2002 Project Summaries
Recipients of Three-year Awards
Bob S. Carter, M.D., Ph.D. The long-term goal of this project is to bring a new, targeted immunotherapy strategy for human glioblastoma to clinical investigation. EGFRvIII is a tumor-specific, ligand-independent, and constitutively active variant of the epidermal growth factor receptor that is expressed in a high percentage of glioblastoma and breast cancer patients. It has not been detected in normal tissues and represents an attractive tumor-specific target for glioblastoma. Our laboratory has engineered chimeric T-cell receptor constructs (cTCRs) consisting of EGFRvIII specific-single chain antibody extracellular domain fused to the cytoplasmic tail of the T-cell receptor CD3 complex zeta chain (MR1scFv:zeta) and has developed methodologies to genetically modify T-lymphocytes with MLV based vectors. Ex vivo MR1scFv:zeta gene-modified primary murine CD8+ cytotoxic T-lymphocytes (CTL) specifically recognize the lyse EGFRvIII expressing cells. The observation raises the potential of using an MR1cTCR ex vivo gene therapy strategy to target certain glioblastoma tumors. We proposed three specific aims: 1) Test the hypothesis that higher binding affinity variants of MR1 coupled with co-stimulatory sequences such as CD28, 41-BB, and OX40 will enhance the overall proliferative and anti-tumor potential of chimeric T-cell receptor modified human lymphocytes. 2) Test the hypothesis that lentiviral transduction can offer improved in vivo survival and function outcome for transduced lymphocytes by using an abbreviated activation protocol. And 3) Test the hypothesis that MR1cTCR-modified lymphocytes from human glioblastoma patients can lyse autologous tumors grown in NOD/SCID mice. We hope these studies will lay a foundation for a Phase I clinical trial for this approach.
Lynda Chin M.D. Alterations in EGFR, PDGF, INK4a, p53 and/or PTEN are among the most common lesions encountered in malignant gliomas. Notably, a significant portion of malignant gliomas do not harbor these signature genetic lesions, implying that many other glioma-relevant mutations remain unidentified. Recent advances in functional genomics have provided new capabilities for the rapid identification and characterization of candidate glioma-relevant genes and their pathways. Taking advantage of the presence of recurring chromosomal alterations associated with amplification or deletion of specific genes in malignant gliomas, array-based CGH technique will be used to compile the profiles of recurrent DNA copy number alterations (CNAs) with high throughput. Using the human genome sequence, we have developed a novel STS-specific quantitative PCR approach to finely map the minimal regions of these CNAs. When combined with bioinformatic and expression-based analyses, aCGH and STS-PCR enable the rapid identification of candidate target genes within these CNAs. Preliminary studies have identified a large number of recurring cancer hotspots, which proves the effectiveness of this combined approach. The dreaded nature of this particular cancer and the current lack of therapeutic options beg for the identification of additional points of pharmacological attack. With seed support from The Goldhirsh Foundation, we will use this approach to expand the repertoire of genes driving glioma genesis and pave the foundation for further functional characterization, genetic validation, and eventual therapeutic development.
Rakesh K. Jain, Ph.D. Adult glioblastoma multiforme (GBM) is a uniformly fatal disease with an average survival rate of less than one year. We propose to develop a novel approach to treating GBM that allows for a rapid translation to the clinic. GBMs have an abundance of blood vessels, which are recruited to the tumor by VEGF (vascular endothelial growth factor). Blocking VEGF signaling has been shown to cause growth suppression of GMB, but no cure. We have recently shown that anti-VEGF therapy combined with fractionated radiation can lead to both growth inhibition and long-term cures in GBMs grown in the legs of mice. While our findings are extremely exciting, we do not know 1) what mechanisms cause the synergism between these two therapies, 2) whether this combination is also effective in GBMs growing in their natural environment in the brain. We hypothesize that judiciously applied anti-VEGF therapy can "normalize" the abnormal GBM vasculature and hence improve the delivery of oxygen, a potent radiation sensitizer. Furthermore, we propose that anti-VEGF treatment will block VEGF action, which protects tumors and their blood vessels from radiation. Thus, our aims are to 1) understand the molecular and physiologic mechanisms of vessel "normalization" (Aim 1, years 1 and 2); and 2) to take advantage of this normalization window of opportunity to optimally combine anti-angiogenic and radiation therapy for GBMs growing in their native site in a relevant animal model (Aim 2, years 2 and 3). We will implant human GBMs that produce various levels of VEGF into the cerebra of mice. Tumor size, vessel size, blood flow, vessel function, tissue oxygen, and VEGF will be measured in vivo using 2-photon microscopy at different doses as well as schedules of antibody treatment to identify the "normalization" window. To obtain molecular insights into the normalization process, we will also measure proliferation apoptosis of endothelial and cancer cells along with the levels of key proteins that are responsible for blood vessel growth and survival. Once the normalization window is identified, we will combine the anti-VEGF therapy with the fractionated radiation treatment. We will measure both the short term (growth delay) and the long term (cure rate) response of tumors. We anticipate that radiation treatment during the normalization window has the highest effectiveness and can improve the long-term cure of GBMs. The resulting information will be used to develop guidelines for combining therapy of GBM in the clinic in collaboration with Dr. Jay Loeffler, Chief of Radiation Oncology Department, MGH. Recipients of One-year Awards
Xandra O. Breakefield, Ph.D. This research will focus on in vivo imaging of gene delivery into brain tumors, and monitoring the therapeutic effect of apoptotic genes on brain tumors. We propose to accomplish our goals by creating amplicon vectors bearing TRAIL, with and without a cell fusion protein, and the only extracellular domain of TRAIL to evaluate the range of apoptosis with membrane-bound and secreted forms of TRAIL. In order to visualize the amplicon vector delivery and monitor tumor regression, dual non-invasive bioluminescense imaging will be used. Aplicon vector delivery will be imaged using Renilla luciferase imaging, whereas tumor regression in the same animal will be monitored using firefly luciferase imaging. This will enable us to monitor HSV-amplicon delivery in addition to monitoring the therapeutic effects of TRAIL in living animals. To explore the possibility of combining protease controlled high-affinity NIRF imaging and tumor cell apoptosis, we will fuse the TRAIL to HIV-1 or HSV-1 protease substrate sequences and the endoplasmic reticulum (ER) retention signal KDEL, in-frame near the C-terminus. This should prevent the TRAIL from reaching the cell surface and restrain its cell-killing properties. Subsequent infection by HIV-1 or HSV-1 protease-bearing vectors should release the TRAIL protein from the ER and result in cell death. We will be able to monitor vector delivery and apoptotic gene expression in vivo by imaging optically quenched NIRF probes. We expect that the proposed experiments will provide the medical community with: 1) a system for imaging gene delivery and expression of a therapeutic transgene suited for us in human trials, and 2) the parameters necessary to specifically image apoptosis and eventually eliminate tumors using viral vectors.
Andrew L. Kung, M.D., Ph.D., Joshua Rubin, M.D., Ph.D. Although new therapies for glioblastoma multiforme (GBM) are urgently needed, the translation of basic laboratory insights into clinically useful therapeutics has been slow. The overall goals of this proposal are to develop methods that will be widely useful in bridging this transition and to use such methods to study several potentially novel brain tumor therapies. Our specific aim is to develop a sensitive and rapid in vivo imaging system to facilitate the study of brain tumor biology as well as the rapid assessment of new therapies in mouse GBM models.
Lois A. Lampson, Ph.D. Our hypothesis is that pirfenidone, a drug that is already used clinically to block invasion in other settings, can also block invasion of malignant astroctoma. Our first aim is to develop a novel mouse model designed for study of anti-invasive therapy in vivo. Our second aim is to use the model to test our hypothesis that pirfenidone can block astrocytoma invasion. Tumor invasion is a key cause of mortality in astrocytoma. Much is known about the biology of invasion, especially from in vitro studies. However, there is no current therapy that can target invasive tumor. The drug we will test, pirfenidone, has anti-invasive properties outside the brain and has been found to be safe in human trials. Now we will ask if pirfenidone can block astrocytoma invasion in the brain. Our approach is built on accumulating evidence that the cytokine, TGF-b1, is a key modulator of glioma invasion and that pirfenidone can block TRG-b-related processes in other contexts. We will use a panel of glioma cell lines engineered to express different levels of TGF-b1, which should show different extents of invasion in vivo. Our first aim completes the characterization of the panel. Our second aim asks if pirfenidone can block invasion of cells that are already characterized. This new project brings together our PI's expertise in invasive brain tumor from the viewpoint of basic immunobiology and the surgical experience and commitment to anti-invasion therapy of our post-doctoral fellow, Dr. Kate Drummond. Methods include tissue culture to grow the tumor cells lines, stereotactic implantation of tumor cell lines, pharmacological treatment of tumor-bearing animals, and histochemical and antibody staining of brain section. Mice will be used.
David N. Louis, M.D. Clinical and research neuro-oncology depends on accurate tumor classification, particularly in the study and treatment of adult astrocytic tumors. We have demonstrated that molecular approaches to classification can greatly augment current pathology-based schemes, with practical implications for adult patients with malignant gliomas. We have recently extended these approaches to expression profiling, and have generated preliminary data that strongly suggests the existence of a novel group of adult malignant astrocytomas. In the present proposal, we will test the hypothesis that this novel subtype of adult astrocytoma is both biologically distinct and clinically relevant. To address this hypothesis, we propose three aims. 1) To develop a practical means for identifying this novel adult astrocytoma subtype, we will develop markers for their identification. This will primarily be based on comparisons of gene expression between the novel group and the other "classic" groups, followed by the creation of either immunohistochemical or RT-PCR assays that would be simple, reproducible, sensitive, and specific. 2) To determine the prognostic and therapeutic relevance of the novel group, we will compare clinical parameters from patients whose tumors are in the novel group with those whose tumors are in the "classic" groups. The endpoints will primarily include overall survival, time to progression, and radiographic response to chemotherapy and/or radiation therapy. These comparisons will be accomplished by consulting the MGH Brain Tumor Center clinical database and our collaborators in neuro-oncology and biostatistics. 3) To characterize the pathway(s) activated in the novel adult astrocytoma subtype, we will begin database analyses to elucidate pathways activated in the novel group in comparison with the other groups. In summary, the present proposal will delineate a novel group of high-grade gliomas, which will eventually lay the foundation for the development of targeted molecular therapies for these neoplasms.
Pamela A. Silver, Ph.D. Recent studies have implicated RNA binding proteins (RBPs) in the pathogenesis of astrocytic tumors based on their specific expression in glioblastoma tissue and glioma cell lines. Current evidence also suggests that receptor tyrosine kinase overexpression, a key event in the malignant transformation of astrocytes, may (in part) result from post-transcriptional modifications including RNA stabilization and alternative splicing. Thus, RBPs and their cognate RNAs may be novel targets for selective drug design. The specific aims of this research are two-fold and have been designed to test our hypothesis that post-transcriptional modifications through the differential expression of RBPs may promote tumor growth. 1) To identify novel proteins that are expressed in astrocyte and glioma cell lines, we will perform a genome-wide screen of approximately 400 RBPs using RT-PCR. All positive signals will be confirmed and quantitiated by real-time PCR to monitor differences in expression between astrocyte and glioma cell lines. Astrocyte- and glioma-specific RBPs will also be mapped in mouse tissue sections by in situ hybridization to confirm their cell-type localizations and the central nervous system. 2) To address the potential mechanisms by which RBPs may contribute to astrocytic tumor growth, we will identify the RNAs bound by each astrocytic-specific RBP using both microarray strategies and novel nanotechnologies. These experiments will provide a molecular profile of the RNA binding proteins expressed in atrocytes and astrocytic tumors as well as the RNAs that are differentially bound by these proteins. |
 |
 |
