1. Natural History, Biology, and Treatment of Dermal Neurofibromas in Neurofibromatosis Type 1 (NF1)

Abstract

Neurofibromatosis type 1 (NF1) is a common multisystem genetic disorder associated with the development of benign and malignant tumors primarily of the nervous system. No standard treatment other than surgery exists for most NF1-associated tumors. NF1 is 100% penetrant and features variable expressivity and essentially no phenotype/genotype correlation. The ability to predict the ultimate severity of disease would have a significant impact on the management and treatment of individuals with NF1. In addition, the development of active medical treatments for tumors associated with NF1 is an important goal given the lack of such treatments (other than surgery) and the morbidity associated with these tumors.

The NCI Pediatric Oncology Branch (POB) has developed a clinical trials program for children and young adults with NF1 and progressive plexiform neurofibromas. The program is based on the application of novel targeted agents (under development for various forms of cancer) to NF1-related tumors. However, many aspects of the natural history of NF1-associated tumors are not fully characterized and thus require study to be able to assess the effects of new treatments in clinical trials. This includes the natural history of dermal neurofibromas, which occur in every individual with NF1 and which are a significant cosmetic problem and a major cause of morbidity.

The proposed project (1) uses an innovative gene expression method to identify genetic modifiers of dermal neurofibroma burden, (2) explores the natural history of dermal neurofibromas in NF1 with new imaging techniques, and (3) outlines a clinical trial with a novel targeted agent in adults with NF1 and dermal neurofibromas. Should the new agent demonstrate significant clinical effect against dermal neurofibromas, we will extend our efforts to identify genetic modifiers of chemosensitivity and to use gene expression profiling to predict response. These goals are built on the expertise in clinical trials development at the POB and in genomic analysis at NHGRI. The NIH CC provides the ideal infrastructure for the conduct of natural history and innovative clinical trials. Given the current absence of therapeutic protocols for adults with NF1 and dermal neurofibromas, this trial will likely generate great interest among affected individuals and have rapid accrual.

2. Development of a Specific Drug Treatment for WHIM Syndrome

Abstract

WHIM syndrome is a rare autosomal-dominant immunodeficiency disorder characterized by the combination of warts, hypogammaglobulinemia, infections, and myelokathexis that is associated with gain of function mutations in the chemokine receptor CXCR4. How WHIM mutations cause disease, particularly the predilection for papillomavirus infection, is poorly understood, and there is no specific treatment. We propose to use the Bench-to-Bedside mechanism to launch a new research initiative at the NIH aimed at filling these gaps. The Bench component involves development of a mouse model of WHIM syndrome, and the Bedside component involves recruitment of WHIM patients (for the first time) to the Clinical Center. Patients and the mouse model will be studied to understand immunopathogenetic mechanisms, particularly in the bone marrow and skin, as a collaborative effort involving experts in immunology, dermatology, signal transduction, hematopoiesis, and chemokine receptors from NIAID and NCI. Since CXCR4 is a major HIV coreceptor and also regulates neutrophil distribution between bone marrow and peripheral blood, the pharmaceutical industry has targeted CXCR4 and antagonists have already been tested in clinical trials for HIV/AIDS patients and for hematopoietic stem cell and neutrophil mobilization in the setting of transplantation and cancer. A long-term goal of this project is to seek collaboration with companies that have developed such agents for preclinical evaluation in the WHIM mouse model followed by clinical trial at the Clinical Center in patients. Although WHIM syndrome is a rare disease, effective treatments might be broadly applied, potentially even as topical agents, for example, in otherwise normal individuals with common warts.

3. Testing Treatment of Hutchinson-Gilford Progeria Syndrome with Farnesyl Transferase Inhibitors

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare and uniformly fatal segmental "premature aging" disease in which all children die as a consequence of severe atherosclerosis at an average age of 13 years (range 8 to 21 years) (1). We have recently discovered that HGPS is a sporadic autosomal-dominant disease caused in most cases by a single base alteration (henceforth designated as G608G) in the LMNA gene, which creates a cryptic splice site and therefore alters the lamin A protein product (2). Lamin A is normally expressed by most differentiated cells and requires posttranslational farnesylation to incorporate into the nuclear membrane. The lamin A C-terminal peptide, including the farnesyl group, is subsequently cleaved, and mature lamin A becomes a prominent component of the nuclear scaffold just internal to the nuclear membrane, affecting nuclear structure and function (3). The defective protein product in HGPS (progerin) lacks the cleavage site for removal of the C-terminal farnesylated peptide and likely produces disease via dominant-negative effects on the nuclear structure and function of various cell types that express lamin A. Both in vitro cellular and in vivo vascular HGPS phenotypes have been established. In vitro, our data demonstrate that HGPS skin fibroblasts, progerin-transfected HeLa cells, and vascular smooth muscle cells (VSMCs) display disruption of nuclear architecture, premature senescence, and apoptosis. In vivo, we have recently developed a mouse model of HGPS by creating a transgenic line that expresses progerin from a human bacterial artificial chromosome (BAC) engineered to carry the HGPS mutation. These animals develop dramatic disappearance of VSMCs in the media of large arteries in a manner highly reminiscent of the autopsy findings in children with HGPS. We propose that these features can be improved or reversed in vitro and in vivo by blocking posttranslational processing via treatment with farnesyl transferase inhibitors (FTIs). We hypothesize that reduction of the quantity of mature progerin will improve the function of HGPS cells and vessels. Preliminary evidence in HEK-293 cells indicates that exposure to FTIs prohibits the interaction between progerin and the nuclear membrane where it normally functions and eliminates nuclear deformity. We will assess the ability of two different FTIs to block the cellular phenotype in HGPS dermal fibroblasts and vascular cell types transfected with the G608G gene defect. We will also treat the G608G progeria mouse line with FTIs and assess whether these drugs prevent progression to vascular disease and/or restore normal vascular anatomy as assessed by physiologic measurements of blood pressure and post-mortem quantitative morphometric and immunohistochemical analysis of the intima and media. In Year 1 of this project, cell culture and mouse experiments will establish whether efficacy of FTI treatment in patients with HGPS is likely. In addition, the FTIs we propose to test are currently being used in phase III clinical trials for cancer (including in children) and would therefore be available for immediate application to HGPS. Therefore, if data generated in Year 1 show promise, we anticipate proceeding with a clinical trial of FTIs in HGPS in Year 2. We will design, submit for IRB approval, and implement recruitment for a clinical trial; possible parameters to follow to assess drug response are already being defined in an ongoing longitudinal study of HGPS at NIH.

4. Analysis of Global Gene Expression Patterns and Mitochondrial DNA Damage in Lymphocytes of Friedreich’s Ataxia Patients Undergoing Idebenone Treatment in a Phase II Double-Blind Placebo-Controlled Study

Abstract

Friedreich’s ataxia (FRDA) is the most common inherited ataxia, with a prevalence of 1:40,000. It is an autosomal-recessive genetic disorder that usually has onset in the first two decades of life with gait disturbance and speech difficulty. Progressive damage to the nervous system leads to muscle weakness, inability to walk, and loss of sensation in the extremities. Other problems that arise in FRDA patients include diabetes, scoliosis, and cardiomyopathy. This disease is caused by a GAA triplet repeat expansion in the first intron of the gene for the protein frataxin, leading to a reduction of mRNA and protein levels. While the exact molecular action of frataxin is not known, it is known to be a mitochondrial protein involved in iron homeostasis and biosynthesis of iron-sulfur cluster containing proteins. This inter-institute collaborative project between a clinical laboratory at NINDS and a basic research laboratory at NIEHS seeks to test the hypothesis that FRDA patients (1) accumulate mitochondrial DNA damage in peripheral lymphocytes, (2) share common gene expression patterns unique to the pathogenesis of the disease, and (3) show diminution of both of these effects by idebenone treatment. This collaborative project advances current Friedreich’s ataxia research by increasing understanding of the disease mechanism and identifying biomarkers for future clinical trials. The project would not be possible without a direct collaboration between these two laboratories.

5. Adoptive Cell Therapy for Ewing’s Sarcoma Using Artificial Antigen Presenting Cells

Abstract

Lymphopenia induces profound changes in T-cell homeostatic controls related, in part, to an increased availability of the T-cell stimulatory cytokine IL7. These changes augment proliferation of adoptively transferred T cells and allow dramatic skewing of the immune repertoire when adoptive T-cell therapy is combined with tumor vaccines in lymphopenic hosts. Standard therapies for Ewing’s sarcoma induce profound lymphopenia and an increased availability of circulating IL7, raising the possibility that adoptive cell therapy administered following standard therapy may improve outcomes in this disease. The use of artificial antigen presenting cells (aAPCs) to expand polyclonal and antigen-specific cytolytic CD8+ T cells represents a promising approach for improving the functionality and reproducibility of cells used for adoptive therapy. Among aAPCs currently under study, very promising results have been observed in preclinical studies using the K562 cell line stably transfected with the high-affinity Fc receptor used to provide anti-CD3 ± anti-CD28 stimulation using moAbs and 4-1BBL, which provides critical costimulation for expansion of cytolytic CD8+ T cells. This project will undertake three specific aims that delineate the steps required to translate these basic insights into a clinical trial of adoptive cellular therapy for Ewing’s sarcoma.

6. Ganaxolone Therapy for Niemann-Pick Type C

Abstract

Niemann-Pick type C (NPC) is an autosomal-recessive, lysosomal storage disorder characterized by accumulation of cholesterol and gangliosides in both peripheral and central nervous system tissues. The major clinical problems in NPC are neurological and include cerebellar ataxia, dysarthria, seizures, supranuclear vertical gaze palsy, motor impairment, dysphagia, psychotic episodes, and progressive dementia. NPC is a progressive, lethal disorder with an estimated prevalence of 1/120,000 to 1/150,000. There is no effective therapy for NPC. Recently, it was reported that NPC mice have a deficiency of the enzymes involved in allopregnanolone synthesis and of allopregnanolone itself. Allopregnanolone is a neurosteroid. Treatment of NPC mice with allopregnanolone delays the onset of neurological symptoms, is neuroprotective, and significantly increases lifespan. Ganaxolone is a methylated derivative of allopregnanolone that has previously been used in pediatric clinical trials for the treatment of infantile spasms. We are proposing to use both mouse and human studies to determine the safety and efficacy of ganaxolone therapy in NPC.

7. UVA Sensitivity in Smith-Lemli-Opitz Syndrome: Possible Involvement of Cholesta-5,7,9(11)-trien-3β-ol

Abstract

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal-recessive, multiple congenital anomaly/mental retardation syndrome due to an inborn error of cholesterol synthesis. Photosensitivity is one clinical problem that is encountered in SLOS. The defect in cholesterol synthesis is due to a deficiency of 7-dehydrocholesterol reductase (DHCR7) activity. DHCR7 catalyzes the reduction of 7-dehydrocholesterol (7DHC) to yield cholesterol. Thus in SLOS patients, there is an accumulation of 7DHC, which may have toxic effects. Oxidation of 7DHC results in the formation of cholesta-5,7,9(11)-trien-3β-ol (9-DDHC), and 9-DDHC has been identified in serum from SLOS patients. We hypothesize that 9-DDHC may be a photosensitizer in SLOS patients, and we further hypothesize that 9-DDHC may have systemic effects. Thus, the first component of this project proposes to measure 9-DDHC levels in the skin of SLOS patients and correlate these levels with symptoms of photosensitivity. The second phase of this project will correlate serum 9-DDHC levels with clinical findings in SLOS patients and determine if treatment with antioxidants is efficacious in reducing 9-DDHC levels. If 9-DDHC is the photosensitizer in SLOS patients, this work may have broader clinical implications. 9-DDHC is present in normal skin; thus, it may be a chromophore involved in skin photoaging and UV-induced skin cancer.

8. Pre-Clinical and Clinical Investigations into the Mechanisms and Efficacy of Extracorporeal Photopheresis (ECP) in the Abrogation of Graft-versus-Host Disease (GVHD) and Facilitation of Graft-versus-Tumor (GVT) Immunity in Pediatric Patients.

Abstract

Extracorporeal photopheresis (ECP) has been effective for treatment of Graft-versus-Host Disease (GVHD) and to prevent solid organ rejection. However, the mechanisms of action of ECP are poorly understood. In particular, it is unclear whether ECP results in global immune suppression or antigen-specific suppression of undesirable immune responses such as those directed against histocompatibility antigens. In addition, although this approach has been used successfully in adult patients, the ability to apply ECP to the pediatric population is limited due to the large extracorporeal volume associated with current technology. The first goal of this proposal is to utilize murine models currently in use in the Pediatric Oncology Branch (POB) to further elucidate the mechanisms by which ECP can inhibit graft-versus-host responses. In particular, these studies will determine whether treatment of mice undergoing minor histocompatibility mismatched bone marrow transplantation with ECP results in diminished GVHD but allows vaccination against a model tumor antigen. This aim will complement ongoing efforts to direct antigen-specific immune responses against tumor antigens in the setting of allogeneic transplantation. DTM has an interest in applying apheresis techniques to small children. The clinical portion of this proposal will involve the development and application of novel ECP technology to pediatric patients with GVHD. Clinical samples will be analyzed for laboratory correlates of ECP using assays currently being employed in existing adult trials being conducted by the LHD. Additional correlates elucidated from the murine studies will also be assessed. It is hoped that both the bench and clinical research components of this proposal will form the basis for the development of future protocols to explore the use of ECP as GVHD prophylaxis combined with dendritic cell-based vaccines in pediatric patients with high-risk malignancies. The overriding goal of this project is to bring together expertise across several NIH Institutes and CC departments for the following purposes:

• To investigate the immunobiologic effects of ECP in murine models and pediatric patients.
  
• To study the effects of ECP on antigen-specific vaccine response and anti-tumor alloreactivity, i.e., graft-versus-leukemia (GVL) effect in murine models.
  
• To study the feasibility, safety, and efficacy of ECP in pediatric oncology patients with cGVHD.

9. Site-Selective cAMP Analogs for Treatment of Carney Complex

Abstract

Carney Complex (CNC), the complex of spotty skin pigmentation that can accompany multiple endocrine neoplasia, is attributed to the mutational loss of PRKARIA, the gene that codes for the Rα subunit of cAMP-dependent protein kinase type I (PKA-I). PKA exists in two isoforms, PKA-I and PKA-II. The PKA isozymes are expressed in a balance of cell growth and differentiation. It has been shown that the PKA-I-to-PKA-II ratio is reversed in primary clinical tumors and transformed tumor cell lines as compared to their normal counterparts and that the experimental approaches that induce PKA-isozyme switching in cancer cells result in tumor cell growth arrest and induction of tumor reversion. The objective of this study is to restore the PKA-I-to-PKA-II ratio in the adrenal glands and other related tumors of Carney complex to that of normal tissue by the following experimental approaches: (1) Use of a site-selective cAMP analog, 8-Cl-cAMP, to restore normal PKA activity in tumors of CNC patients in the setting of an 8-Cl-cAMP phase I clinical study; (2) use of unhydrolyzable Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS with 8-Cl-cAMP to enhance PKA-II activation; and (3) use of the CRE (cAMP response element)-transcription factor decoy to inhibit tumor growth without harming normal cell growth. A long-term goal is the RIα gene transfer/gene therapy of our patients; in the context of this grant we will test this in CNC tumor cell lines.

10. Pathogenesis of and Risk Factors for Autoimmunity in the Wiskott-Aldrich Syndrome

Abstract

Autoimmunity is an increasingly common and difficult to treat manifestation of the primary X-linked immunodeficiency Wiskott-Aldrich syndrome (WAS). Up to 70% of WAS patients affected in recent retrospective cohorts have been found to have autoimmune manifestations. However, little is known about the pathogenesis of autoimmunity in these patients. In particular, whether negative regulatory elements of the immune system such as regulatory T cells or antigen-receptor triggered lymphocyte apoptosis are intact in WASP patients is not known. We will investigate the functioning of these regulatory pathways in WASP-deficient mice and WAS patients enrolled in NIH clinical research protocols and correlate defects found with the genetic lesions and clinical features of each patient. We will attempt to identify particular mutations in WAS and clinical risk factors for the development of autoimmune manifestations through a prospective comprehensive rheumatologic evaluation of each patient seen at the Clinical Center. These studies should provide a clearer picture of the pathogenesis of autoimmunity in WAS and form the basis for a clinical protocol designed to deliver mechanism-based and more effective immunotherapy to WAS patients with autoimmune complications.