, NF1-associated and radiation-induced MPNSTs, and review the challenging pathology of these lesions. The mutations involved in neurofibroma-MPNST progression, including the recent identification of mutations affecting epigenetic regulators, are then considered. Finally, we explore how our current understanding of neurofibroma and MPNST pathogenesis is informing the design of new therapies for these neoplasms.

The Challenge of Cancer Genomics in Rare Nervous System Neoplasms: Malignant Peripheral Nerve Sheath Tumors as a Paradigm for Cross-Species Comparative Oncogenomics.

Comprehensive genomic analyses of common nervous system cancers provide new insights into their pathogenesis, diagnosis, and treatment. Although analogous studies of rare nervous system tumors are needed, there are major barriers to performing such studies. Cross-species comparative oncogenomics, identifying driver mutations in mouse cancer models and validating them in human tumors, is a promising alternative. Although still in its infancy, this approach is being applied to malignant peripheral nerve sheath tumors (MPNSTs), rare Schwann cell-derived malignancies that occur sporadically, after radiotherapy, and in neurofibromatosis type 1. Studies of human neurofibromatosis type 1-associated tumors suggest that NF1 tumor suppressor loss in Schwann cells triggers cell-autonomous and intercellular changes, resulting in development of benign neurofibromas; subsequent neurofibroma-MPNST progression is caused by aberrant growth factor signaling and mutations affecting the p16(INK4A)-cyclin D1-CDK4-Rb and p19(ARF)-Mdm2-p53 cell cycle pathways. Mice with Nf1, Trp53, and/or Cdkn2a mutations that overexpress the Schwann cell mitogen neuregulin-1 or overexpress the epidermal growth factor receptor validate observations in human tumors and, to various degrees, model human tumorigenesis. Genomic analyses of MPNSTs arising in neuregulin-1 and epidermal growth factor receptor-overexpressing mice and forward genetic screens with Sleeping Beauty transposons implicate additional signaling cascades in MPNST pathogenesis. These studies confirm the utility of mouse models for MPNST driver gene discovery and provide new insights into the complexity of MPNST pathogenesis.

Neuronal exosomes reveal Alzheimer's disease biomarkers in Down syndrome.

INTRODUCTION: Individuals with Down syndrome (DS) exhibit Alzheimer's disease (AD) neuropathology and dementia early in life. Blood biomarkers of AD neuropathology would be valuable, as non-AD intellectual disabilities of DS and AD dementia overlap clinically. We hypothesized that elevations of amyloid ß (Aß) peptides and phosphorylated-tau in neuronal exosomes may document preclinical AD. METHODS: AD neuropathogenic proteins Aß1-42, P-T181-tau, and P-S396-tau were quantified by enzyme-linked immunosorbent assays in extracts of neuronal exosomes purified from blood of individuals with DS and age-matched controls. RESULTS: Neuronal exosome levels of Aß1-42, P-T181-tau, and P-S396-tau were significantly elevated in individuals with DS compared with age-matched controls at all ages beginning in childhood. No significant gender differences were observed. DISCUSSION: These early increases in Aß1-42, P-T181-tau, and P-S396-tau in individuals with DS may provide a basis for early intervention as targeted treatments become available.

Transgenic mice overexpressing neuregulin-1 model neurofibroma-malignant peripheral nerve sheath tumor progression and implicate specific chromosomal copy number variations in tumorigenesis.

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFß3 mice) develop MPNSTs. To determine whether P(0)-GGFß3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFß3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFß3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFß3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFß3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

Neuregulin-1 overexpression and Trp53 haploinsufficiency cooperatively promote de novo malignant peripheral nerve sheath tumor pathogenesis.

Malignant peripheral nerve sheath tumors (MPNSTs) are Schwann cell-derived malignancies that arise from plexiform neurofibromas in patients with mutation of the neurofibromin 1 (NF1) gene. We have shown that the growth factor neuregulin-1 (NRG1) also contributes to human neurofibroma and MPNST pathogenesis and that outbred C57BL/6J × SJL/J transgenic mice overexpressing NRG1 in Schwann cells (P0-GGFß3 mice) recapitulate the process of neurofibroma-MPNST progression. However, it is unclear whether NRG1 acts predominantly within NF1-regulated signaling cascades or instead activates other essential cascades that cooperate with NF1 loss to promote tumorigenesis. We now report that tumorigenesis is suppressed in inbred P0-GGFß3 mice on a C57BL/6J background. To determine whether NRG1 overexpression interacts with reduced Nf1 or Trp53 gene dosage to "unmask" tumorigenesis in these animals, we followed cohorts of inbred P0-GGFß3;Nf1+/−, P0-GGFß3;Trp53+/− and control (P0-GGFß3, Nf1+/− and Trp53+/−) mice for 1 year. We found no reduction in survival or tumors in control and P0-GGFß3;Nf1+/− mice. In contrast, P0-GGFß3;Trp53+/− mice died on average at 226 days, with MPNSTs present in 95 % of these mice. MPNSTs in inbred P0-GGFß3;Trp53+/− mice arose de novo from micro-MPNSTs that uniformly develop intraganglionically. These micro-MPNSTs are of lower grade (WHO grade II-III) than the major MPNSTs (WHO grade III-IV); array comparative genomic hybridization showed that lower grade MPNSTs also had fewer genomic abnormalities. Thus, P0-GGFß3;Trp53+/− mice represent a novel model of low- to high-grade MPNST progression. We further conclude that NRG1 promotes peripheral nervous system neoplasia predominantly via its effects on the signaling cascades affected by Nf1 loss.

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models.

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that subsequently transform into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Understanding the process by which a PN transforms into an MPNST would be facilitated by the availability of genetically engineered mouse (GEM) models that accurately replicate the PN-MPNST progression seen in humans with NF1. Unfortunately, GEM models with Nf1 ablation do not fully recapitulate this process. This led us to develop P0-GGFß3 mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 (NRG1) in Schwann cells results in the development of PNs that progress to become MPNSTs with high frequency. However, to determine whether tumorigenesis and neoplastic progression in P0-GGFß3 mice accurately model the processes seen in NF1 patients, we had to first prove that the pathology of P0-GGFß3 peripheral nerve sheath tumors recapitulates the pathology of their human counterparts. Here, we describe the specialized methodologies used to accurately diagnose and grade peripheral nervous system neoplasms in GEM models, using P0-GGFß3 and P0-GGFß3;Trp53+/- mice as an example. We describe the histologic, immunohistochemical, and histochemical methods used to diagnose PNs and MPNSTs, how to distinguish these neoplasms from other tumor types that mimic their pathology, and how to grade these neoplasms. We discuss the establishment of early-passage cultures from GEM MPNSTs, how to characterize these cultures using immunocytochemistry, and how to verify their tumorigenicity by establishing allografts. Collectively, these techniques characterize the pathology of PNs and MPNSTs that arise in GEM models and critically compare the pathology of these murine tumors to their human counterparts.

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Molecular mechanisms promoting the pathogenesis of Schwann cell neoplasms.

Neurofibromas, schwannomas and malignant peripheral nerve sheath tumors (MPNSTs) all arise from the Schwann cell lineage. Despite their common origin, these tumor types have distinct pathologies and clinical behaviors; a growing body of evidence indicates that they also arise via distinct pathogenic mechanisms. Identification of the genes that are mutated in genetic diseases characterized by the development of either neurofibromas and MPNSTs [neurofibromatosis type 1 (NF1)] or schwannomas [neurofibromatosis type 2 (NF2), schwannomatosis and Carney complex type 1] has greatly advanced our understanding of these mechanisms. The development of genetically engineered mice with ablation of NF1, NF2, SMARCB1/INI1 or PRKAR1A has confirmed the key role these genes play in peripheral nerve sheath tumorigenesis. Establishing the functions of the NF1, NF2, SMARCB1/INI1 and PRKAR1A gene products has led to the identification of key cytoplasmic signaling pathways promoting Schwann cell neoplasia and identified new therapeutic targets. Analyses of human neoplasms and genetically engineered mouse models have established that interactions with other tumor suppressors such as TP53 and CDKN2A promote neurofibroma-MPNST progression and indicate that intratumoral interactions between neoplastic and non-neoplastic cell types play an essential role in peripheral nerve sheath tumorigenesis. Recent advances have also provided new insights into the identity of the neural crest-derived populations that give rise to different types of peripheral nerve sheath tumors. Based on these findings, we now have an initial outline of the molecular mechanisms driving the pathogenesis of neurofibromas, MPNSTs and schwannomas. However, this improved understanding in turn raises a host of intriguing new questions.

The RASopathies: Biology, genetics and therapeutic options.

The RASopathies are a group of genetic diseases in which the Ras/MAPK signaling pathway is inappropriately activated as a result of mutations in genes encoding proteins within this pathway. As their causative mutations have been identified, this group of diseases has expanded to include neurofibromatosis type 1 (NF1), Legius syndrome, Noonan syndrome, CBL syndrome, Noonan syndrome-like disorder with loose anagen hair, Noonan syndrome with multiple lentigines, Costello syndrome, cardiofaciocutaneous syndrome, gingival fibromatosis and capillary malformation-arteriovenous malformation syndrome. Many of these genetic disorders share clinical features in common such as abnormal facies, short stature, varying degrees of cognitive impairment, cardiovascular abnormalities, skeletal abnormalities and a predisposition to develop benign and malignant neoplasms. Others are more dissimilar, even though their mutations are in the same gene that is mutated in a different RASopathy. Here, we describe the clinical features of each RASopathy and contrast them with the other RASopathies. We discuss the genetics of these disorders, including the causative mutations for each RASopathy, the impact that these mutations have on the function of an individual protein and how this dysregulates the Ras/MAPK signaling pathway. As several of these individual disorders are genetically heterogeneous, we also consider the different genes that can be mutated to produce disease with the same phenotype. We also discuss how our growing understanding of dysregulated Ras/MAPK signaling had led to the development of new therapeutic agents and what work will be critically important in the future to improve the lives of patients with RASopathies.