2009 Innovative Research Grant 12-Month Progress Reports
An Emerging Tumor Suppressor Pathway in Human Cancer
Fernando D. Camargo, Ph.D., Children’s Hospital Boston
Hippo is a novel biochemical pathway that can regulate organ growth. It seems to work by preventing further cell division once organs have reached their proper size. Our group believes that Hippo signals participate in a novel and powerful “checkpoint” that restricts cell proliferation and activates cell death. Such a checkpoint would normally enable an organ to “know” its size and stop growing when the appropriate dimensions are achieved. A checkpoint mechanism would also serve to suppress the unchecked cell proliferation that characterizes tumor growth. Our proposal aims to study the existence of this checkpoint and to examine its role in tumor growth. In Aim 1, we proposed to test this hypothesis by using genetically engineered mice in which Hippo signaling can be turned on or off. For Aim 2, we proposed to identify novel proteins and small molecules that can modulate Hippo signaling in cultured cells and that could eventually be used to develop therapies for cancer.
For Specific Aim 1, we have found that inactivation of the Hippo pathway in the skin of mice leads to a expansion of the stem cells that normally form and maintain the skin, and this eventually leads to the development of invasive squamous cell carcinomas. We have made similar observations in skeletal muscle, where inactivation of the Hippo pathway leads to the amplification of the muscle-specific stem cells, and the subsequent development of invasive rhabdomyosarcomas. Studies are ongoing now to test whether over-activation of this pathway can lead to the suppression of tumor growth or regression of an established tumor.
For Specific Aim 2, we have finalized a genetic screen to identify kinases or phosphatases (a specific subset of proteins in the human genome) that can control Hippo signaling. We have identified approximately 40 molecules that can have important effects on Hippo activity. We are currently investigating how these proteins modulate Hippo signaling and testing whether these are mutated in human cancer. Additionally, we have started a genome-wide screen, and we have identified one molecule, alpha-catenin, a known tumor suppressor in the skin that is a direct regulator of Hippo signaling. Thus, our results would argue that these tumors would benefit from therapies inhibiting Hippo signaling.
Modeling Ewing Tumor Initiation in Human Neural Crest Stem Cells
Elizabeth R. Lawlor, M.D., Ph.D., Children’s Hospital Los Angeles
Tumors often look like tissues that have not developed normally. In fact, some tumors arise when abnormalities in the DNA of normal stem cells creates changes that lead to the formation of malignant rather than normal tissues. We are studying whether changes in DNA methylation contribute to the genesis and growth of Ewing sarcoma family tumors (ET), highly aggressive tumors that primarily affect children and young adults. Precise control of DNA methylation is essential for the creation of normal adult tissues. We believe that ET arises from normal neural crest stem cells (NCSC) in which the expression of an abnormal gene, EWS-FLI1, induces changes to DNA methylation that result in cancer formation.
Because NCSC are very rare cells very little is known about their DNA methylation profiles. In Aim 1 of our proposal we are studying normal human NCSC before, during and after differentiation. In the first year we developed the means to reproducibly generate high quality NCSC and sufficient numbers of terminally differentiated cells for whole genome DNA profiling. The first set of DNA samples from these studies is currently being processed at the USC Epigenome Center. Replicate studies will be performed in year two and data from these assays used to define what methylation looks like during normal neural crest development.
In our second aim we will assess how EWS-FLI1 alters the normal pattern of DNA methylation. DNA samples from NCSC that were grown for up to 30 weeks in the presence of EWS-FLI1 have been sent to USC for analysis. We are also profiling the whole-genome methylation status of five established ET cell lines along side these NCSC samples. In year two and three of the proposal we will repeat these studies and also test cells that have been grown in low oxygen concentrations, a condition that better represents the environment of tumor cells in the patient. These studies will help us to determine which disruptions to DNA methylation are mediated by EWS-FLI1 and important for the creation of ET cells.
In Aim 3 we are testing whether abnormal DNA methylation in ET cells can be corrected by treatment with decitabine, a drug that inhibits DNA methylation and/or by turning off EWS-FLI1. In year one we performed experiments to determine if decitabine treatment in vitro would lead to re-expression of three abnormally silenced genes. Our data show that, in some cells, decitabine is effective at achieving this. In the final months of year one we initiated in vivo studies in which mice harboring ET tumors were being treated with decitabine. It is our hope that the drug will reverse the abnormal methylation patterns and inhibit tumor growth. Over the next year we will also to test to what extent turning off EWS-FLI1 mimics the effects of decitabine. These studies will determine if how DNA methylation contributes to continued growth of established tumors and whether targeting DNA methylation might be an effective therapeutic option.
Cancer Cell Specific, Self-delivering Pro-Drugs
Matthew Levy, Ph.D., Albert Einstein College of Medicine of Yeshiva University
We are developing a system of nucleic acid-based molecules that can home to and deliver anti-cancer drugs specifically to tumorigenic cells in the body. In our first six months of funding, we synthesized a series of drug- laden oligonucleotides and aptamer molecules with different types of modifications and/or different drugs incorporated within. Over the past year, we have been performing tests to find the optimal drug/vehicle combination using one of two core aptamers as the basis for targeting: one that binds the human transferrin receptor (TfR; a receptor commonly overexpressed on different types of cancer cells) and one that binds the prostate specific membrane antigen (PSMA; a marker of prostate cancer). To ease with synthesis of the drug- bearing aptamers, we minimized the two aptamers to their core functional regions through a series of selection and deletion experiments and binding assays. We confirmed both sets of minimized aptamers for their ability to bind to and internalize into the appropriate receptor-bearing cells.
We have been testing the toxicity of our various drug-bearing oligonucleotides and aptamers on different cancerous cell lines as well as the individual free drugs. Based on some initial results, we have also redesigned and synthesized a number of other constructs along the way. Interestingly, the different drugs we have chosen to use in our assays appear to have markedly different effects on the different types of cancerous cells. As such, we have been preparing new reagents and cell lines in preparation for our in vivo work.
Targeted Inhibition of BCL6 for Leukemia Stem Cell Eradication
Markus Müschen, M.D., Children’s Hospital Los Angeles
Despite significant advances in the treatment of leukemia over the past four decades, the rate of long-term survival has reached a plateau. Large numbers of leukemia patients still die, mostly because of relapse and drug- resistance. These two clinical problems were recently attributed to the persistence of leukemia stem cells. If a therapy succeeds in eradicating leukemia stem cells, renewed initiation of the disease (relapse) is no longer possible. Therapeutic progress in recent clinical trials has likely been stalled, partly because current chemotherapy approaches target proliferating bulk leukemia cells rather than non-dividing leukemia stem cells.
We have now discovered that BCL6, a factor known to play a central role in lymphomas, also plays a key role in the maintenance of leukemia stem cells. Since leukemia stem cells represent the origin of relapse and drug-resistance in leukemia in many cases, the identification of BCL6 as a target for leukemia stem cell eradication holds great promise. BCL6 is a master regulatory factor that controls the production of many different important genes. BCL6 was not previously known to be involved in leukemia. In preliminary studies for this proposal, we have discovered aberrant expression of BCL6 as a central component of a fundamentally novel pathway of leukemia stem cell self-renewal and drug-resistance in a wide array of human leukemias, some of which are still difficult to treat. In these leukemias, drug-treatment results in aberrant production of BCL6 by the leukemia cells, which appears to allow leukemia stem cells to self-renew and become resistant against chemotherapy.
Recently, a drug has been developed that can attach to BCL6 and block its cancer- causing activities. We found that this BCL6 inhibitor, called RI-BPI, has strong cooperative activity when combined with conventional chemotherapy. This opens up a powerful new therapeutic strategy for leukemia stem cell eradication through targeted inhibition of BCL6. Based on the discovery of BCL6 as a key component of a novel pathway of drug-resistance and stem cell self-renewal in a wide array of leukemias, we propose three Aims to develop these findings towards application in patient care: (1) To test the hypothesis that BCL6 is critical for leukemia-initiation and relapse of leukemia, (2) To determine the frequency and appearance of BCL6-dependent leukemia stem cells in human leukemia samples and (3) To validate the role of the BCL6 inhibitor RI-BPI as a novel therapeutic agent for targeted eradication of leukemia stem cells. Since RI-BPI is currently going through the process of approval for use in clinical trials, we expect to be able to test the power of this approach in clinical trials by the end of the funding period.
Identifying Solid Tumor Kinase Fusions via Exon Capture and 454 Sequencing
William Pao, M.D., Ph.D., Vanderbilt-Ingram Cancer Center/Vanderbilt University
Cells rely upon molecular switches to carry out normal functions. One class of switches is called tyrosine kinases (TKs). In a highly simplified model, when TKs are ‘on’, cells divide; when TKs are ‘off’, cells stop dividing. In some cancers, TKs have become altered, leading to abnormal signaling. One major type of alteration is called a ‘fusion’. A fusion ‘fuses’ together a part of a cellular protein (that normally has another function in the cell) with the signaling portion of the TK molecule. Instead of turning ‘off’ and ‘on’ in a tightly regulated manner, TKs fusions are ‘on’ all the time, tricking cells into constantly dividing. If one blocks the abnormal signals from a TK fusion with a drug, cancer cells can die, and patients can enormously benefit. The best example of this idea involves the drug Gleevec, which targets a TK fusion (called BCR-ABL) in patients with a type of leukemia.
Thus far, only a limited number of TK fusion proteins have been identified because experimental procedures for their identification are inadequate, especially in solid tumors that contain cells from multiple tissue types. As part of this grant, we developed a novel method to identify TK fusion proteins using DNA from any type of tumor. After demonstrating the feasibility of this platform on tumor cell lines, we used the results of the pilot study to create an improved more powerful platform with streamlined workflow and computational analysis. Using the newer kinase fusion design, we have screened multiple tumor samples and have identified a novel fusion from one patient sample thus far. We are currently exploring how this TK fusion contributes to the development of cancer and can be treated with existing targeted therapies. The screening of multiple other tumor samples with no known targets for therapy is ongoing. Finally, we have mapped the genomic sequences of multiple known kinase fusions in order to gain further insights into the DNA structures of these cancer-specific alterations.
Therapeutically Targeting the Epigenome in Aggressive Pediatric Cancers
Charles M. Roberts, M.D., Ph.D., Dana-Farber Cancer Institute
Recently there has been a growing realization that some of the critical changes in gene expression required for the development of cancer do not arise via genetic mutations in DNA but rather are ‘epigenetic’ changes that affect gene expression indirectly by affecting DNA packaging. The SWI/SNF complex controls the protein support structure that surrounds key growth genes and is thus at the heart of this epigenetic regulation. Mutations in SNF5, a core subunit of the SWI/SNF complex, are present in the large majority of malignant rhabdoid tumors (MRT), a highly lethal cancer that occurs in kidney, brain, and soft tissues of young children. Inactivating mutations in SNF5 have recently been found to occur in a variety of other cancers as well including epithelioid sarcomas, small cell hepatoblastomas, undifferentiated sarcomas, chondrosarcomas, familial schwannomatosis, and renal medullary carcinomas. Mutation in SNF5 is also the basis of an inherited cancer predisposition syndrome and mutation of another SWI/SNF sub-unit that is frequently found in adult lung cancers. As the cancers that arise following Snf5 loss appear to be largely driven by the epigenetic consequences, we hypothesize that these cancers will be particularly susceptible to drugs that interfere with epigenetic mechanisms of gene regulation. The experiments in this proposal were designed to reveal the underlying mechanisms by which SNF5 loss affects gene expression and thereby causes cancer with a goal of identifying improved therapies that can be rapidly translated into patients.
We have continued to make substantial advances during months seven through twelve of funding from Stand Up 2 Cancer and have met and exceeded our milestones. We have generated an animal model with which we are now specifically testing whether inactivation of a small RNA molecule miR-21 can block the growth of cancers driven by Snf5 loss. Unfortunately, we now have some mice that have developed cancers despite absence of miR-21, suggesting that this may not be sufficient to block tumor formation. We are awaiting further data to determine whether the absence of miR-21 has any effect on the rate of tumor formation caused by Snf5 inactivation. We are in the process of collecting systematic data on the effects of Snf5 loss upon other histone acetylation and DNA methylation so that we can determine whether drugs that target these related modifications may be therapeutically beneficial. Related to this work, we have identified an antagonistic relationship between Snf5 and another epigenetic regulator, Polycomb and have now shown that inactivation of Ezh2 can completely block tumor formation driven by Snf5 loss. We published this finding in Cancer Cell in October and were subsequently contacted by multiple pharmaceutical companies that wish to work with us on this, a collaboration that we are now pursuing.
Endogenous Small Molecules that Regulate Signaling Pathways in Cancer Cells
Rajat Rohatgi, M.D., Ph.D., Stanford University
A major goal in cancer biology is the comprehensive understanding of signals that drive the growth and spread of cancer cells. My long-term goal is to develop methods to isolate new small molecules that play a role in cancer signaling and then to identify the proteins that interact with these small molecules. Such small molecule- protein pairs are likely to be particularly good drug targets in oncology. To develop tools for this endeavor, we are focusing on the identification of small molecules that regulate the “Hedgehog” signaling circuit. Damage to this circuit has been shown to drive the development of a large number of adult and childhood cancers. We have taken three complementary trajectories to tackle this project. First, we are searching for new small molecules present in crude homogenates of cells and tissues that can modulate the Hedgehog pathway. We are also taking advantage of our previous observation that molecules related to cholesterol, called oxysterols, can activate the Hedgehog pathway. To understand how these enigmatic molecules work, we are attempting to find the proteins through which oxysterols function. In addition to continuing to develop the approaches described in the last progress report, we have made substantial progress on understanding how oxysterols function at a molecular level in the Hedgehog pathway. This work will be submitted for publication shortly.
Genetic Approaches for Next Generation of Breast Cancer Tailored Therapies
José M. Silva, Ph.D., Columbia University Medical Center
Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as low toxicity and more efficient alternatives to classical treatments. An alternative promising approach for the design of these personalized therapies is the use of genetic synthetic lethal interactions. These occur when two genetic alterations that are individually innocuous appear in the same cell, causing growth inhibition. This concept can be exploited to identify genes that, when inhibited, exclusively reduce the viability of tumor cells that carry a preexisting genetic lesion.
Recently, RNA interference (RNAi) technology has emerged as a very powerful approach to attenuate the expression of any chosen gene. Thus, we envision using RNAi to identify genes that, when attenuated, exclusively reduce the viability of tumor cells carrying specific genetic lesions without affecting normal cells. During the last years, my group has pioneered the development of RNAi based genetic tools for studies in mammalian cells. This technology represents a unique opportunity to identify synthetic lethal effects with major cancer alterations. In this project we propose to apply our state- of-the-art technology to uncover synthetic lethal interactions with the major breast cancer genes.
Our proposal is divided into three specific aims that represent the transition from target discovery and validation to mechanistic characterization.
Specific Aim 1. Identify genes that interact with the major breast cancer alterations to produce synthetic lethality in vitro (1st year): During the first year of this project, we proposed to complete four genome-wide RNAi screens in vitro to identify target genes that, upon inhibition, reduce the cell viability in breast cancer cells with any of the major breast cancer alterations; ErbB2, c-Myc, Cyclin-D or RB. In our pre-defined milestones we estimated that two of the four RNAi screens would be completed during the first six months and the rest during the second semester of the first year. At this point, the four genome-wide RNAi screens have been completed and analyzed.
Specific Aim 2. Model selected lethal interactions in vivo (1st and 2nd year): Completion of the above mentioned screens has provided us with a list of candidates that will be further validated in vivo by candidate driven RNAi screens in mouse models during the second year of the award.
Specific Aim.3- Initial characterization of the molecular mechanism of the genetic lethality (2nd and 3nd year): Upon completion of Aim 2, we will select the most promising (2-3) target genes to investigate the biology of the lethal phenotype in more detail.
Modulating Transcription Factor Abnormalities in Pediatric Cancer
Kimberly Stegmaier, M.D., Dana-Farber Cancer Institute
The identification of cancer-promoting proteins has advanced at a rapid pace with the development of new genomic technologies. However, the majority of these proteins have been considered “undruggable” by traditional pharmacological approaches. One class of “undruggable” proteins critically important in both pediatric and adult malignancies is proteins that bind to DNA, the so-called transcription factors. One such protein historically considered “undruggable” is the cancer-promoting protein in the pediatric solid tumor Ewing sarcoma, EWS/FLI. Ewing sarcoma is the second most common cause of bone cancer in children and young adults, and the EWS/FLI protein can be identified in over 80% of tumor samples. While progress has been made in treating patients with localized Ewing sarcoma, little advancement has been made for those patients with metastatic or recurrent disease. Current regimens employ cytotoxic chemotherapy, and targeted treatments are only beginning to emerge. New approaches to treating this disease are needed.
One approach to tackling this challenging class of proteins is to develop alternatives to traditional drug discovery. Instead of using standard approaches to chemical screening, we developed a genomic approach using DNA microarrays (“gene chips”) to characterize the genes that are turned on or off in the presence or the absence of the Ewing sarcoma protein. Over the past 12 months, we have developed a 123-gene signature that distinguishes Ewing sarcoma cells with the active versus inactive EWS/FLI protein. We next adapted this signature to a robust assay, which can be measured in 384-well high-throughput format. We confirmed that the signature identifies the active versus inactive EWS/FLI protein across a number of different Ewing sarcoma cell lines with genetic knockdown of EWS/FLI. We next performed a successful pilot screen of 1600 bioactive chemicals and FDA-approved drugs. We identified two chemical hits, which induce the EWS/FLI inactive expression signature. We have just completed a large-scale screen of over 10,000 small molecules. This chemical collection consists of bioactive molecules (including many FDA- approved drugs), natural products, and novel chemicals created by an approach called diversity oriented synthesis. This new approach to chemical synthesis is expected to yield chemicals with greater similarity to those produced in nature, with the hope that they will also have greater biological activity than compounds produced by standard approaches to chemical synthesis. We have prioritized 150 top scoring compounds, which we are actively testing in a secondary screen across multiple doses.
Over the next funding period, we will identify those chemicals which reproducibly induce the EWS/FLI inactive gene signature. For those chemicals confirmed to inactivate the EWS/FLI signature we will next measure their effects on various aspects of Ewing sarcoma cells, including the effects on cell growth/death and the cancerous properties of the tumor cells. Chemicals that emerge from this screen will be used in the laboratory as tools to identify new drug targets for Ewing sarcoma and as lead compounds for clinical trial development. Moreover, if this work is successful, it can be extended to many more tumor types both in adults and in children (e.g. prostate cancer and leukemia) where proteins similar to EWS/FLI promote the development of the tumor.
Non Invasive Molecular Profiling of Cancer via Tumor-Derived Microparticles
Muneesh Tewari, M.D., Ph.D., Fred Hutchinson Cancer Research Center
Obtaining molecular information about an individual cancer patient’s tumor would allow for the development of patient-specific treatment plans. However, obtaining tumor biopsy samples in many cases requires invasive surgery, which can be painful, disfiguring and potentially dangerous. Consequently, alternative non-invasive sampling methods are needed, and could serve as a reliable surrogate for actual tumor tissue. The approach we are pursuing takes advantage of the known fact that cancer cells release “information” into the bloodstream in the form of specific types of RNA and DNA molecules. This information is packaged into what we call tumor-derived microparticles; essentially, small parcels derived from and representative of the contents of tumor cells. The goal of this project is to develop methods to efficiently capture these particles from patient blood samples and decode the information within them in order to gain molecular information about the cancer cells from which they originated. In the recent period, our major progress includes: (i) improvement in our methods for concentrating microparticles that yielded a 400-fold improvement in our ability to see them using electron microscopy, (ii) improvement in methods to detect RNA associated with one class of microparticles, and (iii) use of the improved methods to detect the presence of a specific class of microparticles in ovarian cancer tissue samples. Moving forward we plan to continue to develop the methods needed to capture and decipher the molecular information contained within these particles. Our ultimate goal is to use a blood sample to obtain key information that will inform critical clinical decisions, such as identifying the most effective therapy for an individual patient.
A Transformative Technology to Capture and Drug New Cancer Targets
Loren D. Walensky M.D., Ph.D., Dana-Farber Cancer Institute
The goal of our SU2C project is to create a powerful, new, and versatile approach to identifying and drugging new cancer targets. To that end, we are combining a chemical technology termed “hydrocarbon stapling” that restores natural shape to bioactive peptide alpha-helices with a protein capture technology in order to trap and characterize critical targets involved in defective signaling in cancer. For the first year of funding, our goal was to chemically synthesize a pilot panel of “photoreactive stabilized alpha-helices” or pSAHs, which are the chemical tools designed to capture and characterize new cancer targets. With our first series of pSAHs in hand, we examined and optimized the sensitivity and specificity of these new tool compounds for cross-linking to discrete physiologic targets of the BCL-2 pathway.
In an SU2C-sponsored manuscript published on December 22nd in Cell’s Chemistry and Biology journal, we demonstrate our capacity to successfully and reproducibly generate pSAHs that recapitulate the structure of distinct bioactive domains and deploy them to trap, purify, and identify their natural cellular targets with high fidelity. In addition, we developed and reported a reliable method for inputiing our cross-linking data into a binding site algorithm that employs mass spectrometry and computational docking analysis to calculate model structures of the key-in-Iock binding interfaces we discover. This critical information will provide the basis for validating new protein-protein interactions for drug development and therapeutic targeting. Having defined and published the “rules” for successful production and application of pSAHs based on our year one work, we are currently expanding our arsenal of pSAH constructs to home in on key protein interactions that drive cancer. Indeed, as we enter year two of the funding cycle, the implementation phase of our technology for cancer target discovery is well underway, setting the stage for an exciting year ahead. From a laboratory development standpoint, SU2C funds have enabled us to dramatically expand our pSAH production capacity through the purchase of a Liberty microwave-enhanced peptide synthesizer, which is now installed and fully operational. The success of our proteomic approach has further motivated me to deploy laboratory funds to purchase our own Thermo L TO Orbitrap mass spectrometer, which will enable us to build a state-of-the-art infrastructure for advancing our cancer protein target discovery platform.
Functional Oncogene Identification
David M. Weinstock, M.D., Dana-Farber Cancer Institute
Despite advances in diagnosis and treatment, more than half of adults with cancers of the blood (i.e., leukemia, lymphoma and multiple myeloma) will die from their disease. One of the limitations in our current approach is that most cancer chemotherapy nonspecifically kills all growing cells, and does not target abnormalities unique to the tumor cells. Thus, the identification of specific cancer-associated abnormalities is an essential first step toward newer and more effective therapies. We developed a system to identify new targets for therapy directly from leukemia and lymphoma samples. Briefly, we isolate the many millions of pieces of genetic material from a tumor sample and then individually insert each into cells that can only grow in a special kind of culture. If one of the pieces of genetic material has a cancer-promoting effect, it allows the cells to grow in a normal culture. Thus, any cell that survives in the normal culture must contain a piece of genetic material from the tumor that has a cancer-promoting effect. We can easily identify that piece of genetic material and then confirm that it is important for the tumor’s growth. The system we developed is efficient and can be scaled up to analyze a large number of individual specimens. Using this approach, we have already discovered a new cancer protein called CRLF2 in some cases of acute lymphocytic leukemia.
The overall goal of our Stand Up To Cancer Innovative Research Grant proposal is to identify important alterations that promote the growth of other types of blood cancer. During the initial 12 months of funding, we utilized our approach to screen six types of human leukemia and lymphoma samples for new mutations. From this screen and from sequencing large amounts of DNA in the specimens, we identified multiple mutations that have never been described from tumor specimens. Of particular interest, we identified mutated versions of two proteins called kinases that can be targeted with available drugs. We are in the process of confirming that the mutations we identified contribute to tumor growth. We are also defining the frequency of these mutations in other leukemia and lymphoma specimens. At least one of the mutations is present in multiple different lymphomas of the same type. Once further confirmation is completed, we will test leukemias and lymphomas dependent on these new mutations with a panel of targeted agents to define drugs for further testing in patients. Over the next two years, we will apply the same screening approach to identify targetable aberrations in other blood cancers, including subtypes of leukemia, non-Hodgkin lymphoma and Hodgkin’s disease.
Probing EBV-LMP-1’s Transmembrane Activation Domain with Synthetic Peptide
Hang (Hubert) Yin, Ph.D., University of Colorado at Boulder
Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic core of the phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains with high affinity and selectivity. This study utilizes this cutting edge technology to study the activation mechanism of oncogenic Latent Membrane Protein 1 (LMP-1) of Epstein-Barr virus (EBV). EBV is a human tumor virus associated with a number of malignancies and lymphoproliferative syndromes. EBV’s ability to infect and immortalize B-lymphocytes underlies its contribution to cancers.
EBV’s transforming activity depends on the expression and activity of LMP-1, the viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 functions as a constitutively active Tumor Necrosis Factor Receptor (TNFR) whose activity requires the function of its hydrophobic transmembrane domain. LMP-1 most resembles the TNFR CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1’s activity is constitutive and ligand-independent. Constitutive homo-oligomerization and lipid raft association, activities of LMP-1’s transmembrane domain, play a key role in activation of downstream signaling. This proposal focuses on LMP-1 as a model membrane protein target for the design of peptide inhibitors because of LMP-1’s essential role in EBV-dependent B cell transformation, LMP-1’scontribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and EBV’s dependence on LMP- 1’s hydrophobic transmembrane domain for activity.
This study aims to develop novel prevention and treatment methods to target LMP-1’s transmembrane domain, using CHAMP-designed anti-TMD peptide antagonists as probes to study the contribution of oligomerization to LMP-1 activation, with the goal of inhibiting downstream signaling. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes; will reveal the mechanism of LMP-1’s constitutive activation of signaling; and will be applicable to the future development of novel therapeutics targeting cancers dependent on critical transmembrane proteins.
Specifically, this proposal addresses the following Aims:
1) Develop specific peptide probes targeting the TMD-mediated homo-oligomerization.
2) Answer the question as to what the role of TMD- mediated oligomerization in LMP-1 activation is.