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DUKE BRAIN TUMOR SPORE Duke University Medical Center, Durham, North Carolina Principal Investigator – Darell D. Bigner, M.D., Ph.D. This Duke Brain Tumor SPORE grant consists of four projects, five cores, a Developmental Research Program, and a Career Development Program. The goal of the proposed research is to advance knowledge of brain tumor biology and etiology and to translate these into clinical protocols that represent significant and novel advances in the management of these therapeutically intractable tumors. The grant, as a whole, and each project and core, are led by basic and clinical Principal Investigators. In Project 1, novel therapeutics that target the GSTP1 protein, overexpressed and poor prognostic indicator in malignant gliomas, will be rationally developed and evaluated for clinical efficacy. Project 2 will investigate novel molecular mechanisms of resistance of CNS tumos to Temodar, a clinically active brain tumor agent and evaluate the clinical efficacy of a PARP inhibitor in reversing Temodar resistance. Project 3 will investigate dendritic cell-based vaccination against CMV as a therapeutic modality in malignant glioma and the underlying mechanisms of the anti-tumor response. In Project 4, molecular and epidemiologic studies will examine the etiology of brain tumors in a case-control study. Exposure to putative neurocarcinogens will be examined and polymorphisms in metabolism and DNA repair genes will be examined as a determinant of brain tumor risk and treatment-associated toxicity. The cores include a Tissue Procurement and Genetics Core, an Investigational New Drug Permit Core, a Phase I/II Clinical Trials Core, a Biostatistics and Information Systems Core, and an Administrative Core will provide critical infrastructural and service functions. The Developmental Research Program will consist of pilot projects identified from the entire Cancer center membership at Duke as well as from selected, outside institutions. Emphasis is on investigators who have not previously worked in neuro-oncology. We are submitting nine examples of pilot projects from senior investigators at Duke who have not previously worked on brain tumors. There are three examples of potential Career Development awardees. They include Dr. Hai Yan, Dr. Simon Gregory, and Dr. Duane Mitchell, an African-American minority.
Project 1 In the United States, the incidence of brain tumors continues to increase and, presently, brain tumors are the most common solid tumors of childhood and adolescence. Unfortunately, however, these tumors remain highly intractable to therapy and prognosis remains abysmal, with, generally, no long-term survivors among patients with anaplastic astrocytomas or glioblastoma multiforme. Understanding the biology underlying therapeutic failure in brain tumors and the development of more effective therapies for the disease are thus a matter of urgency, as was recently emphasized in the report of the Brain Tumor Review Group appointed by the Directors of the National Cancer Institute and the NINDS. This proposal builds on previous findings from our laboratory, and other laboratories, that the allelo-polymorphic glutathione S-transferase P1 (GSTP1) gene is frequently over-expressed in human malignant gliomas and is a major determinant of failure of chemotherapy and is associated with poor patient survival. Decreased GSTP1 expression or inhibiting its activity has also been shown to enhance the efficacy of chemotherapy. The GSTP1 protein is a major enzyme of Phase II metabolism and thus is involved in metabolic inactivation of anticancer agents leading to drug resistance. It is also a potent inhibitor of jun N-terminal kinase, and consequently plays a significant role in multiple cell signaling cascades and apoptosis. Our hypothesis is that rationally designed small molecules that bind to the GSTP1 active site and inhibit the GSTP1 protein with high affinity and specificity will have significant antitumor activity against gliomas and will overcome GSTP1-based drug resistance and, thereby, enhance the efficacy of chemotherapy in gliomas. In preliminary studies, we identified a novel class of GSTP1 inhibitors, using a rational strategy of structure-based ligand design, computer modeling, high throughput screening, and in vitro efficacy studies to target the 3-dimensional active site of the GSTP1 protein with high affinity and specificity. In this project, we will further optimize this lead and develop candidates with high in vitro and in vivo antiglioma activity for human clinical trials. The studies are likely to lead to a novel class of therapeutic agents for the treatment of malignant gliomas and other human malignancies that are characterized by high GSTP1 expression. The mechanistic studies will provide important insights into the role of GSTP1 in metabolism and signaling in glioma cells. The absence of GSTP1 expression in the normal tissues from which these tumors arise suggests a potential high therapeutic index of these agents. Our final goal in this project is to conduct a clinical trial of the lead GSTP1 inhibitor from this study, as well as, one that targets the GSTP1 G-site and is already in Phase I/II clinical trial.
Project 2 The prognosis of patients with malignant glioma remains dismal, with conventional treatment with surgery, radiotherapy and alkylnitrosourea-based chemotherapy failing to cure all patients with glioblastoma multiforme and the majority of patients with anaplastic astrocytoma. Review of clinical trials for treatment of malignant glioma indicate that a major impediment to further progress is the emergence of drug-resistant tumor cells. Methylating agents are one of the two “gold” standards (the other being nitrosoureas) for the treatment of malignant glioma. Temodar (temozolomide) is an imidazole tetrazinone whose mechanism of action is similar to that of dacarbazine, specifically via metabolic conversion to a common active intermediate, the methylating agent MTIC (Horspool et al 1990). Clinical trials suggest that Temodar has activity in the treatment of patients with newly diagnosed and recurrent high-grade glioma. Nevertheless, it is clear that a cohort of patients with this tumor will fail Temodar. A series of studies conducted predominantly, but not exclusively, for non-CNS tumors has demonstrated that at least two mechanisms of resistance appear to be operational in mediating resistance to Temodar, O 6-alkylguanine-DNA alkyltransferase (AGT) and DNA mismatch repair deficiency. The hypothesis of this proposal is that: mechanisms (discrete from AGT or DNA mismatch repair deficiency) involving DNA base excision repair and alterations in cell signaling mediate Temodar resistance in malignant glioma and medulloblastoma. The specific aims of this proposal are: 1) to define the relative importance of novel mechanisms (per Specific Aims 2 & 3) of resistance to Temodar in human glioma and medulloblastoma cell lines, xenografts and clinical tumor samples by quantitating the role of AGT, AGT mutations, and DNA mismatch repair deficiency; 2) to define the role of adduct repair in mediating resistance to Temodar in human glioma and medulloblastoma cell lines, xenografts and clinical tumor samples; 3) to define the role of alterations in cell signaling following Temodar induced DNA methylation in mediating resistance to Temodar in human glioma and medulloblastoma cell lines, xenografts and clinical tumor samples; 4) to conduct Phase 1 and 2 trials of Temodar in combination with inhibitors of DNA repair in patients with malignant glioma and medulloblastoma.
Project 3 Malignant gliomas (MGs) are universally fatal, and effective therapy is limited by collateral damage to normal tissue. Immunotherapy directed against tumor-specific antigens may allow neoplastic cells to be targeted more precisely, and o ur dendritic cell (DC)-based vaccinations targeting of a mutated tumor-specific epidermal growth factor receptor have produced immunologic and radiographic responses in patients with MGs. The discovery that MGs, but not surrounding normal brain, serve as a refuge for Cytomegalovirus (CMV) reactivation provides an unparalleled opportunity to subvert, as a tumor-specific antigen, the highly immunogenic CMV protein, pp65. Despite the numerous advantages of targeting CMV antigens in MGs with DC-based vaccines, a number of factors clearly limit antitumor immune responses in these patients. Innovative complementary strategies that eliminate CD25+ regulatory T cells or block cytotoxic T lymphocyte antigen-4-induced T cell tolerance may enhance such immune responses, but the indiscriminate application of these potent adjuvants carries the risk of inducing autoimmune encephalomyelitis. In order to understand the limitations and risks of targeting CMV antigens in MGs, we have developed a novel murine astrocytoma cell line that supports infection with murine CMV and is tumorigenic in syngeneic mice. Our preliminary murine studies demonstrate that these tumors in the brain can be targeted with RNA-loaded DCs. We have also shown that DCs from patients with MGs that are loaded with pp65mRNA, induce interferon- g production from CD4+ and CD8+ T-cells in an antigen-specific manner and incite T-cells to kill malignant astrocytes infected with human CMV. Interestingly, we have also found that CMV-specific T-cells preferentially accumulate at the tumor site in patients with MGs. We believe that our murine model system and the complementary human studies proposed will allow selection and translation of the most effective strategies for targeting CMV-associated antigens in patients with MGs, without the induction of autoimmunity. In this project, we will use the murine model, in combination with in vitro human studies to evaluate the safety of, and to gain a better understanding of the mechanisms involved in the therapeutic targeting of CMV-associated proteins in malignant gliomas. The results will then be used to rationally design and conduct a clinical CMV-targeted clinical trial.
Project 4 The purpose of this case-control study is to assess the contributions of neurocarcinogen exposure and of glutathione S-transferase, cytochrome P-450, and DNA repair genes to the development and genetic susceptibility of brain tumors and to study the effect of these polymorphisms on treatment outcome and toxicity. Studies to date suggest a moderate to large effect for GSTT1 in selected tumor subtypes. We are targeting obtaining data on ~ 1000 cases and controls, with an adequate number of glioblastomas (n=430), astrocytomas (n=300), oligodendrogliomas (n=100) and meningiomas (n=150) to evaluate this hypothesis with reasonable power for relative risks previously reported for GSTT1. All study subjects will be asked to complete a web-based self-administered or telephone-based questionnaire and to provide blood and/or buccal samples for DNA analysis. Research nurses at Duke University Cancer Center (DUCC)/ Evanston Hospital will recruit ~ 770/220 recently diagnosed cases (within 3 months) over a 4-year period. An equal number of friend controls will be identified by asking cases to distribute packets of information to 5 friends of the same age (± 5 yrs), gender and race. Controls will return interest cards or phone the research nurse to express interest, then complete the protocol at a clinic appointment or using a distance based protocol. Medical records for cases will be reviewed to obtain clinical and treatment information for the survival and toxicity analyses. Linkage with the National Death Index in Year 4 will obtain vital statistics for up to 3 years of follow-up time to assess outcomes. Study coordination and survey data management will be completed at UIC; molecular analysis will be conducted at DUCC. DUCC will facilitate the neuropathology review, provide management support and some local data collection, and store the remaining specimens in their central repository. Data analysis files will be compiled at Duke University and distributed to the PI’s at Duke and UIC. Statistical analyses will use conditional logistic regression models appropriate to each specific aim.
Core 1 We hypothesize that a tissue bank organized around an integrated histologic and molecular diagnostic laboratory with all research and clinical information available from a constantly updated database is an essential element to the success of this SPORE. To test this hypothesis we propose the following 4 Specific Aims:
Core 2 The role of Core 2is threefold: 1) the preclinical evaluation and analysis of drugs, peptides, MAbs and derived constructs, and activated immune cells for clinical application in terms of toxicity, stability, in vivo localization and therapeutic effect, generated in Projects 1, 2, and 3 of this SPORE and implemented clinically in Core 3; 2) the production of clinical grade MAb reagents; and 3) the preparation and submission of Investigational New Drug (IND) permit applications for all reagents of potential clinical utility. This Core provides a continuum from bench to clinical administration and assures the orderly progression and quality control of reagents in development that have characterized the development and implementation of our currently successful MAb reagents in Phase I and II clinical trials. The Functions/Specific Aims of Core 2 are as follows: Specific Aim 1. Pre-clinical evaluation of intact, engineered, conjugated, and modified constructs of MAbs and immunoreagent batchescurrently under investigation in Core 3 of this SPORE in terms of in vivo stability, localization, and therapeutic effect in immunocompetent and athymic rodent tumor xenograft models, including subcutaneous and intracranial models. Specific Aim 2. Pre-clinical analysis of derived peptides, drugs, and other moieties in terms of in vivo stability, localization, and therapeutic effect in animal models as described in Aim 1 for any reagents of promise identified in Projects 1, 2, and 3. Specific Aim 3. Pre-clinical evaluation and quality certification of all reagents (drug, peptide, MAb, and derived constructs) generated in Projects 1, 2, and 3 and under investigation in Core 3 of this SPORE. a. Quality control by performance of, or contract for, all assays for bacterial, fungal, viral, pyrogen, and DNA contamination, and general safety tests in vivo (Core 3 immunoreagents). b. Toxicity testing and analysis of drug, peptide, MAb-toxin conjugates, nuclide-MAb conjugates and their derivatives, and effector cells in animal models (normal and athymic rodents; Projects 1, 2 and 3; Core 3). c Determination of the maximum tolerated doses (MTD) for all substances showing no toxicity and maximal stability and therapeutic effect in athymic and normal rat and mouse model systems (subcutaneous, intracranial, CNS tumor xenografts: Projects 1 and 2). Specific Aim 4. Production of clinical grade reagents: Production of clinical grade MAb, antibody fragments, constructs, and conjugates in sufficient quantity for clinical trials conducted in Core 3. Small molecule inhibitors from Project 1 will be contracted out to GMP producers or the NCI RAID Program. Specific Aim 5. Preparation and submission of IND permit applications for all proposed therapies developed in Projects 1, 2,and 3. As the pre-clinical development Core, Core 2 has fundamental support functions for Projects 1, 2 and 3, and Cores 1 and 3.
Core 3 The outcome for patients with primary malignant brain tumors remains dismal with over 90% of patients dying within 2 years of diagnosis. In this application we will focus on two major roadblocks to successful treatment for patients with these tumors. Response to chemotherapy is either non-existent or short-lived indicating that de novo or acquired resistance to chemotherapy is a critical contributor to poor outcome. In addition, the vast majority of patients recur locally indicating that local control remains elusive and represents a critical step to achieve a cure. To build upon our successful efforts to overcome chemoresistance mediated by AGT using the competitive inhibitor O 6-BG, we will focus on two additional important mediators of chemoresistance including the DNA protectant glutathione-S-transferase (GSTP1) and the nuclear DNA repair enzyme poly(ADP-ribose) polymerase (PARP). In addition, our encouraging results with radiolabeled MAbs such as the anti-tenascin MAb 81C6 or the EGFR-targeting toxin-conjugate TP-38, confirm that innovative tumor-targeting therapeutics can improve local control and improve overall outcome. We therefore will also evaluate additional novel therapeutics targeting recently identified tumor-associated markers such as EGFRvIII, glycoprotein NMB, multidrug resistant protein 3, the glioma-associated cell-surface gangliosides 3'-isoLM1 and 3',6'-isoLD1, human CMV and the poliovirus receptor CD155. Our HYPOTHESIS is that rationally designed, novel therapeutic strategies that either block key mediators of chemoresistance or that augment local control, will improve the survival of patients with malignant brain tumors while preserving an optimal quality of life. The Specific Aims of this proposal are: Specific Aim 1. To conduct Phase I and II clinical trials to assess the anti-tumor activity and safety of inhibitors of chemoresistance mediated by GSTP1 and PARP. Specific Aim 2. To conduct Phase I and II clinical trials to assess the anti-tumor activity and safety of innovative therapeutics designed to improve local control including radiolabeled MAbs, MAb fragments, toxin conjugates, DCs against tumor-associated CMV antigens and an oncolytic polio/rhinovirus recombinant. Specific Aim 3. To determine the impact of these therapeutic agents on overall quality of life for patients with malignant brain tumors.
Core 4 The Biostatistics and Informatics Core for the Duke SPORE in Brain Cancer supports the statistical, bioinformatics and information systems needs of all projects and cores in the SPORE. The Core was developed as a collaboration between Cancer Center Biostatistics and Cancer Center Information Systems (CCIS). Dr. Stephen L. George, Ph.D., Director of Cancer Center Biostatistics and Group Statistician for the CALGB and Kimberly Johnson, Director of Cancer Center Information Systems are Co-Directors of the Core. Dr. George will direct biostatistics and bioinformatics initiatives in this area, while Ms. Johnson will direct information systems initiatives and assist Dr. George in administering the Core. CCIS falls under the administrative oversight of Dr. George within the Cancer Center. Additional staff for this Core includes statisticians and programmers dedicated to each project and core based on their area of expertise and past associations with individual projects. In addition to the core directors, partial support is requested for three statistical personnel (.70 FTE total), one bioinformatician (.10 FTE), two information systems personnel (.55 FTE total) and one full-time data manager. This Core will provide assistance in the statistical analysis of clinical, pre-clinical and laboratory studies, as well as genetic investigations. In addition, the Core will support the development of research database structures and data management activities. The Core will use existing computing infrastructure resources as an efficient means to support SPORE research and will build on past successes in infrastructure development. Resources available to this Core include 25 servers supported by CCIS, offering over 20GB of memory and 2 TB of disk space with multi-processing capabilities and a variety of software applications and compilers.
Core 5 In organizing complex multidisciplinary research, the Administrative Core will provide the structures to ensure that all the activities and processes vital in such an interdisciplinary program will occur. The Administrative Core is designed to ensure the translational nature of all projects. The significance of the Administrative Core is to promote scientific research advances, interdisciplinary communication, to provide fiscal accountability, and to communicate and coordinate information arriving from the activities of the SPORE. New initiative development, collaboration with outside investigators, proper administration of Pilot Projects and the Developmental Research Program, and providing guidance and quality assurance for the Career Development Program are all scientific goals of the Administrative Core. Day-to-day administration of the Duke Brain Tumor SPORE will be done by the Principal Investigator and the Administrative Assistant. The Core will schedule all meetings of all committees of the SPORE; it will arrange the annual SPORE retreat, research dinner meeting, schedule seminars with invited speakers, monitor research progress, foster continuous improvement and set priorities. Handling of requests for the use of human specimens developed in the Tissue Procurement and Molecular Genetics Core will be coordinated by the Administrative Core and lastly, it will foster participation by patient and family advocates in planning and educational activities of the SPORE. The Administrative Core will also enhance participation by minorities and women in all aspects of the SPORE. This will be expedited by the leadership of Dr. Francis Ali-Osman, SPORE Co-P.I. and Co-Leader of the Administrative Core who is an African-American. The leadership of the Administrative Core will be the leadership of the SPORE itself; that is, with Dr. Darell Bigner as Administrative Core Leader and Dr. Francis Ali-Osman as Administrative Core Co-Leader. Travel for 10 investigators to attend the annual SPORE Meeting in Washington will be coordinated as well as the travel for the Principal Investigator to attend the SPORE Principal Investigator’s Meeting. |
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