Wolfram E. Samlowski - US grants

Leukemia, aplastic anemia, congenital immunodeficiency, as well as a variety of other hematologic and neoplastic conditions are currently being successfully treated by bone marrow transplantation (BMT). The BMT-procedure is, however, associated with significant morbidity and mortality due to severe alterations of host cellular and humoral immune functions. Some of these BMT-related immunologic deficits, such as in secretory immunity and contact hypersensitivity responsiveness, may last for years following successfully hematopoietic reconstitution. The current research effort to understand depressions in post- BMT immunity has focused primarily on the consequences of graft-versus-host disease. Due to this concentrated effort, significant progress has been made in the prevention and suppression of this once-feared consequence of transplantation. Even in the absence of graft-versus-host disease, it has been noted that the timecourse of immunologic reconstitution within human twin and autologous BMT-recipients is remarkably similar to that observed in allogeneic BMT. We have therefore proposed the hypothesis that endothelial cell toxicity of the preparative regimen contributes to the observed long-term immunologic deficits. Microvascular endothelial cells are known to represent a critical interface between circulating cells of the immune system and the tissues which they must protect. A number of specific lymphocyte surface molecules exist that allow organ-specific adhesion to the microvascular endothelium and subsequent extravasation of these cells. Our studies have identified gamma- irradiation-induced microvascular lesions within the peripheral lymph nodes of mice, which result in a decreased capacity for lymphocyte localization into these organs. Since lymph nodes are a major site of antigen-specific lymphocyte stimulation, as well as amplification and regulation of immunologic responses, alterations in lymphocyte localization to these organs may have significant functional consequences. Our proposed investigations compare three clinically used BMT- preparative agents (gamma-irradiation, busulfan, and etoposide) to establish their relative toxicity to the lymphocyte recirculation process and the eventual immunologic reconstitution of transplanted mice. One of the aims is to analyze changes in the anatomy and function of the lymphocyte receptive regions of the microvascular endothelium following exposure of mice to a given preparative protocol. The overall goal of these studies will be to determine: a) the bone marrow ablative agents least toxic to the lymphocyte localization process, and b) the relationship between lymphocyte recirculation and immunologic recovery of the host following BMT. These studies should have clinical relevance in guiding clinicians as to which BMT preparative regimen might allow the most rapid or complete immunologic recovery following human BMT.

The current proposal represents closely integrated basic science, preclinical and clinical investigations of the role of cytokine-inducible nitric oxide synthesis as an effector mechanism during cancer immunotherapy with interleukin-2 (IL-2). Since glutathione appears to be a cofactor for the nitric oxide synthase, the goal of the proposed studies is to develop a better understanding of the interrelationship of glutathione biosynthesis with IL-2 induced macrophage and lymphocyte antitumor mechanisms. In Program 1 we will develop cellular and basic science models to evaluate the role of sulfhydryl compounds, such as glutathione and cysteine in regulating the function of the L-arginine dependent pathway of nitric oxide synthesis. These studies will also focus on developing a better understanding of the effector mechanisms of this pathway on a molecular level. Using the cysteine analogue N-acetyl cysteine (NAC) we hope to increase glutathione synthesis in cells and in vivo, and to increase the activity of the cytokine inducible nitric oxide synthase. Since NAC unexpectedly augmented lymphocyte activation by IL-2, as well as improving therapeutic responses to IL-2 in a subcutaneous murine tumor model, we will also evaluate the effects of this agent on cytotoxic lymphocyte function. In Program 2 we will perform the initial toxicity testing of NAC in high-dose IL-2 treated patients. This agent has already been safely used in patients to counteract drug and heavy metal intoxications. The proposed studies will evaluate the maximum tolerated dose of NAC, as well as the dose limiting toxicity. During this Phase IA trial, we will also evaluate the pharmacokinetics of NAC during IL-2 administration. The effect of NAC on the cytokines that induce NO. synthase, the metabolic intermediates of the L-arginine dependent pathway, as well as on the effector mechanisms of this pathway will be evaluated during the course of the clinical study. This combined program should contribute to a better understanding of the role of nitric oxide as an effector mechanism in IL-2 therapy and its role in IL-2 related toxicity. The long term goal of these studies is increase the effectiveness of IL-2 therapy by modulating the activity L-arginine dependent pathway via cysteine analogues.

Objectives of our program are: To conduct multidisciplinary collaborative clinical studies in the management of malignant disease in humans. This program involves participants in medical oncology, surgical oncology, radiation oncology and pathology. The Southwest Oncology Group Program at the University of Utah has effectively continued over the past four years with minimal support from NCI by extensively using institutional funds to maintain a full level of participation. Several faculty changes have led to a reorganization of our SWOG leadership. The investigators participating in this program represent the strongest areas of the clinical investigations at the University of Utah. As these areas develop, we plan to introduce the resulting new programs into the Southwest Oncology Group. Specifically, these include: the development of an active bone marrow transplant program; in surgical oncology, new leadership in cancer immunotherapy which supplements prior programs in IL-2 with vaccine therapy; continuing developments in neuro- oncology focusing on the search for gene alterations correlating with therapy outcome; in pathology, a significant group effort in the biology of leukemias emphasizing correlations to disease response which will be supplemented by laboratory studies at Utah measuring cytidine triphosphate synthetase activity; and in radiation oncology, a substantial expertise in hyperthermia combined with radiation and chemotherapy. The institutional strengths in genetics and epidemiology involving colon cancer and familial polyps as well as proliferative breast disease and familial prostate cancer will be integrated into proposed chemoprevention studies in the group.

The University of Utah Cancer Center Flow Cytometry Facility is severely limited by an antiquated optical bench, which is urgently in need of replacement. Current problems include an inability to expand to 4-5 color fluorescence detection, inadequately powered laser for current needs, inefficient sorting, and non-availability of parts and service contract on our current outdated instrument We are applying through the Shared equipment grant mechanism to replace the main flow cytometer in the University of Utah Cancer Center sponsored Flow Cytometry Facility with a state of the art instrument The University of Utah will provide technician support and pay for maintenance of service contracts on the optical bench, computer and lasers. The University is also recruiting a nationally recognized Flow Cytometry facility director to oversee the function of this core facility. A large number of currently funded investigators working in several different departments at the University have applications for this instrument in their work. A diverse spectrum of research topics are represented among users, including cell biology (apoptosis, cell adhesion, proteolysis, extracellular matrix, signal transduction, peptide regulatory factors, cell polarization), immunology (lymphocyte and macrophage cytotoxicity, cytokine biology, demyelinating diseases, arthritis, thermal injury, antigen presentation, bone marrow transplantation ), microbiology, and molecular genetics (regulation of DNA replication, antibody repertoire and diversity, embryogenesis, somatic mutation, genetic therapy). Improvement of the Cancer Center Flow Cytometry facility would greatly enhance the ability of these investigators in performing their peer-reviewed and well funded research.

Evalution of the toxicity of continuous infusion N-acetyl acysteine (NAC) in conjunction with high dose i.v. bolus interleukin-2 (IL-2) in the treatment of metastatic or unresectable renal cell carcinoma and malignant melanoma. This phase 1A trial will attempt to define the dose-limiting toxicity and maximal tolerated dose of NAC in conjunction with IL-2.

Cytokine activated macrophages have potent effects against tumor cells in vitro, including inhibition of mitochondrial respiration and DNA synthesis, via high-output nitric oxide (NO.) synthesis. It is not yet known whether NO can act as a host defense against cancers in vivo. Our studies have demonstrated that NO. synthesis is activated within many murine tumors in vivo, and may serve to decrease tumor growth. Furthermore, treatment of mice or humans with IL-2 strongly induces NO. synthesis. Recent murine studies have suggested that the induction of NO. synthesis during lL-2 treatment can mediate tumor regression and improved survival. Exposure of tumor cells to NO. may also induce tumor cell apoptosis (programmed cell death ), a novel cytolytic mechanism. The goal of the current application is to further evaluate the mechanisms by which cytokine induced NO. synthesis acts as an anticancer effector mechanism in vivo, using lL-2 treatment of tumor hearing mice as a paradigm. A better understanding of the role of NO. synthesis during immune response to tumors is necessary to determine whether future attempts should he made to augment or block the function of iNOS enzyme with pharmacologic agents during cytokine treatment of human cancers. In Specific Aim 1 we propose to characterize the time and dose relationships of IL-2 induced NO. production by serial measurements of serum and urine nitrite and nitrate excretion. The induction of NO. synthesis within tumors in IL-2 treated mice will be-evaluated by analyzing target cell nitrosylation by electron paramagnetic resonance spectroscopy. Further experiments will evaluate iNOS mRNA and protein induction in tumor specimens using RT-PCR and immunoblotting techniques. We have obtained a strain of C57/B16 mice that have had the iNOS gene inactivated by homologous recombination. These mice will be used to further evaluate whether NO. synthesis during IL-2 treatment is derived from the inducible or constitutive enzyme isoforms. In Specific Aim 2 we plan to identify cellular sources of cytokine induced NO. synthesis within tumors and normal tissues of normal mice treated with IL-2. The proposed experiments will employ immunohistology, flow cytometry, and immunomagnetic separation techniques to identify activated cell populations within tumors and in normal tissues of wild type and iNOS knockout mice. A specific focus will be to determine if iNOS is expressed by tumor infiltrating macrophages or neoplastic cells (or both). Experiments using B-16 melanoma (a syngeneic tumor containing an intact iNOS gene) implanted into C57/B16 iNOS knockout mice will be used to evaluate the role of cytokine-induced iNOS production by tumor cells. In Specific Aim 3 we will evaluate potential cellular mechanisms by which activated macrophages may mediate cytotoxicity against cancers. Proposed studies will evaluate-target cell damage by reactive oxygen and nitrogen intermediates using assays for hydrogen peroxide, superoxide, peroxynitrites, as well as nitric oxide. The role of NO. induced apoptosis during IL-2 treatment will be evaluated using TdT mediated nick-end labeling assays, as well as DNA electrophoresis on agarose gels.

Evalution of the toxicity of continuous infusion N-acetyl acysteine (NAC) in conjunction with high dose i.v. bolus interleukin-2 (IL-2) in the treatment of metastatic or unresectable renal cell carcinoma and malignant melanoma. This phase 1A trial will attempt to define the dose-limiting toxicity and maximal tolerated dose of NAC in conjunction with IL-2.

DESCRIPTION (provided by applicant): High-dose IL-2 produces complete remissions in a small percentage (5-10%) of patients with metastatic renal carcinoma or melanoma. It is intriguing that many of the IL-2 induced complete remissions have proven durable, with up to 18-year follow-up. The clinical use of IL-2 is limited by severe side effects, particularly hypotension and "vascular leak syndrome"(VLS). Patients treated with high-dose IL-2 require hospitalization for close monitoring and administration of fluids, colloids and pressor medications to treat low blood pressure that occurs in a dose dependent manner. A substantial fraction (1/3 to 1/2) of planned IL-2 doses is usually omitted due to cardiovascular toxicity, which may compromise effectiveness. Our laboratory and clinical studies have shown that synthesis of nitric oxide (NO) is strongly induced following IL-2 treatment. NO is an important mediator of hypotension and vascular leak. We have recently identified a promising new agent, PHP, a pyridoxalated hemoglobin polyoxyethylene conjugate, that appears to be safe in humans and has the potential to prevent both IL-2 induced hypotension and VLS due to its ability to catabolize NO, as well as other potentially injurious oxidative radicals. This grant application is designed to support a Phase I clinical investigation of PHP in conjunction with high-dose IL-2 treatment to evaluate safety of this compound and to establish an effective dose. In Specific Aim 1 we will assess the safety of PHP in patients with metastatic renal carcinoma or malignant melanoma. This study will establish dose-limiting toxicity (if any) of this agent in combination with high dose IL-2, and define a biologically effective dose for reversal of IL-2 induced hypotension. In Specific Aim 2 we will determine the effect of PHP on hemodynamic function in IL-2 treated patients, including assessment of blood pressure, systemic vascular resistance, cardiac index, pulmonary artery pressure and capillary wedge pressure and the total dose of pressors required during each course of high-dose IL-2. In Specific Aim 3, we will assess the potential usefulness of intermediate biomarkers for activation of the nitric oxide synthesis pathway in patients treated with IL-2 with or without PHP, to develop assays for phase II testing. It should be stressed that knowledge gained from evaluation of mechanisms of IL-2 toxicity is likely to be broadly relevant to understanding the pathophysiology of hypotension and vascular leak induced by other cytokines, such as IL-1, TNF, VEGF and IL-12.

[unreadable] DESCRIPTION (provided by applicant): Aberrant hypermethylation of CpG islands within promoter regions of DNA modifies expression of genes involved in diverse cellular processes that impact tumor cell growth and behavior. Recent studies have shown that promoter methylation is important in silencing DNA repair enzymes, and inducing expression of drug sensitivity or resistance phenotypes. For example, cancer cell sensitivity to interferon-a is believed to be regulated by promoter methylation of key genes. The nucleoside analogue 5-aza-2'- deoxycytidine (decitabine) is a potent pharmacological inhibitor of DNA methylation in vitro. Decitabine-induced DNA hypomethylation, gene reactivation, and effects on cell function require incorporation into DNA followed by several cellular division cycles in vitro. We previously performed a phase I trial of decitabine, which established that 2mg/m2/day, given as a 168h continuous i.v. infusion resulted in significantly decreased MAGE-1promoter-specific and total genomic DNA methylation with minimal toxicity. Further preclinical studies suggested that the interferon-signaling pathway is reactivated following decitabine exposure of cancer cells. We propose to build on our previous preclinical experiments to perform a phase I clinical trial to test whether a decitabine infusion can sensitize cancer cells to interferon-a in vivo. In Specific Aim1 we plan to perform a phase I clinical trial to assess the toxicities of a continuous intravenous infusion of decitabine with escalating doses of subcutaneous PEG-IFN in patients with refractory metastatic cancer. The clinical protocol is designed to identify the dose limiting toxicity and the maximum tolerated dose of PEG-IFN in this combination. In Specific Aim 2 we will perform molecular correlation studies to evaluate pretreatment and post treatment samples of blood, skin and tumor from patients to identify changes in global (genomic) DNA methylation, as well as evaluating promoter-specific methylation . Additional experiments will evaluate interferon signaling and DNA damage response pathways in normal and neoplastic cells. [unreadable] [unreadable] [unreadable]