1. Exploring the Anti-Tumor Effects of in vitro Expanded Natural Killer (NK) Cells against Renal Cell Carcinoma Sensitized to NK-TRAIL Cytotoxicity with Bortezomib

Abstract

The inability to expand NK cells in vitro to sufficient numbers required for their adoptive infusion in humans and the inactivation of NK cells as a consequence of tumor killer IgG-like receptor (KIR) ligands have precluded investigators from testing the therapeutic potential of NK cells in humans with cancer. Over the past 2 years, the group has developed a method to expand by 10⁴–10⁵ fold NK cells in vitro. Further, they have discovered that renal cell carcinoma (RCC) and other tumor cells can be sensitized to the cytotoxic effects of NK cells by pretreating them with bortezomib or depsipeptide through their upregulation of TRAIL death receptors on the tumor surface. This sensitization significantly enhances NK cell cytotoxicity against tumor cells in vitro and, importantly, appears to override NK cell inhibition mediated through the KIR pathway. A patent application on the expansion technology and the method to potentiate NK cell cytotoxicity against cancer, both developed in this laboratory, was filed with the U.S. Patent and Trademark Office on November 2, 2005. The group now proposes to evaluate the therapeutic potential of this method to potentiate NK cell anti-tumor activity in humans with advanced RCC.

Based on their data, the investigators propose the development of an immunotherapy trial conducted in the hematology branch at the NHLBI in collaboration with the Departments of Transfusion Medicine at the CC and the Medical and Urologic Oncology Branches of the NCI that explores the anti-neoplastic effects of adoptively infused NK cells into patients with advanced RCC. Specifically, this trial will be a phase I/II study evaluating the safety and anti-tumor efficacy of escalating doses of adoptively infused in vitro expanded autologous NK cells in RCC patients, with and without tumor sensitization using bortezomib (depsipeptide will not be used because it is not approved by the FDA). This trial will be conducted in collaboration with members of the Department of Transfusion Medicine, Medical Oncology Branch and the Urologic Oncology Branch of the NCI.

Patients with metastatic RCC will undergo a 10–15 liter apheresis procedure. Peripheral blood mononuclear cells (PBMCs) will be enriched for NK cells using the Miltenyi NK cell enrichment process that uses immuno-magnetic beads and magnetic columns. Enriched NK cells will be expanded over 3–4 weeks in vitro using an irradiated EBVLCL line generated at the NIH that has been certified for use under GMP conditions as a universal cell line. RCC patients will then receive adoptive NK cell infusions in a phase I dose escalating fashion, initially with NK cells alone, and if no response is observed, with the combination of bortezomib (to enhance susceptibility to NK cell TRAIL-mediated killing) and NK cells. Data regarding NK cell safety and anti-tumor efficacy with and without bortezomib will be gleaned from this study. Five dose levels accruing up to a maximum of 6 RCC patients in each dose level are proposed (i.e., a maximum of 30 patients to be enrolled on study).

a. Year 1: (1) Establish master EBVLCL cell bank and (2) perform initial developmental studies using Wave bioreactor to establish clinical scale NK cell culture parameters including cell concentration, media and other reagents, timing, and sampling for assays. (This will require gaining familiarity with this system and approximately five NK cell selections and culture events.) (3) Develop and submit clinical protocol to the NHLBI IRB.
b. Year 2: (1) Validate and establish final clinical scale NK cell generation procedure and release assays for GMP production, using clinical-grade reagents and cells from master cell bank. (This will require at least three NK cell selections and culture events, but also assay development.) (2) Submit IND, including NK generation.

2. Therapeutic Approaches for Cancer Stem Cells in Small Cell Neuroendocrine Carcinomas

Abstract

Small cell carcinomas (SCC) are rare neuroendocrine (NE) tumors that occur in multiple organs and follow distribution of NE cells. The cell of origin is not well defined and presumes that NE cells are both neural and endocrine in origin, thereby bridging two classically distinct body systems. The NE tumors are rare and include small cell carcinoma of the lung, uvea, and GI and GU tract. Regardless of their anatomic site, these are aggressive tumors with poor survival and ineffective therapeutic options. Because the majority of SCC present with disseminated disease and rapidly relapse with drug-resistant disease, we hypothesize that these tumors harbor a cancer stem cell population with a drug-resistance phenotype. The investigators characterized a population of SCC cells based on immunophenotypic markers and Verapamil-sensitive extrusion of Hoechst 33342 dye. As few as 50 of these cells are capable of self-renewal and reproduce tumors when injected subcutaneously into NOD/SCID mice. The investigators will develop novel therapeutic regimens that specifically target the stem cell population. They will generate tumors from isolated SCC stem cells that are capable of self-renewal in vivo. These valuable preclinical models will be used as a tool for testing drugs that target this cell population and to guide future clinical trials. The investigators will also develop clinical trials with a novel P-glycoprotein inhibitor, Tariquidar, and identify and characterize in vitro and in vivo the SCC stem cell population from these patients. Analysis will include the evaluation of the role of the stem cell population in response to therapy, thus completing the bench-to-bedside cycle. This approach provides the basis for development of clinical protocols for these rare tumors that otherwise have few medical options.

3. A New Global Function for a Rare Disease Gene: Clinical Significance of the Regulation of Mitochondrial Respiration by Tumor Suppressor p53 in Li-Fraumeni Syndrome

Abstract

The investigators have recently found that p53, one of the most frequently mutated genes in human cancers, dose dependently modulates the balance between the utilization of oxidative and glycolytic pathways for energy generation. Importantly, though morphologically indistinguishable from their wild-type littermates, mice deficient in p53 display a prominent gene dose-dependent decrease in aerobic exercise capacity, implying that p53 has functions beyond its well-characterized cell cycle activities. Their current findings have opened a new dimension for the function of tumor suppressor p53 with broad implications in fields ranging from cancer and aging research to cardiovascular physiology.

In the Li-Fraumeni familial cancer syndrome, carriers harbor one copy of a p53 germline mutation; therefore, they are p53 heterozygotes with reduced wild-type p53 activity. The investigators hypothesize that the carriers will have a metabolic deficiency in aerobic capacity and metabolism that previously has not been appreciated. This bench-to-bedside project translates the experimental observation to human subjects in collaboration with an extramural group that is currently evaluating and following Li-Fraumeni syndrome families. The results may not only help clarify why mutations of p53 gene are so common in cancers by conferring metabolic advantages in tumorigenesis, but they may also provide an opportunity to understand a fundamental regulatory mechanism in cellular energy generation relevant to diverse processes. The findings may also reveal new pathways or strategies for modulating bioenergetic abnormalities in cancers and other disorders such as the metabolic syndrome and for potentially improving cardiovascular function.

4. Pilot Trial of Intravenous Nitrite for Sickle Cell Vaso-occlusive Pain Crisis

Abstract

Sickle cell disease is an autosomal recessive disorder and the most common genetic disease affecting African Americans. Approximately 0.15% of African Americans are homozygous for sickle cell disease, and 8% have sickle cell trait. Hemoglobin S polymerization leads to red cell rigidity, microvascular obstruction, inflammation, and end-organ ischemia-reperfusion injury and infarction. Previously published data indicate that up to 50% of sickle cell patients have endothelial dysfunction due to impaired bioavailability of endogenous nitric oxide (NO) due in large part to scavenging of NO by cell-free plasma hemoglobin. These data suggest that therapies directed at restoring NO bioavailability might prove beneficial. The investigators have recently discovered that the nitrite anion, available currently for human use as a component of the cyanide antidote kit, is a vasodilator in vivo by generating NO in tissues with lower oxygen tension and pH. The mechanism involves a novel physiological function of human hemoglobin as an oxygen- and pH-dependent nitrite reductase. To date the team has observed that nitrite infusions in animal models significantly reduce liver and cardiac ischemia-reperfusion injury and infarction in mouse and canine models, prevent cerebral vasospasm after subarachnoid hemorrhage in primates, and decrease pulmonary hypertension in newborn hypoxic sheep. This protocol is designed as an animal study overlapping with a clinical trial in humans. The animal study will evaluate nitrite levels and pharmacokinetics in the sickle cell mouse. The human trial will be a pilot clinical trial of systemic intravenous nitrite in sickle cell vaso-occlusive inpatient pain crisis, with the hypothesis that this agent will reduce the duration of vaso-occlusive pain crisis; secondary outcome measures will include amount of opioid analgesic usage during the hospitalization, oxidation of plasma cell free hemoglobin to methemoglobin, and decrease in plasma NO consumption activity.

5. High-density Genotyping in Diffuse Large B-cell Lymphoma (DLBCL) and Follicular Lymphoma—Translating Etiologic Clues to Prognosis Relevance in the NCI-SEER NHL Case Control Study

Abstract

Recent investigations from the NIH and elsewhere reveal strong evidence that immunogenetic variations influence the risk of developing non-Hodgkins lymphoma (NHL). Specially, the investigators have demonstrated that a promoter variation within the pro-inflammatory cytokine, tumor necrosis factor (TNF), doubles the risk for major lymphoma subtype, diffuse large B-cell lymphoma (DLBCL)¹ . They have further confirmed this relationship in pooled analysis within the InterLymph Consortium² . Having recently linked the population registry (SEER) case-control study to survival outcomes (R01, PI: Jems Cerhan), investigators have discovered that among the etiology-based genes we investigated, immunogenetic variations and DNA repair genes also demonstrate prognostic significance³. In a multidisciplinary collaboration, they are currently determining the relevance of their findings to recently recognized molecular subtypes of DLBCL that have markedly distinct clinical prognosis⁴. At present, the investigators are the first to incorporate survival-based molecular pathology delineations in a population study. They believe a study specifically aimed at identifying genetic predictors of lymphoma survival is warranted. Within their population-based study, they are poised to quickly translate their knowledge of etiology and molecular pathology into clinical relevance for prognosis and survival outcomes. The completion of Phase II of the International HapMap Project (www.hapmap.org) now allows the investigators to thoroughly interrogate genetic variations within genes, chromosomal regions, and biologic pathways of highest interest. They propose to enrich the coverage of the genetic regions that have shown strong evidence of importance (e.g., immune genes) in lymphomagenesis and to fully interrogate critical pathways (e.g., DNA repair, drug metabolism) of highest relevance for survival outcomes with high-density genotyping of 6,000 well-chosen single nucleotide polymorphisms (SNPs) in the 2 major lymphoma subtypes (282 DLBCL, 190 follicular lymphomas, 282 controls). This study uniquely combines epidemiology, molecular pathology, genetics, and clinical response. In light of the converging evidence for a central role of genetics in lymphomagenesis, investigators can accelerate the understanding of this disease and translate science into saving lives.

¹ Wang SS, Cerhan JR, Hartge P, Davis S, Cozen W, Severson R, Chatterjee N, Yeager M, Chanock S, Rothman N. Common genetic variants in pro-inflammatory and innate immune genes and the risk for non-Hodgkin lymphoma. (submitted)
² Rothman N, Skibola CF, Wang SS, Morgan G, Lan Q, Smith MT, Spinelli JJ, Willett E, Sanjose SD, Cocco P, Berndt SI, Brennan P, Brooks-Wilson A, Wacholder S, Becker N, Hartge P, Zheng T, Roman E, Holly EA, Boffetta P, Armstrong B, Cozen W, Linet M, Bosch FX, Ennas MG, Holford TR, Gallagher RP, Rollinson S, Bracci PM, Cerhan JR, Whitby D, Moore PS, Leaderer B, Lai A, Spink C, Davis S, Bosch R, Scarpa A, Zhang Y, Severson RK, Yeager-Jeffery M, Chanock S, Nieters A. Genetic variation in TNF and IL10 and risk of non-Hodgkin lymphoma: A report from the Interlymph Consortium. Lancet Oncology 2005 [Epub].
³Cerhan JR, Habermann TM, Morton LM, Maurer MJ, Geyer SM, Allmer C, Cozen W, Davis S, Severson RK, Chanock SJ, Rothman N, Hartge P, Wang SS. Immunogenetics and survival: the example of non-Hodgkin Lymphoma. AACR Special Conference—New Developments in the Epidemiology of Cancer Prognosis: Traditional and Molecular Predictors of Treatment Response and Survival. Abstract #4277.
⁴Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, Moore T, Hudson J Jr, Lu L, Lewis DB, Tibshirani R, Sherlock G, Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Warnke R, Levy R, Wilson W, Grever MR, Byrd JC, Botstein D, Brown PO, Staudt LM. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000 403(6769):503-11.

6. Immunotherapy for Myelodysplastic Syndrome

Abstract

Myelodysplastic syndromes (MDS) are a group of heterogeneous disorders showing varying degrees of bone marrow failure. Because of the advanced age of many of these patients and their significant co-morbidities, current therapies are tolerated poorly and most lack efficacy. To clarify the cellular and molecular mechanisms in MDS, our laboratory has focused on syndromes that are defined by specific cytogenetics. In previous studies we demonstrated that CD34 cells from patients with trisomy 8 MDS are distinguished from some other MDS and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We subsequently demonstrated significant upregulation of cyclin D1 (CD1) by microarray analysis and realtime PCR in these and other MDS patients. Overexpression of CD1 would be expected to act to increase the proliferation and survival of the dysplastic clone and could explain the apparent survival advantage of trisomy 8 cells in particular in a bone marrow under immune attack. Styryl sulfones have proved to be significant inhibitors of cyclin D1 and successfully inhibit growth of mantle cell lymphoma in culture where they are currently in phase I clinical trials demonstrating significant activity and little toxicity. In this bench-to-bedside grant we will examine the activity of this novel agent against the dysplastic clone in vitro and initiate a clinical trial if significant in vitro activity is seen.

7. A Nutrigenomics Intervention for the Study of the Role of Dietary Sitosterol on Lipid, Glucose, and Energy Metabolism

Abstract

Mounting evidence indicates that the relative distribution of the various amounts of sterols in the cell membranes and lipid droplet membrane of the adipocyte plays an important role in lipid metabolism and energy homeostasis. Sitosterolemia, a rare autosomal recessive disease, is caused by a defective ABC transporter in the gut and biliary tract, which results in increased absorption and decreased excretion of plant sterols, ultimately leading to accelerated atherosclerosis and premature death.

Very recently, a mutation of the ABCG8 gene (G574R), very rare in the general population, has been described in 4% of the Old Order Amish, a well-characterized founder population in Lancaster County, Pennsylvania. The proband was homozygous for this mutation and died in his teens of premature atherosclerosis. Interestingly, otherwise healthy carriers (heterozygotes) of the mutation showed, as compared to controls, reduced body mass index, more large buoyant LDL cholesterol, decreased carotid intima media thickness (IMT), and a trend toward lower insulin and glucose levels, consistent with an improved “metabolic syndrome profile.” These data suggest that a mild excess in plant sterols caused by ABCG8 haploinsufficiency in these heterozygotes could play a role in the modulation of the energy metabolism and is consistent with some studies that show that increased dietary sitosterol (e.g., Benechol) may improve lipid profile and other aspects of the metabolic syndrome in genetically normal subjects.

In order to characterize mechanistically the effects of sitosterol, the most abundant plant sterol in the diet, on the development of the metabolic syndrome the investigators will study in greater detail the G574R heterozygotes, “experiments of nature,” that will provide the opportunity to glean new insights into dietary sitosterol and its role in lipid and energy metabolism. They hypothesize that sitosterol in the diet will affect metabolic syndrome indices differentially in G574R carriers compared to noncarriers.

To address this hypothesis, investigators will perform a nutrigenomics intervention on ABCG8 G574R carriers and sex-matched unaffected siblings (age ± 5 years). They will be treated with high-, low-, and normal-sitosterol iso-caloric diets. Study subjects will be then evaluated in the NIH Clinical Center for changes in the following parameters: circulating lipids and free fatty acids, glucose disposal, resting energy expenditure and RQ, isoproterenol-induced lipolysis, and endothelial vascular function. The Division of Endocrinology, Diabetes, and Nutrition at the University of Maryland School of Medicine will carry out the genotyping, study volunteer recruitment, cell membrane lipid content analysis, and ex vivo adipocyte analysis.

Specifically, investigators hypothesize that carriers of the ABCG8 G574R gene mutation will further improve metabolic syndrome indices when challenged with a high-sitosterol diet and will regress toward the non-carrier controls when treated with a low-sitosterol diet. Noncarrier controls will remain unchanged or show modest improvement in metabolic syndrome indices when challenged with a high-sitosterol diet that will worsen, compared to baseline and carrier sibs, when treated with low-sitosterol diet.

These clinical data, combined with the in vitro analysis of the effects of sitosterol on cell membranes and adipocyte metabolism, will advance knowledge in the field of lipid metabolism on the relation of lipid composition to common disorders such as the metabolic syndrome. Data obtained from this project could then easily be translated into nutritional and therapeutic interventions in the general population.

8. Novel Suicide Gene-Modified Donor Th2 Cells for GvHD Prevention

Abstract

Graft-versus-Host Disease (GvHD) remains the main cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Because the incidence and severity of GvHD increases as the HLA disparity between donor and host increases, potentially curative transplantation is generally limited to those subjects who have a suitable HLA-matched sibling donor. Allogeneic T cells contained in the stem cell inocula mediate GvHD but also mediate beneficial transplant effects, including immune reconstitution, graft-versus-tumor (GvT) effects, and the prevention of graft rejection. As such, a need exists to develop new strategies that allow beneficial allogeneic T-cell effects with reduced GvHD. To this end, investigators will utilize novel suicide gene insertion into allogeneic Th2 cells as an approach to prevent graft rejection with reduced GvHD. The scope of this work will include murine modeling, preclinical human studies, submission of an Investigational New Drug (IND) application to the FDA, and design of a pilot clinical trial to evaluate the safety and efficacy of this approach to GvHD prevention. Within the NCI Experimental Transplantation and Immunology Branch, we have developed a strategy of allograft T cell engineering that utilizes ex vivo-generated donor CD4+ T cells of Th2 cytokine phenotype. In a murine breast cancer model, we have shown that Th2 cells permit GvT effects with reduced GvHD; furthermore, in a fully MHC-disparate stem cell graft rejection model, we have shown that Th2 cells abrogate rejection with reduced GvHD. At the NIH Clinical Center, the investigators have completed a phase I/II clinical trial of Th2 cell therapy after HLA-matched sibling allogeneic HSCT (NIH protocol 99-C-0143) and have initiated a second-generation clinical trial of enhanced Th2 cells that are expanded in the presence of the immune modulation agent rapamycin (NIH protocol 04-C-0055). Current clinical trial results indicate that allogeneic Th2 cells are feasible to generate and safe to administer, promote Th2 cytokines post transplant, and represent a promising strategy for the mediation of beneficial transplant responses with reduced GvHD.

In this new initiative, the investigators seek to further enhance Th2 cell therapy for GvHD prevention through incorporation of a novel suicide gene into the Th2 cells. They hypothesize that suicide gene-modified Th2 cells will abrogate graft rejection with reduced GvHD relative to unmanipulated allogeneic T-cells. Subsequently, after facilitation of engraftment, the possibility of ongoing Th2 cell-mediated GvHD will be prevented through suicide gene activation of exogenously administered prodrug, with consequential Th2 cell clonal deletion. The investigators will employ a novel suicide gene therapy strategy incorporating a vector that engineers expression of a mutated version of human thymidylate kinase (TMPK) that is advantageous relative to conventional suicide genes as it has reduced immunogenicity, a 200-fold increase in enzyme efficiency, and utilizes AZT rather than gancyclovir as prodrug. Furthermore, they will use a recombinant lentiviral vector that will allow gene delivery to nearly 100% of allogeneic Th2 cells; as an additional step to ensure efficient gene delivery, investigators will utilize a bi-cistronic vector that incorporates both the engineered TMPK suicide gene and a truncated version of the cell surface marker CD19, which will allow for antibody-based purification of obligate vector-transduced Th2 cells prior to infusion.

In FY 2005, ORD co-funded with NIH Institutes 10 Bench-to-Bedside grants for 2-year periods at the NIH Clinical Center. The Bench-to-Bedside Awards Program encourages new collaborations between basic and clinical investigators across the NIH Institutes. Intramural researchers translate scientific findings into clinical applications.

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.