2006 Project Summaries
Recipients of Three-year Award
Robert Darnell, M.D., Ph.D. This proposal is based on a series of striking of clinical observations we have made on patients a rare set of human diseases over the past 13 years. We have been studying a rare group of cancer patients with paraneoplastic neurologic degenerations (PND) who recognize their cancers as foreign, mount an immune response them, and do extremely well with them clinically. In fact, most of these patients do not know that they have cancer until their tumor immune response develops into an autoimmune disorder, allowing them to present clinically, and exposing the otherwise invisible phenomenon of successful immune recognition of cancer. In our view PND patients provide the best known examples of humans with effective, naturally occurring, tumor immunity. This is a proposal to take two elements of what we have learned from studying these patients—that their immune response is mediated at least in part by classical α/β T cells, and that this immune response can be triggered through a naturally occurring pathway in which tumor cells undergoing apoptotic death are scavenged by antigen presenting cells to activate the T cell response—and apply them to brain tumor patients in a Phase I clinical trial. Tumor material obtained at surgical resection from patients with primary brain tumors will be treated to undergo apoptotic death, co-cultured with autologous dendritic cells ex vivo, and injected subcutaneously to stimulate a T-cell anti-tumor immune response. Patients will be monitored for safety and immunogenicity. This will be the first rigorous test of this approach in humans, and will form the platform for combining this strategy with other immune adjuvants. Recipients of One-year Awards
Arnab Chakravarti, M.D. Glioblastoma (GBM) is the most common primary brain tumor of adults, and is among the most lethal of all cancers. Despite aggressive multimodality management with surgery, radiation (RT), and temozolomide (TMZ)-based chemotherapy, the median survival times of GBM patients still range from 12 to 15 months. Clearly, new approaches to the understanding and treatment of GBM are needed. Molecular and genetic mechanisms that serve to enhance the malignant phenotype of these tumors are becoming better understood. As RT+TMZ represent the current standard of care for GBM, identifying mechanisms of resistance to RT+TMZ is critical in improving outcomes of GBM patients. Our laboratory was the first to identify Survivin, a 16.5 kDa member of the inhibitor of apoptosis (IAP) family of proteins as an adverse prognostic marker and a potentially important mediator of resistance to RT+TMZ in GBMs. Survivin is expressed most strongly in the G2/M phases of the cell cycle in tumor cells, but is absent from most normal adult differentiated tissues. Preliminary data from the Chakravarti laboratory has also revealed clinically-relevant ways of downregulating Survivin through targeting the IGFR1-PI3K-AKT signaling pathway. Accordingly, our hypothesis is that the therapeutic ratio of RT+TMZ in GBM patients can be significantly enhanced through these novel Survivin targeting strategies, which will be examined in the proposed study during the first year. Additionally, in future years, the prognostic and predictive value of Survivin will be validated in a larger clinical dataset using precious tissue specimens from the Radiation Therapy Oncology Group (RTOG), an NCI-sponsored cooperative group. Our aims are 1) To determine the efficacy of these novel Survivin targeting strategies in enhancing the therapeutic ratio of RT+TMZ in GBMs. 2) To overcome resistance to kinase inhibitors targeting the EGFR/PI3K pathway through the use of novel targeting strategies in Aim#1 3) To investigate the prognostic and predictive value of Survivin in newly-diagnosed GBM patients treated on RTOG clinical studies involving RT alone or RT+Gefitinib.
Lynda Chin, M.D. The hallmarks of glioblastoma (GBM) include rapid progression, neovascularization, necrosis and intense apoptotic resistance, the molecular correlates of these phenotypic features, however, are not well understood. With the support of a previous Goldhirsh Award, oncogenomic analyses of a large panel of human GBMs by array-CGH and expression profiling were performed in an effort to gain a more comprehensive view of the genetic events underlying GBM development. In the course of characterization of a region of gain on chr19, we identified Bcl2L12 as a glioma oncogene that functions to block apoptosis signal at the distal end of the caspase cascade. Bcl2L12 (for Bcl2-Like12) is a Bcl-2-family member that is overexpressed in virtually all GBM samples, yet low or absent in low-grade disease and normal tissue. Extensive biochemical studies have established that Bcl2L12 functions as an anti-apoptotic and pro-necrotic protein in primary astrocytic cultures by inhibiting postmitochondrial caspase-3 and caspase-7 activation. These studies demonstrated that Bcl2L12 modulates key signaling pathways central to gliomagenesis, establishing it as a factor conferring hallmarks of GBM. These results have motivated a preliminary compound library screen which identified several candidate molecules capable of enhancing caspase activities in presence of Bcl2L12 activity. The goal of this project is to further characterize the anti-Bcl2L12 activities of lead compounds in human cell system as well as in a preclinial in vivo Bcl2L12-driven glioma model and to identify the compounds’ mechanism of action through a detailed multi-level characterization of glioma-relevant signaling pathways.
Samira Guccione, Ph.D. The primary objective of our research effort is based on the development of new targeted imaging and therapeutic agents for cancer with particular application in brain tumors for clinical translation. We have developed a vascular-targeted nanoparticle platform that allows the delivery of chemo, radiation, and/or genes to the tumor vasculature (NanoVast). The multifunctional capabilities of this agent allows for combined imaging and therapy applications as well as the delivery of multiple therapeutics as a cocktail, in vivo. This nanoparticle is 40 nm in diameter and targets the integrin αvβ3. Integrin targeted delivery of a plasmid encoding for a mutated Raf protein, results in extensive apoptosis and a reduction in tumor mass. The specific aims of this proposal are designed to evaluate the therapeutic efficacy and the associated functional imaging changes in the rat RT2 model for GBM. In vivo functional imaging evaluation will include metabolic (PET), vascular permeability (MRI), and biodistribution (SPECT) to establish the dynamic changes in response to this anti-angiogenic therapy. Additionally, temporal assessment of changes associated with the destruction of the tumor endothelium is valuable for optimizing therapeutic combinations this and other anti-angiogenic agents enter clinical trials.
Alonzo Ross, Ph.D. Recently, several laboratories have proposed the existence of cancer stem cells (CSC). This model is based on the observation that tumor cells vary greatly in their proliferative capacity and posits that tumors are organized in a lineage, i.e., a minority of stem-like cells gives rise to the bulk of the tumor cells. The CSCs have strong self-renewal and proliferative capacities. CSCs may change the way that we treat cancer. Currently, clinical trials are geared toward reductions in tumor volume. However, the death of the cells derived from the CSCs may cause only a transient effect on tumor volume. Killing the CSCs is essential to prevent future tumors and actually cure the patient. It may be that CSCs are relatively resistant to chemotherapy. We hope to reverse this situation and develop drugs that preferentially eliminate CSCs. Unfortunately, we do not know whether there are signaling pathway selectively important for CSCs. Identification of CSC-selective pathways is the overall goal of this proposal. To identify pathways required for self-renewal in CSCs, we will screen a subset of short hairpin RNA (shRNA) library. The University of Massachusetts Medical School has purchased a shRNA library developed by Gregory Hannon at Cold Spring Harbor Laboratory and set up a facility to allow UMASS investigators access to individual clones or the entire library at a reasonable price. The library consists of 61,000 plasmids encoding shRNAs. We will transfect neural stem cells and cancer stem cells from our brain tumor model with a mix of the shRNA clones for the 540 mouse kinases. We have chosen this subset of clones for our initial screen because kinases are key regulators for all signaling pathways and, therefore, it is very likely that we will identify shRNA clones that inhibit self-renewal. In addition, kinases are productive targets for drug development, and therapeutic applications will require a small organic inhibitor. We will select clones that preferentially target CSCs and not normal stem cells. By real-time PCR, we will verify the decreased kinase mRNA levels for those shRNAs that reduce self-renewal. We will test control shRNAs with point mutations or scrambled nucleotides that eliminate reduction of kinase levels. If possible we will transfect cells with both the shRNA plasmid and a kinase expression vector to test whether the phenotype can be rescued by expression of the kinase protein. In summary, the CSC model is changing the way that we think about cancer and, hopefully, will lead to new therapeutic approaches. We are in an excellent position to pursue this project because we routinely culture neural stem cells and CSCs and have a quantitative assay for self-renewal. Finally, we have ready access to the shRNA library through the University of Massachusetts Facility. Devanand Sarkar, M.B.B.S., Ph.D. Malignant glioma is one of the most virulent neoplastic diseases, with a median survival rate of only 10 to 12 months, because of its profound resistance to conventional therapies such as chemo- and radiotherapy. As such detailed understanding of the molecular pathogenesis of the disease and development of targeted more efficacious therapy based on the garnered knowledge is mandatory for improving long-term survival of the patient with the ultimate aim of establishing a “cure”. We recently observed that Astrocyte Elevated Gene-1 (AEG-1), a HIV-I- and TNF-α- induced gene, is overexpressed in >95% of human malignant glioma samples when compared with normal human brain. AEG-1 cooperates with Ha-Ras to augment transformed phenotype of normal immortal cells. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. These findings indicate that AEG-1 might play a crucial role in the pathogenesis of malignant glioma. The augmentation of tumor migration and invasion is mediated by activation of NF-κB signaling pathway by AEG-1. AEG-1 is predominantly localized in the endoplasmic reticulum. Upon TNF-α treatment and when overexpressed AEG-1 translocates to the nucleus where it interacts with the p65 subunit of NF-κB. AEG-1 protein contains three putative nuclear localization signals. However, the importance of the nuclear translocation and p65 interaction in mediating the tumor-promoting effects of AEG-1 remains to be determined. The long-term objective of the present proposal is to unravel the molecular mechanism of malignant glioma generation and progression and to employ the garnered information for developing novel therapeutic strategies to accelerate progress toward more effective treatment of malignant diffuse glioma tumors. The immediate objective of the present proposal is to elucidate in detail the molecular mechanism of AEG-1 function, especially in the context of NF-κB activation and develop a gene therapy approach for malignant glioma by targeted inhibition of AEG-1 expression. The central hypothesis of the present proposal is that translocation of AEG-1 to nucleus and interaction of AEG-1 with p65 subunit of NF-κB is crucial in mediating its tumor progression properties and direct inhibition of AEG-1 expression might be an effective approach for gene therapy of malignant glioma. The hypotheses will be evaluated by two specific aims: (1) To identify the domains of AEG-1 that are crucial in mediating its function and interaction with NF-κB p65. Different deletion and mutant constructs of AEG-1 will be generated and their ability to interact with p65 and augment growth and invasion will be analyzed. (2) To develop a gene therapy approach for malignant glioma by targeted inhibition of AEG-1. A lentivirus expressing AEG-1 siRNA will be created and its efficacy in inhibiting growth of human malignant glioma xenografts will be evaluated in athymic nude mice. Successful completion of our proposed studies will provide better insights into the molecular mechanism of AEG-1 fuction and will significantly extend the mission of Goldhirsh Foundation by opening up new and effective avenues for malignant glioma treatment. Charles Stiles, Ph.D. The most common form of malignant glioma in humans is termed “primary glioblastoma”. These tumors are characterized by amplified/mutated EGFR but are generally wild type with respect to P53 status. The genetic integrity of P53 in primary glioma is at odds with the aggressive growth of these tumors and their notorious resistance to radiation and cytotoxic drugs. In preliminary studies, we have found a potential resolution to this paradox. We show i) that essentially 100% of the CD133 positive stem cells that underlie malignant glioma in humans express the bHLH transcription repressor OLIG2, ii) that OLIG2 is essential for growth in a “genetically relevant” murine model of primary glioma and iii) that OLIG2 suppresses expression of P21 - a key cell cycle inhibitor gene that is an inducible target gene of P53. Collectively, these observations suggest that expression of OLIG2 in glioma stem cells may be a surrogate for the genetic deletions of P53 that are seen in a wide variety of other solid tumors in adults. This hypothesis makes a series of testable predictions that we would like to address in studies with human tumor stem cells and genetically accessible murine models of human glioma. As a first step in our analysis of this potential “OLIG2:P53 signaling axis” we want to define the molecular mechanism whereby P53 suppresses expression of P21 and display the functional consequences of P21 suppression. Using P21 reporter constructs we will map the OLIG2 binding site by deletion analysis and validate the binding site by mutational analysis. Using P21 knockout mouse strains, we will test the prediction that P21 is epistatic to OLIG2 for replication competence of tumor stem cells.
Wei Zhang, Ph.D. Gliomas are diseases with multiple genetic and epigenetic alterations. These changes work in concert in a coordinated fashion in cancer development and progression. Genomic systems biology is an emerging discipline in which genomic biology and engineering approaches are integrated in order to provide a coherent understanding of the underlying interactions among the myriad components. This new discipline promises to transform the practice of medicine from a reactive one to a predictive one. In this regard, we have recently developed a mathematical model termed Probabilistic Boolean Networks (PBNs) and constructed a gene network using glioma transcriptome data. In the proposed research program, we will use the RCAS-tva mouse model system to functionally validate the relationships revealed by our mathematical modeling analysis between IGFBP2 and ILK. Specifically, we will test the hypothesis that ILK functions in the same IGFBP2 pathway in gliomagenesis. We will investigate whether ILK activation is required for IGFBP2 function in gliomagenesis when combined with either PDGFb or K-ras. Through these studies, we will gain deeper knowledge into a key pathway of gliomagenesis and potential targets for intervention. We will also gain insight into the validity of mathematical modeling by PBN analysis, and the level of confidence that we can place on the new relationships revealed by modeling. We envision a new paradigm through which mathematical modeling provides cancer biologists with functional maps to follow in their effort to understand biological systems and the ways in which these systems are deregulated in cancer and the key nodes in the systems for therapeutic intervention.
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