David Zagzag, MD PhD - US grants

DESCRIPTION (provided by applicant): There is a dire need for novel therapy for brain tumors. Diffuse glioma cell invasion into surrounding brain tissue and angiogenesis are fundamental features of gliomas that have not been successfully addressed by conventional therapy to control tumor growth in brain tumor patients. Current aggressive local treatments such as surgery, radiotherapy and chemotherapy are effective at controlling the localized mass of tumor cells in the brain. This success is only temporary, because tumor cells that have already invaded into the surrounding brain and serve as seeds by which the tumor re-grows. The process by which these tumor cells invade into the brain is not well understood. New treatment approaches that are capable to control these invading tumor cells can have a dramatic impact on patient outcome. We have developed assays in vitro and in vivo that allow us to study invading glioma and endothelial cells. Our long-term goal remains to interfere with tumor invasion and angiogenesis to diminish tumor growth. To this end we propose i) to determine mechanisms involved in the migration of both endothelial and glioma cells; and ii) to investigate the mechanism and therapeutic potential of two geldanamycin analogues on glioma in vitro and in vivo. Our proposal seeks to take advantage of new molecular biologic techniques to improve our understanding of this process. Collectively, the proposed experiments will analyze the cellular and molecular pathways necessary for tumor invasion and angiogenesis. Results from these experiments will lead to future strategies designed to inhibit specific molecular components implicated in these critical processes.

Aftercare; AMD3100; Anatomy; angiogenesis; Angiogenesis Inhibitors; Animal Model; Animals; Antibodies; antitumor drug; Apoptosis; bevacizumab; Binding (Molecular Function); Biological; Blood Vessels; Brain; Brain Neoplasms; brain tissue; Bromodeoxyuridine; Cells; Cerebrospinal Fluid; Clinical; clinically relevant; Complex; CXCR4 gene; Data; DC101 Monoclonal Antibody; Development; Disease Progression; Drug Delivery Systems; drug efficacy; efficacy testing; Extracellular Matrix; Freezing; Gelatinase A; Glioma; Goals; Green Fluorescent Proteins; Harvest; Human; human VEGF protein; Hypoxia; hypoxia inducible factor 1; Immunohistochemistry; improved; In Situ Nick-End Labeling; In Vitro; in vivo; in vivo Model; Infiltrative Growth; Intracranial Neoplasms; Invaded; Knowledge; Malignant Glioma; Methods; migration; Modeling; Molecular; mouse model; Mus; neoplastic cell; neovascularization; neovasculature; nestin protein; Neuraxis; neutralizing antibody; Noscapine; novel; novel therapeutic intervention; novel therapeutics; Oral; Outcome; Patients; Pattern; Pharmaceutical Preparations; Property; research study; Route; small molecule; subcutaneous; System; Therapeutic Agents; Treatment Protocols; Trigeminal nerve structure; tumor; Tumor Angiogenesis; Tumor Cell Invasion; tumor growth; tumor progression; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors

DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common and malignant primary intracranial human neoplasm. Anti-(R) angiogenic therapy with bevacizumab (Avastin) has become standard therapy in recurrent high-grade gliomas in adults. Patients at New York University Langone Medical Center and elsewhere continue to receive bevacizumab because of (i) marked improvement in quality of life, (ii) although transient, a demonstrable increase in progression-free survival and overall survival compared to historical controls, and (iii) relief from steroid dependence due to diminished tumor edema (Narayana, 2009). We and others have observed that the pattern of relapse in bevacizumab-treated GBM patients is often characterized by local, as well as distant infiltration of the brain by the tumor. We have conducted a proof-of-concept experiment with mouse bevacizumab (anti-VEGF antibody B20-4.1.1 from Genentech) to determine whether bevacizumab induces invasive growth of GL261 glioma cells in the brain of mice. GL261 gliomas treated with mouse bevacizumab showed increased infiltration of the brain highly similar to that observed in human patients receiving the humanized bevacizumab antibody. The chemokine receptor CXCR4 plays a critical role in glioma invasion. We intend to use two experimental in vivo murine glioma models (GL261 and CT-2A) to screen novel and extremely potent CXCR4 inhibitors (POL5551 and POL6326) developed by Polyphor Ltd. for their efficacy in blocking bevacizumab-induced glioma dissemination. CXCR4 antagonists are being used increasingly in the clinic for cancer therapy (Wong, 2008) and could potentially control the invasive behavior of CXCR4- positive glioma cells, prolonging bevacizumab's efficacy and improving the prognosis of glioma patients. PUBLIC HEALTH RELEVANCE: (R) Anti-angiogenic therapy with bevacizumab (Avastin) has become standard therapy in recurrent high-grade gliomas in adults. We will use novel and potent CXCR4 antagonists POL5551 and POL6326 to block bevacizumab-induced glioma dissemination.

Glioblastoma (GBM) is the most common and malignant primary intracranial human neoplasm. Anti-(R) angiogenic therapy with bevacizumab (Avastin) has become standard therapy in recurrent high-grade gliomas in adults. Patients at New York University Langone Medical Center and elsewhere continue to receive bevacizumab because of (i) marked improvement in quality of life, (ii) although transient, a demonstrable increase in progression-free survival and overall survival compared to historical controls, and (iii) relief from steroid dependence due to diminished tumor edema (Narayana, 2009). We and others have observed that the pattern of relapse in bevacizumab-treated GBM patients is often characterized by local, as well as distant infiltration of the brain by the tumor. We have conducted a proof-of-concept experiment with mouse bevacizumab (anti-VEGF antibody B20-4.1.1 from Genentech) to determine whether bevacizumab induces invasive growth of GL261 glioma cells in the brain of mice. GL261 gliomas treated with mouse bevacizumab showed increased infiltration of the brain highly similar to that observed in human patients receiving the humanized bevacizumab antibody. The chemokine receptor CXCR4 plays a critical role in glioma invasion. We intend to use two experimental in vivo murine glioma models (GL261 and CT-2A) to screen novel and extremely potent CXCR4 inhibitors (POL5551 and POL6326) developed by Polyphor Ltd. for their efficacy in blocking bevacizumab-induced glioma dissemination. CXCR4 antagonists are being used increasingly in the clinic for cancer therapy (Wong, 2008) and could potentially control the invasive behavior of CXCR4- positive glioma cells, prolonging bevacizumab's efficacy and improving the prognosis of glioma patients.

DESCRIPTION (provided by applicant): Patients with the genetic syndrome Neurofibromatosis type 2 (NF2) develop multiple intracranial and spinal tumors, including vestibular schwannomas (VS) and meningioma that lead to morbidity and early mortality. These tumors are the defining criteria for NF2, but are also among the most common brain tumors worldwide in non-NF2 patients. There are no known effective medical therapies for either VS or meningiomas. This proposal brings together a multi-institutional team of leading clinical and basic science experts in VS, meningiomas and NF2 to test the hypothesis that the mTOR (mammalian target of rapamycin) inhibitor RAD001 reaches meaningful intratumoral concentrations and shows evidence of mTOR pathway inhibition as well as biological activity in VS and meningioma in patients. VS and meningiomas are deficient in the protein Merlin, the gene product of the NF2 gene. Merlin deficiency may result in abnormal activation of the mTOR molecular signaling pathway and that inhibition of mTOR may stop tumor growth. We therefore hypothesize that treatment with mTOR inhibitors, such as RAD001, may be effective in the treatment of patients with VS and meningiomas. RAD001 is a well-tolerated oral drug with a favorable safety profile and anti-tumor activity against several types of cancer. It has recently shown remarkable efficacy in a genetic tumor syndrome similar to NF2 called Tuberous Sclerosis. In other tumor types, such as malignant cancers with Merlin loss, the anti-tumor efficacy of RAD001 may be limited due to disinhibition of a feedback mechanism, leading to the upregulation of AKT. At the present time, we do not know if this escape mechanism is operational in VS and meningiomas in vivo. To assess the intratumoral activity of RAD001 in patients in vivo, participants with and without NF2 scheduled for resection of a VS or meningioma who agree to participate, will take RAD001 for 7 days preceding surgery. At the time of surgery, blood and tumor tissue will be collected. RAD001 levels in the blood and tumor will be analyzed. We will also assess the effects of the drug on mTOR pathway signaling and tumor growth within the tumor cells and local parenchyma. The results of this study will provide important biological data that will inform further, rational development of mTOR-targeted therapies in VS and meningiomas. If biologically active drug concentrations of RAD001 that inhibit the primary molecular drug target can be achieved in VS and meningiomas, further development of RAD001-based therapies is warranted. If we find molecular escape mechanisms to be operational, combination therapies, such as combined mTOR and PI3K/AKT blockade should be explored. The identification of an effective medical therapy for VS and meningiomas would represent a breakthrough in neuro-oncology and enhance the quality of life and survival in patients with these tumors.

PROJECT SUMMARY Histologically characterized by neoplastic stromal cells and abundant vasculature, hemangioblastomas (HBs) occur as sporadic lesions (~70%, predominantly in the cerebellum) and in familial forms associated with von Hippel-Lindau (VHL) disease, an inherited multisystem tumor disorder characterized by HBs and other benign and malignant visceral neoplasms (e.g., clear cell renal cell carcinoma (RCC)). Central nervous system (CNS) HBs, which are frequently multiple or recurrent in VHL disease, may not be resectable, and an effective drug treatment is not available. Although biallelic inactivation of the VHL tumor suppressor gene by pathogenic mutations, promoter hypermethylation and/or loss of VHL-bearing chromosome 3p25 have been identified in 47% to 64% of both familial (germline mutations) and sporadic (somatic mutations) CNS HBs, the underlying pathogenic mechanisms responsible for HBs remain incompletely understood. By whole exome sequencing of archived VHL disease-associated and sporadic cerebellar HBs and matched cerebellum and/or blood cells (n=23, age 24-63), we found 314 pathogenic and/or likely deleterious mutations (both germline and somatic). In a significant number of cases (14/23, 61%), we identified the germline fibroblast growth factor receptor 4 (FGFR4) p.G388R variant, 8 of which were VHL wild-type and 2 had multiple/recurrent cerebellar HBs; the other 6 cases had both FGFR4 p.G388R and VHL mutations, 1 of which had multiple/recurrent cerebellar HBs and RCC. FGFR4 p.G388R is a pathogenic activating mutation known to enhance basal signal transducer and activator of transcription 3 (STAT3) signaling, resulting in increased HIF-1? mRNA transcription, STAT3- and HIF-1 target gene expression, angiogenesis, and possibly increased tumor susceptibility. We hypothesize that in addition to VHL inactivation, a significant number of VHL disease-associated and sporadic cerebellar HBs harbor germline FGFR4 p.G388R variant that activates STAT3 signaling and target gene expression in these tumors. We also hypothesize that gene promoter hypermethylation (e.g. VHL) and/or chromosomal alterations (e.g., EGFR amplification) may coexist with FGFR4 p.G388R variant and could together contribute to VHL disease-associated and sporadic cerebellar HB pathogenesis. Based on our initial results, we propose (Aim 1a) to validate in a larger cohort of archived cerebellar HBs our novel finding that a significant number of both VHL disease-associated and sporadic HBs harbor germline FGFR4 p.G388R. We also propose (Aim 1b) to define whether FGFR4 p.G388R activates the JAK-STAT-HIF signaling pathway in these tumors and (Aim 2) to demonstrate whether gene promoter hypermethylation and/or chromosomal alterations may co-exist with FGFR4 p.G388R variant and could together contribute to VHL disease-related and sporadic cerebellar HB pathogenesis. We anticipate that our proposed work could have a significant impact on the genetic testing and counseling of patients living with HBs. If successful, our work could also demonstrate a novel biomarker for CNS HB patient classification and potentially targetable mechanisms against CNS HBs.