- A Pain in the Neuroma: Using a New Pain Model to Find Ways to Stop the Vicious Cycle
- Molecular Mechanism of NF1 Vasculopathy
- Neurofibromin Ubiquitin Ligase: Regulation and Function
- New Hope from a Glimmer: Photodynamic Therapy for the Treatment of Plexiform Neurofibromas
- Functional Role of Merlin in Meningioma Cells
- Using Modified Oncolytic Herpes Simplex Virus (HSV) to Treat NF2 Tumors
- Understanding the Pathogenesis of Skeletal Manifestations of NF1 Patients
- Functional Analysis of Drosophila NF1
While surgical removal of a neurofibroma relieves the pain for most NF1 patients, some, unfortunately, experience an increase in pain. This is due to the formation of a neuroma, a jumbled mass of nerve fibers and connective tissues, at the end of the nerve that was cut during the surgery. Palpating the tissue overlying a neuroma evokes tingling or prickling in the distribution of the injured nerve. Surgical removal of the neuroma may provide temporary relief, but the pain often recurs following the inevitable evolution of a new neuroma at the nerve end. Previous animal models of neuropathic pain have focused on the increased sensitivity to pain that develops at a location distant from the site of injury and not on the pain from direct stimulation of the neuroma.
Through his Fiscal Year 2005 Therapeutic Development Award, Dr. Allan Joel Belzberg of Johns Hopkins University developed a new animal model of neuroma pain, the tibial neuroma transposition (TNT) model, in which the neuroma is located in a position that is accessible to mechanical testing and outside of the immediate area of the injured nerve. Mechanical stimulation of the neuroma produced a profound withdrawal behavior that could be distinguished from the increased sensitivity to pain that developed on the mouse's hind paw. This allows testing of pain in response to mechanical stimulation of the neuroma independent of pain due to mechanically increased sensitivity to pain. Dr. Belzberg was able to use the TNT model to show that both lidocaine injections and proximal tibial nerve transaction reversed the neuroma tenderness. Testing of the suicide transport molecule, Ricin, supported the idea that such molecules could eliminate the pain signaling nerve endings from a neuroma and lead to a significant reduction of neuroma tenderness.
Dorsi M, Chen L, Murinson B, Pogatzki-Zahn EM, Meyer RA, and Belzberg AJ. 2008. The tibial neuroma transposition (TNT) model of neuroma pain and hyperalgesia.Pain134(3):320-334.
Neurofibromatosis often results from mutations within the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Vasculopathies are an acceleration of blood vessel degeneration, constituting a major source of morbidity for neurofibromatosis (NF1) patients. Under the most serious of circumstances, this condition can even result in death. The role of neurofibromin in regulating blood vessel cell function is understood only at a fundamental level. Advances in understanding the biology of adult endothelial cell and vascular smooth muscle cell function are necessary to effectively treat the morbidity associated with vasculopathies in NF1 patients. Through a Fiscal Year 2007 Investigator-Initiated Research Award, Dr. David Ingram will investigate the molecular mechanisms behind the role of NF1 in vascular smooth muscle and endothelial cell functions in vivo. Based on existing preliminary data in the field, Dr. Ingram has carefully designed a plan to genetically engineer several animal models. Each model will simulate the pathobiology of this condition in NF1 deficient mice. These experiments will provide critical clues regarding the molecular pathways that initiate the formation of vasculopathies. Furthermore, the data will elucidate the nature of each pathway's relationship to neurofibromin. This project will provide the necessary tools through which the molecular pathogenesis of vasculopathies can be better understood and may pave the way for promising therapeutics or diagnostic agents.
Very little is currently known about how the NF1 protein neurofibromin normally functions to control cell growth. Interestingly, Dr. Cichowski and colleagues have shown that neurofibromin is regulated by a specific ubiquitin ligase. This ligase facilitates the rapid degradation of neurofibromin, resulting in the activation of cell growth mediated by Ras. The molecular activation of Ras by ligase-mediated neurofibromin degradation represents a landmark discovery in understanding the underpinnings of this disease and its progression. Using this key observation, Dr. Karen Cichowski plans to research ways to block this process through funding from a Fiscal Year 2007 Investigator-Initiated Research Award. Dr. Cichowski will study and identify other proteins that regulate the function of neurofibromin and the associated ubiquitin ligase. In researching this fundamental process, Dr. Cichowski hopes to find druggable targets to slow or even halt the degradation of neurofibromin. These findings may serve as a platform by which future therapeutics can be rapidly tested for efficacy ahead of clinical trials, an additional benefit to the NF1 community.
Plexiform neurofibromas (PN) occur with great frequency in patients with neurofibromatosis type 1 (NF1). These tumors can result in disfiguration and functional impairment, and they can even be life-threatening. While surgical intervention is sometimes effective, in most cases it is not a viable option. In addition, current therapeutics may arrest PN growth, but they are not likely to shrink large PNs. Through funding from a Fiscal Year 2007 New Investigator Award, Dr. Michael Fisher of the Children's Hospital of Philadelphia is investigating the feasibility of a new type of therapy in children suffering from PNs. His study will use photodynamic therapy (PDT), or light therapy, which employs a photosensitizer that, upon interacting with a particular wavelength of light, can kill nearby cells through the production of reactive oxygen species. This study will test the safety and effectiveness of using the photosensitizer LS11 to treat PNs in children and young adults. The small implantable light source being used in the study is designed to limit the activation of LS11 to the confines of the tumor. It is anticipated that this treatment will induce tumor shrinkage and subsequently reduce the morbidities associated with PNs.
Dr. Anita Lal at the University of California researches Neurofibromatosis 2 (NF2), a cancer predisposition syndrome phenotypically characterized by the presence of multiple benign brain tumors, primarily schwannomas and meningiomas. Unfortunately, most studies evaluating the mechanism of action of merlin, the NF2 gene product, have used cell lines unrelated to NF2 target cells. Through a Fiscal Year 2005 New Investigator Award, Dr. Lal isolated and immortalized human meningioma cell lines, and with these, engineered merlin-deficient or merlin-expressing meningioma cells. Dr. Lal used these cells to characterize the phenotypic effects of merlin loss in this more accurate NF2 environment. Dr. Lal determined that the loss of merlin in meningioma cells caused those cells to neglect to undergo the normal contact inhibition of growth. With the loss of merlin, those cells also exhibited enhanced anchorage-independent growth in soft agar. Merlin loss was found to alter the cell growth cycle, causing an increase in S-phase entry, and a concomitant decrease in G0-G1 and G2 populations. Dr. Lal determined that merlin loss in this cell population was associated with an increase in transcript and protein levels of cyclin E1, a regulator of the G0-G1 to S transition and potential downstream target of merlin. Merlin loss was also found to affect the apoptotic rates in these meningioma cell lines, causing a decrease in spontaneous apoptotic rates and increasing resistance to Stsp-mediated apoptosis. Dr. Lal intends to investigate these results further, along with exploring for other downstream transcriptional effectors of meningiomas. This more accurate NF2 model will be useful in the future to test potential novel therapeutics against NF2.
Using Modified Oncolytic Herpes Simplex Virus (HSV) to Treat NF2 Tumors
Posted May 2, 2008
Robert L. Martuza, M.D., Xandra Breakefield, Ph.D, Samuel Rabkin, Ph.D., Massachusetts General Hospital, Boston, Massachusetts
Schwannomas and meningiomas are tumors that form in neurofibromatosis 2 (NF2) patients by overgrowth of cells that support neurons in the nervous system. Such tumors can cause pain, paralysis, seizures, hearing loss, and death. Through his Fiscal Year 2003 Therapeutic Development Award, Dr. Robert L. Martuza and his colleagues Dr. Xandra Breakefield and Dr. Samuel Rabkin sought to develop safe, targeted gene delivery methods to treat and evaluate responses in Schwannomas and meningiomas. Dr. Martuza and colleagues tested the use of oncolytic herpes simplex virus (HSV) type 1 vectors as a new treatment for NF2-associated tumors. Oncolytic viruses (viruses that can infect and kill cancer cells while leaving normal cells alone) are not 100% efficient when used in therapies. Dr. Martuza and colleagues attempted to improve efficiency by arming the oncolytic viruses with therapeutic transgenes to allow the vector to attack the tumor in multiple ways. They found that the oncolytic HSV vector G47D decreased cell viability of a human schwannoma cell line 2.5-fold. Dr. Martuza and colleagues tested G47D in a spontaneous NF2 schwannoma mouse model and found that all tumors treated with G47D decreased in size by over 60%. They then added a transgene to an HSV vector that would specifically kill schwannoma cells. In a schwannoma tumor model, three injections of this vector led to almost complete regression of tumors during treatment. The tumors only resumed growth 7 days after the third and final vector treatment.
Neurofibromatosis type 1 (NF1) is a common, autosomal-dominant disorder caused by mutations in the NF1 tumor suppressor gene. At least 50% of NF1 patients have one or more skeletal abnormalities, including kyphoscoliosis, pseudoarthrosis, and osteoporosis, but only limited efforts have been focused on understanding the underlying molecular mechanisms. Dr. Feng-Chun Yang received a Fiscal Year 2007 New Investigator Award to identify the mechanisms underlying the skeletal manifestations of NF1 in murine and human systems. Dr. Yang plans first to evaluate the skeletal phenotype in a murine model of NF1. In collaboration with a skeletal biologist, who has experience in the use of biomechanical engineering and bioimaging methods, Dr. Yang will perform detailed structural studies of bone and examine the role of Nf1 in bone repair and remodeling. Dr. Yang will also begin to dissect the intracellular signaling cascades that control the alterations osteoblast differentiation while collaborating with a clinical geneticist who is focusing on the diagnosis and treatment of the skeletal dysplasias in NF1.
Neurofibromatosis type 1 (NF1) patients are predisposed toward developing a variety of symptoms, including areas of abnormal skin pigmentation, benign tumors associated with peripheral nerves, termed neurofibromas, and developmental abnormalities such as skeletal defects and short stature. NF1 is caused by loss of the neurofibromin protein (NF-1), which includes a segment related to the catalytic domains of Ras-specific GTPase-Activating Proteins (GAPs). The Ras signaling pathway controls several cellular processes including cytoskeletal integrity, proliferation, cell adhesion, apoptosis, and cell migration. Abnormalities of the Ras pathway have been implicated in several types of cancers. Evidence from some in vitro and in vivo systems supports the notion that the ability of neurofibromin to inactivate Ras plays a critical role in the development of NF1-associated tumors. In most Drosophila models, however, NF-1 ortholog phenotypes, including an overall growth defect, are not readily modified by manipulating Ras, but are restored by increasing signaling through the cyclic AMP-dependent protein kinase A (cAMP/PKA) pathway. This information suggests that NF-1 may have distinct Ras- and cAMP-related functions.
Dr. Andre Bernards, recipient of a Fiscal Year 2003 Neurofibromatosis Research Program Investigator-Initiated Research Award, has been working to elucidate how the Drosophila NF-1 ortholog protein intersects with the cAMP/PKA pathway by evaluating whether this pathway represents a valid therapeutic target for human patients with NF1. Performing a comprehensive structure-function analysis of the NF-1 protein in vivo in Drosophila melanogaster, Dr. Bernards found that expression of the NF-1 protein in a subset of Ras2-expressing larval neurons could rescue the overall NF1 growth defect, and that a functional NF-1-GAP catalytic domain is necessary and sufficient for growth restoration. The researcher discovered that the NF-1 protein has a non-cell-autonomous, potentially neuroendocrine function in organism growth control. In contrast to previous assumptions, Dr. Bernards found the involvement of the cAMP/PKA pathway in the NF1 phenotypes to be secondary to one or more Ras signaling defects, and he suggests that aberrant Ras-mediated signaling in larval neurons is the primary cause of the NF1 growth deficiency. These results would suggest that the cAMP/PKA-induced restoration of growth involves a function downstream in the Ras signaling pathway, as opposed to a separate, Ras-independent signaling event. Ongoing work focused on confirming these observations would help to focus the search for potential therapeutic targets in this pathway. Dr. Bernards' studies are advancing our understanding of the function of NF-1 protein while laying the groundwork for possible future therapeutics, giving new hope to those affected by NF1.
Bernards A, Walker J. 2007. Drosophila melanogaster neurofibromatosis-1: ROS, not Ras? Nature Genetics 39:443-445.
Walker JA, Tchoudakova AV, McKenney PT, et al. 2006. Reduced growth of Drosophila neurofibromatosis 1 mutants reflects a non-cell-autonomous requirement for GTPase-Activating Protein activity in larval neurons. Genes & Development 20:3311-3323.