whole-central-and-peripheral-nervous-system-mice-dissection

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

University of Texas Southwestern Medical Center at Dallas

The human disease von Recklinghausen's neurofibromatosis (Nf1) is one of the most common genetic disorders. It is caused by mutations in the NF1 tumor suppressor gene, which encodes a GTPase activating protein (GAP) that negatively regulates p21-RAS signaling. Dermal and plexiform neurofibromas as well as malignant peripheral nerve sheath tumors and other malignant tumors, are significant complications in Nf1. Neurofibromas are complex tumors and composed mainly of abnormal local cells including Schwann cells, endothelial cells, fibroblasts and additionally a large number of infiltrating inflammatory mast cells. Recent work has indicated a role for the microenvironment in plexiform neurofibroma genesis. The emerging evidence points to mast cells as crucial contributors to neurofibroma tumorigenesis. Therefore, further understanding of the molecular interactions between Schwann cells and their environment will provide tools to develop new therapies aimed at delaying or preventing tumor formation in Nf1 patients.

Tumor microenvironment and neurofibromatosis type I: connecting the GAPs.

Stem cells are under strict regulation by both intrinsic factors and the microenvironment. There is increasing evidence that many cancers initiate through acquisition of genetic mutations (loss of intrinsic control) in stem cells or their progenitors, followed by alterations of the surrounding microenvironment (loss of extrinsic control). In neurofibromatosis type 1 (NF1), deregulation of Ras signaling results in development of multiple neurofibromas, complex tumors of the peripheral nerves. Neurofibromas arise from the Schwann cell lineage following loss of function at the NF1 locus, which initiates a cascade of interactions with other cell types in the microenvironment and additional cell autonomous modifications. In this study, we sought to identify whether a temporal "window of opportunity" exists during which cells of the Schwann cell lineage can give rise to neurofibromas following loss of NF1. We showed that acute loss of NF1 in both embryonic and adult Schwann cells can lead to neurofibroma formation. However, the embryonic period when Schwann cell precursors and immature Schwann cells are most abundant coincides with enhanced susceptibility to plexiform neurofibroma tumorigenesis. This model has important implications for understanding early cellular events that dictate neurofibroma development, as well as for the development of novel therapies targeting these tumors.

Susceptible stages in Schwann cells for NF1-associated plexiform neurofibroma development.

Neurofibromatosis type I (NF1) is an autosomal disorder that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. Patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (MPNST), a main cause of mortality. Currently, surgery is the primary course of treatment for MPNST, but with the limitation that these tumors are highly invasive. Radiotherapy is another treatment option, but is undesirable because it can induce additional mutations. Patients with MPNST may also receive doxorubicin as therapy, but this DNA-intercalating agent has relatively low tumor specificity and limited efficacy. In this study, we exploited a robust genetically engineered mouse model of MPNST that recapitulates human NF1-associated MPNST to identify a novel small chemical compound that inhibits tumor cell growth. Compound 21 (Cpd21) inhibits growth of all available in vitro models of MPNST and human MPNST cell lines, while remaining nontoxic to normally dividing Schwann cells or mouse embryonic fibroblasts. We show that this compound delays the cell cycle and leads to cellular apoptosis. Moreover, Cpd21 can reduce MPNST burden in a mouse allograft model, underscoring the compound's potential as a novel chemotherapeutic agent.

Preclinical therapeutic efficacy of a novel pharmacologic inducer of apoptosis in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that develop sporadically or in neurofibromatosis type 1 (NF1) patients. There is no effective treatment for MPNSTs and they are typically fatal. To gain insights into MPNST pathogenesis, we utilized an MPNST mouse model that allowed us to study the evolution of these tumors at the transcriptome level. Strikingly, in MPNSTs we found upregulation of a chromatin regulator, Brd4, and show that BRD4 inhibition profoundly suppresses both growth and tumorigenesis. Our findings reveal roles for BET bromodomains in MPNST development and report a mechanism by which bromodomain inhibition induces apoptosis through induction of proapoptotic Bim, which may represent a paradigm shift in therapy for MPNST patients. Moreover, these findings indicate epigenetic mechanisms underlying the balance of anti- and proapoptotic molecules and that bromodomain inhibition can shift this balance in favor of cancer cell apoptosis.

BET bromodomain inhibition triggers apoptosis of NF1-associated malignant peripheral nerve sheath tumors through Bim induction.

Neurofibromatosis type 1 is a tumor-predisposing genetic disorder. Plexiform neurofibromas are common NF1 tumors carrying a risk of malignant transformation, which is typically fatal. Little is known about mechanisms mediating initiation and identity of specific cell type that gives rise to neurofibromas. Using cell-lineage tracing, we identify a population of GAP43(+) PLP(+) precursors in embryonic nerve roots as the cells of origin for these tumors and report a non-germline neurofibroma model for preclinical drug screening to identify effective therapies. The identity of the tumor cell of origin and facility for isolation and expansion provides fertile ground for continued analysis to define factors critical for neurofibromagenesis. It also provides unique approaches to develop therapies to prevent neurofibroma formation in NF1 patients.

Cells of origin in the embryonic nerve roots for NF1-associated plexiform neurofibroma.

Cutaneous neurofibromas (cNF) are a nearly ubiquitous symptom of neurofibromatosis type 1 (NF1), a disorder with a broad phenotypic spectrum caused by germline mutation of the neurofibromatosis type 1 tumour suppressor gene (NF1). Symptoms of NF1 can include learning disabilities, bone abnormalities and predisposition to tumours such as cNFs, plexiform neurofibromas, malignant peripheral nerve sheath tumours and optic nerve tumours. There are no therapies currently approved for cNFs aside from elective surgery, and the molecular aetiology of cNF remains relatively uncharacterised. Furthermore, whereas the biallelic inactivation of NF1 in neoplastic Schwann cells is critical for cNF formation, it is still unclear which additional genetic, transcriptional, epigenetic, microenvironmental or endocrine changes are important. Significant inroads have been made into cNF understanding, including NF1 genotype-phenotype correlations in NF1 microdeletion patients, the identification of recurring somatic mutations, studies of cNF-invading mast cells and macrophages, and clinical trials of putative therapeutic targets such as mTOR, MEK and c-KIT. Despite these advances, several gaps remain in our knowledge of the associated pathogenesis, which is further hampered by a lack of translationally relevant animal models. Some of these questions may be addressed in part by the adoption of genomic analysis techniques. Understanding the aetiology of cNF at the genomic level may assist in the development of new therapies for cNF, and may also contribute to a greater understanding of NF1/RAS signalling in cancers beyond those associated with NF1. Here, we summarise the present understanding of cNF biology, including the pathogenesis, mutational landscape, contribution of the tumour microenvironment and endocrine signalling, and the historical and current state of clinical trials for cNF. We also highlight open access data resources and potential avenues for future research that leverage recently developed genomics-based methods in cancer research.

Cutaneous neurofibromas in the genomics era: current understanding and open questions.

A group of experts in dermatology, genetics, neuroscience, and regenerative medicine collaborated to summarize current knowledge on the defined factors contributing to cutaneous neurofibroma (cNF) development and to provide consensus recommendations for future research priorities to gain an improved understanding of the biology of cNF.

The biology of cutaneous neurofibromas: Consensus recommendations for setting research priorities.

Neurofibromatosis type 1 (NF1) is a cancer predisposition disorder that results from inactivation of the tumor suppressor neurofibromin, a negative regulator of RAS signaling. Patients with NF1 present with a wide range of clinical manifestations, and the tumor with highest prevalence is cutaneous neurofibroma (cNF). Most patients harboring cNF suffer greatly from the burden of those tumors, which have no effective medical treatment. Ironically, none of the numerous NF1 mouse models developed so far recapitulate cNF. Here, we discovered that HOXB7 serves as a lineage marker to trace the developmental origin of cNF neoplastic cells. Ablating  in the HOXB7 lineage faithfully recapitulates both human cutaneous and plexiform neurofibroma. In addition, we discovered that modulation of the Hippo pathway acts as a "modifier" for neurofibroma tumorigenesis. This mouse model opens the doors for deciphering the evolution of cNF to identify effective therapies, where none exist today. SIGNIFICANCE: This study provides insights into the developmental origin of cNF, the most common tumor in NF1, and generates the first mouse model that faithfully recapitulates both human cutaneous and plexiform neurofibroma. The study also demonstrates that the Hippo pathway can modify neurofibromagenesis, suggesting that dampening the Hippo pathway could be an attractive therapeutic target..

Spatiotemporal Loss of  in Schwann Cell Lineage Leads to Different Types of Cutaneous Neurofibroma Susceptible to Modification by the Hippo Pathway.

Neurofibromatosis type 1 (NF1) is an autosomal genetic disorder. Patients with NF1 are associated with mono-allelic loss of the tumor suppressor gene NF1 in their germline, which predisposes them to develop a wide array of benign lesions. Intriguingly, recent sequencing efforts revealed that the NF1 gene is frequently mutated in multiple malignant tumors not typically associated with NF1 patients, suggesting that NF1 heterozygosity is refractory to at least some cancer types. In two orthogonal mouse models representing NF1- and non-NF1-related tumors, we discover that an Nf1 microenvironment accelerates the formation of benign tumors but impairs further progression to malignancy. Analysis of benign and malignant tumors commonly associated with NF1 patients, as well as those with high NF1 gene mutation frequency, reveals an antagonistic role for NF1 heterozygosity in tumor initiation and malignant transformation and helps to reconciliate the role of the NF1 gene in both NF1 and non-NF1 patient contexts.

NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation.

Skin epithelium is constituted by heterogeneous keratinocytes in different body areas. In this issue, Zhou et al. used transcriptome analysis to investigate the site-specific epidermal cell identity on volar skin. Keratin 9 is highly enriched in volar keratinocytes, and its expression is dependent on low expression of the double-stranded RNA receptor DDX58, suggesting that double-stranded RNA sensing could allow a potential approach to modulate skin thickness and durability.

Double-Stranded RNA Sensing Determines Epithelial Cell Identity.

BET bromodomain inhibitors have emerged as a promising therapy for numerous cancer types in preclinical studies, including neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumor (MPNST). However, potential mechanisms underlying resistance to these inhibitors in different cancers are not completely understood. In this study, we explore new strategy to overcome BET inhibitor resistance in MPNST. Through modeling tumor evolution by studying genetic changes underlying the development of MPNST, a lethal sarcoma with no effective medical treatment, we identified a targetable addiction to BET bromodomain family member BRD4 in MPNST. This served as a controlled model system to delineate mechanisms of sensitivity and resistance to BET bromodomain inhibitors in this disease.

Overcoming BET Inhibitor Resistance in Malignant Peripheral Nerve Sheath Tumors.

Hereditary cancer syndromes are typically caused by mutations of a tumor suppressor gene that lead to the early development of multifocal benign neoplasms followed by their malignant progression. However, the term 'hereditary cancer syndrome' may be misleading, as a large subgroup of syndromes are characterized by highly penetrant benign tumors. The reason why these cardinal tumors rarely progress to malignancy has been an elusive question in cancer biology. In this opinion article, we propose a framework where a heterozygous tumor suppressor gene microenvironment has antagonistic roles in tumorigenesis, by accelerating development of benign tumors while restraining further progression to malignant cancers.

Heterozygous Tumor Suppressor Microenvironment in Cancer Development.

Neurofibromatosis type 1 (NF1) is a hereditary tumour syndrome that predisposes to benign and malignant tumours originating from neural crest cells. Biallelic inactivation of the tumour-suppressor gene NF1 in glial cells in the skin, along a nerve plexus or in the brain results in the development of benign tumours: cutaneous neurofibroma, plexiform neurofibroma and glioma, respectively. Despite more than 40 years of research, only one medication was recently approved for treatment of plexiform neurofibroma and no drugs have been specifically approved for the management of other tumours. Work carried out over the past several years indicates that inhibiting different cellular signalling pathways (such as Hippo, Janus kinase/signal transducer and activator of transcription, mitogen-activated protein kinase and those mediated by sex hormones) in tumour cells or targeting cells in the microenvironment (nerve cells, macrophages, mast cells and T cells) might benefit NF1 patients. In this review, we outline previous strategies aimed at targeting these signalling pathways or cells in the microenvironment, agents that are currently in clinical trials, and the latest advances in basic research that could culminate in the development of novel therapeutics for patients with NF1.

Translating current basic research into future therapies for neurofibromatosis type 1.

Neurofibromatosis Type I (NF1) is a neurocutaneous genetic syndrome characterized by a wide spectrum of clinical presentations, including benign peripheral nerve sheath tumor called neurofibroma. These tumors originate from the Schwann cell lineage but other cell types as well as extracellular matrix (ECM) in the neurofibroma microenvironment constitute the majority of the tumor mass. In fact, collagen accounts for up to 50% of the neurofibroma's dry weight. Although the presence of collagens in neurofibroma is indisputable, the exact repertoire of ECM genes and ECM-associated genes (i.e. the matrisome) and their functions are unknown. Here, transcriptome profiling by single-cell RNA sequencing reveals the matrisome of human cutaneous neurofibroma (cNF). We discovered that classic pro-fibrogenic collagen I myofibroblasts are rare in neurofibroma. In contrast, collagen VI, a pro-tumorigenic ECM, is abundant and mainly secreted by neurofibroma fibroblasts. This study also identified potential cell type-specific markers to further elucidate the biology of the cNF microenvironment.

Human cutaneous neurofibroma matrisome revealed by single-cell RNA sequencing.

Neurofibromatosis Type 1 (NF1) is one of the most common inherited neurological disorders and predisposes patients to develop benign and malignant tumors. Neurofibromas are NF1-associated benign tumors but can cause substantial discomfort and disfigurement. Numerous studies have shown that neurofibromas arise from the Schwann cell lineage but both preclinical mouse models and clinical trials have demonstrated that the neurofibroma tumor microenvironment contributes significantly to tumorigenesis. This offers the opportunity for targeting new therapeutic vulnerabilities to treat neurofibromas. However, a translational gap exists between deciphering the contribution of the neurofibroma tumor microenvironment and clinically applying this knowledge to treat neurofibromas. Here, we discuss the key cellular and molecular components in the neurofibroma tumor microenvironment that can potentially be targeted therapeutically to advance neurofibroma treatment.

Tumorigenesis in neurofibromatosis type 1: role of the microenvironment.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease and one of the most common inherited tumor predisposition syndromes, affecting 1 in 3000 individuals worldwide. The NF1 gene encodes neurofibromin, a large protein with RAS GTP-ase activating (RAS-GAP) activity, and loss of NF1 results in increased RAS signaling. Neurofibromin contains many other domains, and there is considerable evidence that these domains play a role in some manifestations of NF1. Investigating the role of these domains as well as the various signaling pathways that neurofibromin regulates and interacts with will provide a better understanding of how neurofibromin acts to suppress tumor development and potentially open new therapeutic avenues. In this review, we discuss what is known about the structure of neurofibromin, its interactions with other proteins and signaling pathways, its role in development and differentiation, and its function as a tumor suppressor. Finally, we discuss the latest research on potential therapeutics for neurofibromin-deficient neoplasms.

Neurofibromin and suppression of tumorigenesis: beyond the GAP.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, invasive cancer that comprise around 10% of all soft tissue sarcomas and develop in about 8-13% of patients with Neurofibromatosis Type 1. They are associated with poor prognosis and are the leading cause of mortality in NF1 patients. MPNSTs can also develop sporadically or following exposure to radiation. There is currently no effective targeted therapy to treat MPNSTs and surgical removal remains the mainstay treatment. Unfortunately, surgery is not always possible due to the size and location of the tumor, thus, a better understanding of MPNST initiation and development is required to design novel therapeutics. Here, we provide an overview of MPNST biology and genetics, discuss findings regarding the developmental origin of MPNST, and summarize the various model systems employed to study MPNST. Finally, we discuss current management strategies for MPNST, as well as recent developments in translating basic research findings into potential therapies.

Article	Year
Tumor microenvironment and neurofibromatosis type I: connecting the GAPs. Oncogene\| PubMed ID: 17297459	2007
Susceptible stages in Schwann cells for NF1-associated plexiform neurofibroma development. Cancer research\| PubMed ID: 21551250	2011
Preclinical therapeutic efficacy of a novel pharmacologic inducer of apoptosis in malignant peripheral nerve sheath tumors. Cancer research\| PubMed ID: 24285727	2014
BET bromodomain inhibition triggers apoptosis of NF1-associated malignant peripheral nerve sheath tumors through Bim induction. Cell reports\| PubMed ID: 24373973	2014
Cells of origin in the embryonic nerve roots for NF1-associated plexiform neurofibroma. Cancer cell\| PubMed ID: 25446898	2014
Cutaneous neurofibromas in the genomics era: current understanding and open questions. British journal of cancer\| PubMed ID: 29695767	2018
The biology of cutaneous neurofibromas: Consensus recommendations for setting research priorities. Neurology\| PubMed ID: 29987131	2018
Spatiotemporal Loss of in Schwann Cell Lineage Leads to Different Types of Cutaneous Neurofibroma Susceptible to Modification by the Hippo Pathway. Cancer discovery\| PubMed ID: 30348677	2019
NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation. Nature communications\| PubMed ID: 30479396	2018
Double-Stranded RNA Sensing Determines Epithelial Cell Identity. The Journal of investigative dermatology\| PubMed ID: 30579424	2019
Overcoming BET Inhibitor Resistance in Malignant Peripheral Nerve Sheath Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 30796033	2019
Heterozygous Tumor Suppressor Microenvironment in Cancer Development. Trends in cancer\| PubMed ID: 31474359	2019
Translating current basic research into future therapies for neurofibromatosis type 1. British journal of cancer\| PubMed ID: 32439933	2020
Human cutaneous neurofibroma matrisome revealed by single-cell RNA sequencing. Acta neuropathologica communications\| PubMed ID: 33413690	2021
Tumorigenesis in neurofibromatosis type 1: role of the microenvironment. Oncogene\| PubMed ID: 34345017	2021
Neurofibromin and suppression of tumorigenesis: beyond the GAP. Oncogene\| PubMed ID: 35066574	2022
Malignant peripheral nerve sheath tumor: models, biology, and translation. Oncogene\| PubMed ID: 35393544	2022

Lu Q. Le

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