Henry S. Friedman, MD, Upstate Medical University, Syracuse, NY 1977 - US grants

The resistance of medulloblastoma to current therapy is a multifactorial process, due in part to the heterogeneity of this tumor, the lack of an appropriate system to identify active compounds, and the potentially limited access of active compounds to the tumor site. These studies are designed to utilize our in vitro/in vivo models for human medulloblastoma to enhance an understanding of this tumor and its response to single and combination therapy. The specific aims are: 1) to use our present models of human medulloblastoma (TE-671 and D283 Med) and additional ones we will establish to study the in vitro and in vivo response of this tumor to chemotherapeutic agents (particularly classical alkylators and agents altering glutamine and glutamate metabolism) and radiation, determining the classes, properties and schedules of the most effective compounds; 2) to use these results to evaluate combination and combined modality therapy of human medullolastoma; 3) to apply these results to clinical trials. In vitro studies will be performed by using a double layer soft agar clonogenic assay to study sensitivity of the human medulloblastoma cell lines to chemotherapeutic agents and radiation. In vivo studies will be performed by studying the sensitivity of the cell lines growing subcutaneously or intracranially in nude athymic mice or rats. Subcutaneously growing tumors will be treated when the median tumor volume exceeds 200 mm3. Chemotherapeutic agents will be given i.p. at the LD10. Radiation therapy will be performed on a 60Co unit. Response will be assessed by comparing growth delay, per cent regressions, and tumor volume ratios between treated and untreated animals. Intracranial tumors will be treated on day 11 after tumor implantation (see enclosure) and response assessed by the comparison of median survival time and long-term survivors (greater than 60 days) between treated and control groups. These results will be used to define the therapeutic sensitivity of medulloblastoma allowing the rational design of chemotherapeutic and combined modality regimens.

The hypotheses of this proposal are: 1) our models for human medulloblastoma consisting of the continuous human medulloblastoma cell lines and transplantable xenografts TE-671, D283MED and additional lines presently under development, will allow the unique opportunity to analyze specific mechanisms of drug sensitivity, delivery, and resistance in this tumor, and 2) L-phenylalanine mustard (melphalan) and other phenylalanine mustard isomers represent bifunctional alkylating agents highly cytoxic to human medulloblastoma which may be rationally modulated to increase drug delivery and overcome tumor resistance. Accordingly, the specific aims of this proposal are: 1) define the mechanisms that influence melphalan cytotoxicity in human medulloblastoma cell lines in a clonogenic assay, subcutaneous and intracranial human medulloblastoma xenografts in athymic mice and patient medulloblastoma specimens. Specific mechanisms to be examined include: melphalan transport kinetics, total glutathione and glutathione-S-transferase levels, and melphalan-induced DNA damage and repair; 2) to define the effects of agents that alter glutathione metabolism on melphalan by examining (a) cytotoxicity in human medulloblastoma cell lines in a clonogenic assay, (b) cytotoxicity in subcutaneous and intracranial human medulloblastoma xenografts and (c) toxicity in human bone marrow cells(CFU-C) and athymic mice; 3) to determine the potential therapeutic advantage of intra-arterial vs intravenous delivery of melphalan and the cyclophosphamide derivatives. 4-hydroperoxycyclophosphamide and phenylketocyclophosphamide in intracranial human medulloblastoma xenografts in athymic rats. These studies will have direct application to the design of future clinical trials for human medulloblastoma.

The resistance of medulloblastoma to current therapy is a multifactorial process, due in part to the heterogeneity of this tumor, the lack of an appropriate system to identify active compounds, and the potentially limited access of active compounds to the tumor site. These studies are designed to utilize our in vitro/in vivo models for human medulloblastoma to enhance an understanding of this tumor and its response to single and combination therapy. The specific aims are: 1) to use our present models of human medulloblastoma (TE-671 and D283 Med) and additional ones we will establish to study the in vitro and in vivo response of this tumor to chemotherapeutic agents (particularly classical alkylators and agents altering glutamine and glutamate metabolism) and radiation, determining the classes, properties and schedules of the most effective compounds; 2) to use these results to evaluate combination and combined modality therapy of human medullolastoma; 3) to apply these results to clinical trials. In vitro studies will be performed by using a double layer soft agar clonogenic assay to study sensitivity of the human medulloblastoma cell lines to chemotherapeutic agents and radiation. In vivo studies will be performed by studying the sensitivity of the cell lines growing subcutaneously or intracranially in nude athymic mice or rats. Subcutaneously growing tumors will be treated when the median tumor volume exceeds 200 mm3. Chemotherapeutic agents will be given i.p. at the LD10. Radiation therapy will be performed on a 60Co unit. Response will be assessed by comparing growth delay, per cent regressions, and tumor volume ratios between treated and untreated animals. Intracranial tumors will be treated on day 11 after tumor implantation (see enclosure) and response assessed by the comparison of median survival time and long-term survivors (greater than 60 days) between treated and control groups. These results will be used to define the therapeutic sensitivity of medulloblastoma allowing the rational design of chemotherapeutic and combined modality regimens.

Medulloblastoma, the most common malignant brain tumor in childhood, continues to represent a therapeutic challenge. Despite treatment with increasingly sophisticated surgical and radiotherapeutic intervention, the majority of children with this tumor will ultimately die of progressive disease. Although the role of chemotherapy in the treatment of medulloblastoma remains poorly defined, recently clinical and laboratory studies support the activity of the classical bifunctional alkylating agents against these tumors. Studies in our laboratory have documented the activity of a series of bifunctional alkylating agents including cyclophosphamide and melphalan in the treatment of a panel of medulloblastoma cell lines and transplantable xenografts. Clinical trials have confirmed the activity of cyclophosphamide and melphalan in the treatment of patients with recurrent medulloblastoma. Unfortunately, meaningful increases in the survival of patients with medulloblastoma have not yet resulted from the use of alkylating agents, a limitation due in large part to intrinsic or acquired alkylator resistance in a subset of cells in these tumors. The hypothesis of this proposal is that modulation of glutathione metabolism can enhance bifunctional alkylating agent therapy of sensitive and resistant tumor cells. The specific aims of this proposal are: 1) To define the role and mechanism(s) of glutathione in modulating the activity of melphalan and cyclophosphamide (4- hydroperoxycyclophosphamide) in alkylator-sensitive and -resistant human medulloblastoma cell lines and xenografts in athymic nude mice, 2) to define the effects of gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase inhibitors on the activity and toxicity of nitrogen mustard- based alkylating agents in the treatment of alkylator-sensitive and - resistant human medulloblastoma cell lines and xenografts in athymic nude mice, 3) to define the effects of BSO-mediated glutathione depletion on the activity and toxicity of intrathecal malphalan in the treatment of leptomeningeal human medulloblastoma in athymic nude rats.

Primary brain tumors, including malignant gliomas and ependymomas, are second only to leukemia as a cause of childhood cancer. Advances in chemotherapy and combined modality regimens, though dramatically successful in the treatment of other pediatric malignancies, have not been translated into effective therapy for most pediatric brain tumors. Although the role of chemotherapy in the treatment of malignant gliomas and ependymomas remains poorly defined, recent clinical and laboratory studies support the activity of the classical bifunctional alkylating agents against these tumors. Nevertheless, substantial increases in patient survival as a result of adjuvant use of these agents remain to be demonstrated, and an understanding of the mechanisms responsible for drug failure is critical for the design of optimal chemotherapeutic intervention. Successful establishment of cell line and xenograft models of childhood high grade glioma and ependymoma now provide the biological tools to facilitate an understanding of alkylator resistance in these tumors. Resistance to alkylating agents, including cyclophosphamide and melphalan, is multifactorial, with a diverse spectrum of mechanisms observed in murine and human neoplasia. Mechanisms of resistance to cyclophosphamide include increased aldehyde dehydrogenase activity., increased glutathione-S- transferase activity, elevated levels of glutathione, and a presently undefined mechanism in medulloblastoma. Similarly, mechanisms of resistance to malphalan include decreased cellular transport, increased intracellular glutathione levels protective of critical cellular targets, cellular detoxification and enhanced capacity to repair damaged DNA. These studies may not be relevant to the mechanisms of resistance operational in childhood malignant glioma and ependymoma. The hypothesis of this proposal is: definition and modulation/bypass of alkylator resistance in childhood high grade glioma and ependymoma will allow selection of alkylator regimens active in the treatment of these tumors and increase survival of children with these neoplasms. The specific aims of this proposal are: 1) To continue to establish childhood high grade glioma and ependymoma cell lines and transplantable xenografts in athymic mice with de novo clinical, acquired clinical and laboratory-generated cyclophosphamide and melphalan resistance. 2) To define the mechanisms of resistance to cyclophosphamide and melphalan of childhood high grade glioma and ependymoma cell lines and xenografts with de novo, acquired clinical, and laboratory-generated resistance. 3) To define modulation effective in bypassing/reversing cyclophosphamide and melphalan resistance in childhood high grade glioma and ependymoma cell lines and xenografts and 4) To define the role of L- amino acid oxidase-mediated depletion of plasma large neutral amino acids to enhance delivery and activity of melphalan in the treatment of subcutaneous and intracranial childhood high grade glioma and ependymoma xenografts in athymic mice.

DESCRIPTION: Medulloblastoma, the most common malignant brain tumor in childhood, continues to represent a therapeutic challenge. Although the majority of children with standard-risk (localized resectable tumors without metastases) are cured with surgical and radiotherapeutic intervention, 30 percent of these patients relapse and die. Children with high-risk medulloblastoma (extensive or metastatic tumors) do far worse, with the majority failing treatment with surgery, radiotherapy and chemotherapy. Demonstration of the activity of bifunctional alkylators such as cyclophosphamide in the treatment of medulloblastoma has provided the opportunity for advances in the treatment of high- risk medulloblastoma, reduction of the dose (and presumably neurotoxicity) of radiation used to treat standard-risk medulloblastoma, and the salvage of patients with recurrent medulloblastoma. Review of recent clinical trials using cyclophosphamide for treatment of medulloblastoma now indicates two major impediments to further progress: emergence of drug-resistant tumor cells and spread of tumor cells to the leptomeninges. Resistance to alkylating agents, including cyclophosphamide, is multifactorial, with a diverse spectrum of mechanisms observed in murine and human neoplasia, including increased aldehyde dehydrogenase activity, increased GST activity and elevated levels of GSH. Mechanisms of tumor resistance to 4- hydroperoxycyclophosphamide (4-HC) have been studied using a panel of human medulloblastoma cell lines with either laboratory-generated resistance to 4-HC or established from tumors showing clinical resistance to cyclophosphamide. Recent studies indicate preferential repair of DNA interstrand crosslinks (ICL) in a resistant line compared to a parental line, supporting further studies designed to define the scope and relevance of DNA ICL repair as a mechanism underlying resistance of medulloblastoma to cyclophosphamide. Medulloblastoma has a marked tendency for subarachnoid dissemination, with the incidence of leptomeningeal involvement at diagnosis approximately 30 percent using both cytologic analysis of cerebrospinal fluid and radiographic imaging. Recent studies demonstrating the activity of busulfan (as opposed to other alkylators) against 4-HC-resistant medulloblastoma cell lines, coupled with initial experiments indicating that busulfan can be administered intrathecally producing CSF levels > 3 logs higher than that produced by oral administration at a dose used for bone marrow transplantation suggest that intrathecal busulfan may prove effective in prophylaxis or treatment of leptomeningeal medulloblastoma. The specific aims of this proposal are: 1) To construct a phosphoramide mustard- induced single DNA ICL in a defined position of a plasmid vector; 2) To define the molecular events mediating repair of phosphoramide mustard-induced DNA ICL; 3) To define the role of repair of phosphoramide mustard-induced DNA ICL in mediating cyclophosphamide resistance in medulloblastoma.

DESCRIPTION: Malignant human gliomas constitute a group of highly lethal neoplasms. Chloroethylnitrosoureas (CENUs) are moderately effective in the treatment of gliomas in adults. They produce cytotoxicity by alkyl substitution of DNA guanine residues, with subsequent formation of DNA intrastrand cross-links. The alkyl group can be removed from the O6 position by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT), with restoration of an intact guanine. Resistance and susceptibility to nitrosoureas and methylating agents such as procarbazine can be mediated by this protein, which is expressed in the majority of tumors studied to date, including those arising in the central nervous system. The observation that depletion of AGAT with alkylguanines or methylating agents sensitizes cells to the cytotoxic action of CENUs provides the opportunity for the use of combination therapy designed to restore sensitivity to tumor cells resistant to the CENU carmustine (BCNU). Recent studies have demonstrated enhancement of nitrosourea activity in human medulloblastoma and glioblastoma multiforme xenografts growing subcutaneously and intracranially in athymic nude mice by O6-benzylguanine-mediated depletion of AGAT. The hypotheses of this project are: 1) High grade glioma AGAT levels and cellular distribution determine response to nitrosoureas and 2) inhibition of AGAT activity by O6- benzylguanine can reverse nitrosourea resistance in high grade gliomas without an unacceptable increase in toxicity. The specific aims of the project are: 1) To define the optimal doses of the combination of O6-benzylguanine and BCNU in the treatment of patients with recurrent anaplastic astrocytoma or glioblastoma multiforme and 2) To define the activity of the combination of O6-benzylguanine and BCNU in the treatment of patients with recurrent anaplastic astrocytoma or glioblastoma multiforme.

Primary brain tumors, including malignant gliomas and ependymomas, are second only to leukemia as a cause of childhood cancer. Advances in chemotherapy and combined modality regimens, though dramatically successful in the treatment of other pediatric malignancies, have not been translated into effective therapy for most pediatric brain tumors. Although the role of chemotherapy in the treatment of malignant gliomas and ependymomas remains poorly defined, recent clinical and laboratory studies support the activity of the classical bifunctional alkylating agents against these tumors. Nevertheless, substantial increases in patient survival as a result of adjuvant use of these agents remain to be demonstrated, and an understanding of the mechanisms responsible for drug failure is critical for the design of optimal chemotherapeutic intervention. Successful establishment of cell line and xenograft models of childhood high grade glioma and ependymoma now provide the biological tools to facilitate an understanding of alkylator resistance in these tumors. Resistance to alkylating agents, including cyclophosphamide and melphalan, is multifactorial, with a diverse spectrum of mechanisms observed in murine and human neoplasia. Mechanisms of resistance to cyclophosphamide include increased aldehyde dehydrogenase activity., increased glutathione-S- transferase activity, elevated levels of glutathione, and a presently undefined mechanism in medulloblastoma. Similarly, mechanisms of resistance to malphalan include decreased cellular transport, increased intracellular glutathione levels protective of critical cellular targets, cellular detoxification and enhanced capacity to repair damaged DNA. These studies may not be relevant to the mechanisms of resistance operational in childhood malignant glioma and ependymoma. The hypothesis of this proposal is: definition and modulation/bypass of alkylator resistance in childhood high grade glioma and ependymoma will allow selection of alkylator regimens active in the treatment of these tumors and increase survival of children with these neoplasms. The specific aims of this proposal are: 1) To continue to establish childhood high grade glioma and ependymoma cell lines and transplantable xenografts in athymic mice with de novo clinical, acquired clinical and laboratory-generated cyclophosphamide and melphalan resistance. 2) To define the mechanisms of resistance to cyclophosphamide and melphalan of childhood high grade glioma and ependymoma cell lines and xenografts with de novo, acquired clinical, and laboratory-generated resistance. 3) To define modulation effective in bypassing/reversing cyclophosphamide and melphalan resistance in childhood high grade glioma and ependymoma cell lines and xenografts and 4) To define the role of L- amino acid oxidase-mediated depletion of plasma large neutral amino acids to enhance delivery and activity of melphalan in the treatment of subcutaneous and intracranial childhood high grade glioma and ependymoma xenografts in athymic mice.

Malignant central nervous system (CNS) tumors are perhaps the most difficult and frustrating neoplasms to treat, in large part due to the eloquent site in which these lesions arise and grow. Medulloblastoma, the most common malignant brain tumor in childhood, continues to represent a therapeutic challenge. The prognosis for most patients with primary malignant gliomas remains even worse. Demonstration of the activity of bifunctional alkylators such as cyclophosphamide in the treatment of medulloblastoma and glioma has provided the opportunity for significant advances in the treatment of these tumors. Resistance to cyclophosphamide is multifactorial, with a diverse spectrum of mechanisms observe in murine and human neoplasia, including increased aldehyde dehydrogenase activity, increased GST activity and elevated levels of GSH. More recent studies indicate preferential repair of DNA interstrand crosslinks (ICL) in a 4-HC resistant medulloblastoma line compared to a parental line, supporting further studies designed to define the scope and relevance of a DNA ICL repair as a mechanism underlying resistance of medulloblastoma and glioma to cyclophosphamide. However, several considerations need to be addressed in analyzing the role of crosslink repair in alkylator resistance (Chaney and Sancar 1996). Not only will it be necessary to demonstrate an increased removal of adducts of crosslinks but also to demonstrate which repair pathway(s) is (are) involved. Demonstration of repair enzymes at increased levels in resistant cell lines must be followed by studies demonstrating that these enzymes are rate limiting for the repair pathway. The ultimate proof of principle will be the demonstration that the repair pathway is more active in the resistant cells. These studies will require technological approaches/reagents not currently available until now. The hypothesis of this proposal is that: repair of DNA ICL is a major mechanism of resistance to cyclophosphamide in medulloblastoma and glioma. The specific aims of this proposal are 1) to define the molecular events mediating repair of phosphoramide mustard-induced DNA ICL by human cell extracts; 2) to define the role of repair of phosphoramide mustard-induced DNA ICL in mediating cyclophosphamide resistance in medulloblastoma and malignant glioma; and 3) to define the pathways operational in the repair of phosphoramide mustard-induced DNA ICL in cyclophosphamide resistant medulloblastoma and glioma.

The goals of the Brain Tumor Center at Duke are to foster interaction among a group of basic, translational, and clinical scientists to improve diagnosis and treatment of primary and metastatic brain tumors in adults and children and to train new basic, translational, and clinical investigators to work in Neuro-Oncology. The Brain Tumor Center at Duke (also called the Neuro-Oncology Program) is one of the oldest organ-site cancer programs at Duke and it predates the Cancer Center by a generation. The clinical and basic investigation in neuro- oncology at Duke began in 1937 under the leadership of the late Barnes Woodhall, M.D., first Professor of Neurosurgery at Duke and the second Chancellor of the Medical Center. Subsequent leaders of the Program were Guy L. Odom, M.D., James B. Duke Professor of Neurosurgery, and the late M. Stephen Mahaley, Jr., M.D. Dr. Darell Bigner has led the Program since its formal status as a Cancer Center Program was established more than 15 years ago when Core Grants initiated program status. The Neuro- Oncology Program is composed of 21 senior faculty who hold 23 peer-reviewed grants relative to neuro-oncology from the National Institute of Neurological Diseases and Stroke. The most significant progress in the ability to conduct clinical trials in adult and pediatric brain tumor patients at Duke was administrative reorganization of Clinical Neuro-Oncology to effectively merge surgical and medical care of both pediatric and adult patients. This important reorganization was envisioned and successfully negotiated with Duke Hospital, the relevant Department Chairs, and the Chancellor by the new Director of the Duke Comprehensive Cancer Center, O. Michael Colvin, M.D. First, in 1996, a Division of Pediatric Neuro-Oncology was created with Dr. Henry S Friedman as Chief with two additional pediatric neuro-oncology faculty. Dr. Friedman was then given appointments in both the Departments of Medicine and Surgery, and he and Dr. Allan Friedman, Chief of Neurosurgery, were made Co-Directors of Clinical Neuro-Oncology at Duke. The offices, data management staff, physician extenders, and medical and pediatric faculty staff were physically consolidated. With this new administrative structure 450 new adult patients, and 100 new pediatric patients were seen in 1997. There were 400 craniotomies for brain tumor performed in 1997, and approximately 600 adults and 400 children with primary brain tumors are being followed in 1998. The specific aims of this proposal are to 1) design innovative clinical trials for children with brain tumors based on in vitro and preclinical in vivo laboratory studies and 2) to conduct these trials with appropriate patient accrual, data management and monitoring.

high performance liquid chromatography; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology

DESCRIPTION (Provided by applicant): Despite decades of intensive nervous system (CNS) neoplasms remains very poor. Median survival for adults with the most common form of CNS tumor, the cerebral glioblastoma, is 8-12 months after diagnosis. Occasional responses to single or multiple agent chemotherapy are seen in the setting of recurrent tumor, but these responses are generally of short duration, and cures are rare. Identification of agents active against glial malignancies is challenging, with no drug tested to date reliably producing responses in a majority of treated patients. Gene amplification, related to increasing grade of glioma malignancy, has been found to occur in approximately 50 percent of all glioblastoma multiforme (GBM) cases. Although amplification of N-myc and gli (2-4 percent overall) has been reported by different groups, amplification of these genes and c-myc or K-ras are considered sporadic as compared to the amplification of c-erb 1, or the epidermal growth factor (EGFR) gene. The EGFR gene, 110 kb in size, 26 exons in organization, is localized to chromosome arm 7pll-13. Beginning with the initial description of EGFR gene amplification by Libermann et al (1985), subsequent studies have confirmed that approximately 37-58 percent of GBMs, but only isolated anaplastic astrocytomas, amplify the EGFR gene. ZD 1839 is a potent inhibitor in vitro of EGFR tyrosine kinase, competitive with ATP, and noncompetitive with peptide substrate. ZD 1839 inhibits the proliferation of EGF-stimulated KB oral squamous carcinoma cells. This effect is readily reversible on removal of the compound. Enzyme inhibition appears to be selective, with little activity against other kinases tested. Growth inhibition in vivo of a wide variety of human tumour xenograft models in nude mice was demonstrated at a range of once daily, oral doses between 12.5 and 200 mg/kg per day for up to 4 months. In some already established tumours treatment with ZD 1839 produced significant regressions. From the xenograft studies, it is not yet clear if there is a correlation between the level of EGFR expression and antitumor response. The specific aims of this proposal are: 1) To identify the activity and toxicity of ZD 1839 in the treatment of adults with glioblastoma multiforme in first relapse; 2) to determine if qualitative and quantitative levels of genotypic and phenotypic EGFR expression predict response of GBM to ZD 1839.

DESCRIPTION: (provided by applicant) Central nervous system (CNS) neoplasms which either arise in the brain or metastasize from an extraneural primary site, are highly malignant tumors refractory to all conventional therapy. Similarly, patients with neoplastic meningitis from virtually any tumor such as melanoma, sarcoma or breast carcinoma do poorly, with mean survival following leptomeningeal spread measured in months. The major impediment to successful treatment is de novo or acquired resistance to chemotherapy. Temozolomide is an imidazole tetrazinone similar to dacarbazine, requiring conversion to the active methylating agent MTIC. Methylating agents, including temozolomide, produce cytotoxicity due to a lethal cycle of mismatch repair following cellular misrecognition of O(6)-methylguanine. Recent preclinical and clinical studies have confirmed the activity of temoxolomide in the treatment of malignant glioma. Unfortunately, the majority of patients ultimately display resistance to temozolomide. The two primary mechanisms of resistance to temozolomide and other alkylating agents are the enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) and a deficiency in the DNA mismatch repair pathway. Of these two mechanisms, AGT plays a primary role in resistance to temozolomide by removing the alkyl groups from the O(6) position of guanine, in effect reversing the cytotoxic lesion of temozolomide. The sensitivity of tumor cell lines to temozolomide and the alkylating agent BCNU can be correlated with AGT levels. Regional therapy of CNS parenchymal or leptomeningeal neoplasms with intratumoral or intrathecal administration respectively, offers the potential benefit of enhancing delivery to the target neoplasm while minimizing delivery and hence toxicity to systemic organs. We have previously demonstrated the activity and modest toxicity of intrathecal temozolomide in the treatment of athymic rats bearing subarachnoid AGT-human malignant gliom xenografts. We have extended these results and demonstrated the activity and safety of temozolomide delivered by intracerebral microinfusion in the treatment of malignant gliomas intracranially in athymic nude rats. The specific aims of this proposal are: 1. To define the role of intratumoral O(6)-BG and other AGT inhibitors in enhancing systemic or intratumoral temozolomide therapy of malignant glioma; 2. To define the role of intrathecal AGT inhibitors in enhancing system or intrathecal temozolomide therapy of neoplastic meningitis.

meningitis; neoplasm /cancer chemotherapy; metastasis; medulloblastoma; infection related neoplasm /cancer; camptothecin; drug screening /evaluation; central nervous system neoplasms; glioma; topotecan; lomustine; antineoplastics; drug administration routes; pharmacokinetics; drug adverse effect; drug resistance; analog; neoplasm /cancer transplantation; xenotransplantation; disease /disorder model; nonhuman therapy evaluation; subarachnoid space; human tissue; athymic mouse;

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. 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, O6-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, xenografls 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.

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. 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 DMA mismatch repair deficiency. The hypotheses of this proposal are 1) AGT and DNA mismatch repair deficiency play a role in mediating Temodar resistance in malignant glioma and medulloblastoma; 2) other mechanisms are also critical in mediating this resistance; and 3) inhibition of base excision repair can enhance Temodar activity. The specific aims of this proposal are: 1) to define the role of AGT and AGT mutations in mediating resistance in tumors resected from patients with Temodar resistant malignant glioma; 2) to define the role of DNA mismatch repair deficiency in tumors resected from patients with Temodar resistant malignant glioma; and 3) to define the role of base excision repair (BER) in mediating resistance to tumors resected from patients with Temodar resistant malignant glioma and the role of inhibition of BER in enhancing Temodar activity in malignant glioma.