Ileana Cristea - US grants

17-1A; 2H-1,3,2-Oxazaphosphorin-2-amine, N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide; 2H-1,3,2-oxazaphosphorin-2-amine, N,N-is(2-chloroethyl)tetrahydro-,2-oxide; Advanced Cancer; Advanced Malignant Neoplasm; Biological; CRISP; CTCAE; CTX; CYCLO-cell; Carloxan; Cell Surface Glycoprotein TROP-2; Ciclofosfamida; Ciclofosfamide; Cicloxal; Clafen; Claphene; Clinical; Common Terminology Criteria for Adverse Events; Common Toxicity Criteria; Computer Retrieval of Information on Scientific Projects Database; Cycloblastin; Cycloblastine; Cyclophospham; Cyclophosphamide; Cyclophosphamidum; Cyclophosphan; Cyclophosphane; Cyclophosphanum; Cyclostin; Cyclostine; Cytophosphan; Cytophosphane; Cytoxan; Dose; Dose-Limiting; Drug Kinetics; Drugs; ECAM; EGP-2; EGP40; EGP40 protein, human; EPG-2 antigen, human; Endoxan; Endoxana; Enduxan; Ep-CAM; Ep-CAM protein, human; EpCAM; EpCAM protein, human; Epithelial Cellular Adhesive Molecule; Epithelial Glycoprotein 40; Fosfaseron; Funding; GA733-1 Antigen; GA733-1 antigen, human; GA733-2; GA733-2 Antigen; GA733-2 antigen, human; Gastrointestinal Tumor-Associated Antigen 1; Gastrointestinal Tumor-Associated Antigen 2; Genoxal; Genuxal; Grant; Institution; Investigators; KSA Glycoprotein; KSA antigen, human; Label; Ledoxina; M1S1 Protein; M1S1 protein, human; M4S1 Protein; M4S1 protein, human; MK-1 antigen, human; MK1 antigen, human; MOv-16 protein, human; Medication; Membrane Component, Chromosome 4, Surface Marker 1; Mitoxan; NCI; NCI Organization; NIH; NR-LU-10 Antigen; National Cancer Institute; National Institutes of Health; National Institutes of Health (U.S.); Neosar; Pancreatic Carcinoma Marker Protein; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacokinetics; Phase; Procytox; Protocol; Protocols documentation; Reaction; Research; Research Personnel; Research Resources; Researchers; Resources; Safety; Sendoxan; Source; Syklofosfamid; T-16 antigen, human; TACSTD1; TACSTD1 protein, human; TACSTD2; TACSTD2 protein, human; TROP-1; TROP-1 protein, human; TROP-2; TROP-2 protein, human; TROP1 antigen, human; TROP1 protein, human; TROP2 protein, human; Toxic effect; Toxicities; Tumor-Associated Calcium Signal Transducer 1; Tumor-Associated Calcium Signal Transducer 2; United States National Institutes of Health; Zytoxan; cluster-2 antigen, human; drug/agent; epithelial cell adhesion molecule; epithelial glycoprotein 40, human; epithelial glycoprotein-2, human; human TACSTD1 protein; human TACSTD2 protein; tumor-associated calcium signal transducer 1, human; tumor-associated calcium signal transducer 2, human

DESCRIPTION (provided by applicant): With a genetic capacity of only 104 bp, HIV has triggered the devastating acquired immunodeficiency syndrome (AIDS) epidemic. In large part, the refined mechanisms that control the fate of cells invaded by the pathogen remain unknown. To understand the disease at the molecular level and develop therapies we are developing new approaches to reveal the nature of these temporal and spatial protein interactions. These methods entail a rapid cryolysis-based strategy that uses a single fluorescent tag (GFP) to both visualize viral proteins in live cells and capture their interacting partners during the course of infection. Our preliminary work on HIV-1 host protein interactions demonstrates that chromatin remodeling complexes are among other cellular functions modulated by the virus. This finding suggests that viruses have developed mechanisms to control host gene expression by usurping these complexes. Indeed, recent reports have shown that histone deacetylases may mediate the HIV latency. We propose to initiate a comprehensive analysis of the known eleven histone deacetylases during the course of HIV-1 infection. To gain insights into the location, composition, structure, and function of these multi-protein complexes, we will merge novel proteomic approaches with molecular biology, virology, and biochemistry methodologies. Using human cell lines expressing HDAC fused to GFP, we will visualize and isolate these chimeras, first in uninfected cells, and then upon HIV-1 infection. The state-of-the-art mass spectrometric configuration in my laboratory is the first of its kind, combining speed of analysis with the robustness of MALDI (matrix assisted laser desorption ionization), the enrichment in the linear ion trap, and the high accuracy measurement in the Orbitrap. This configuration is perfectly suited for the analysis of macromolecules. This comprehensive multidisciplinary analysis of HDAC complexes will provide insights into the host and viral processes that determine the fate of a cell and the ultimate outcome of infection.

DESCRIPTION (provided by applicant): Dengue and yellow fever virus are significant human pathogens that cause hemorrhagic fever and fatalities. Currently, there are no approved antivirals for these viruses. The development of antivirals for RNA viruses has proven to be particularly difficult due to the rapid emergence of drug resistant viruses. This is primarily due to the error prone nature of the polymerase, which results in rapid evolution of the virus under selective pressure. Targeting a host factor for which the virus does not have any genetic control over should decrease or prevent development of resistant mutants either because the virus cannot function without the host factor or because more mutations are required to confer resistance. Towards this goal we have developed a robust method to identify host factors that are bound to the viral RNA during viral amplification i cell culture. We will use this method to identify host factors that are bound to the dengue virus and yellow fever virus RNA. We will identify which host factors will be good targets to develop antiviral targets. In addition, we will also determine the step in the viral life cycle the host fators participate in and the protein-protein and protein-viral RNA interactions for that host factor during viral infection. These studies will address the mechanism of the host factor in viral amplification and enable development of a high-throughput assay fro inhibitors of the host factor.

? DESCRIPTION (provided by applicant): The ability of the mammalian immune system to recognize pathogenic DNA, such as DNA from viruses, is essential for the onset of intrinsic and innate immune responses. This recognition is accomplished by specialized cellular proteins called DNA sensors, which bind foreign DNA and elicit the secretion of cytokines to alert neighboring cells and inhibit the spread of infection. Until recently, the sensing of foreign DNA was thought to occur only in subcellular compartments normally devoid of DNA. However, this long-standing belief failed to explain how the cell detects nuclear-replicating DNA viruses. Our lab's recent characterization of the first identified nuclear DNA sensor, IFI16, has helped to firmly establish the concept of nuclear sensing. We demonstrated that IFI16 functions to sense herpesviruses, including the important pathogens, human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) (Li et al. PNAS 2012; Li et al. Cell Host Microbe 2013). The discovery that sensing can occur in the nucleus opens a new direction for research in immunity that will increase our understanding of how balanced immune responses work to maintain a healthy system and how their misregulation leads to immune disorders, cancers, and virus-induced morbidity and mortality. However, a fundamental question that has yet to be answered is how the immune signal is propagated from the nucleus. We found that, while IFI16 remains nuclear during the early stages of HCMV and HSV-1 infections, the IFI16-mediated induction of antiviral cytokines requires the endoplasmic reticulum adapter protein STING-a hub for DNA sensing pathways. The requirements for eliciting an immune response from the nucleus and the mechanism of immune signal propagation to the cytoplasm remain elusive. Our proposal will address these important questions. First, we will characterize the mechanism by which IFI16 rapidly co-localizes with viral DNA in the nucleus. We have discovered that IFI16 is recruited to promyelocytic leukemia (PML) nuclear bodies (NBs) following infection. PML-NBs have been implicated in antiviral response and shown to localize to origins of replication of herpesviruses. Using a multidisciplinary approach, we will test our hypothesis that PML-NBs function as interaction centers where IFI16 concentrates its binding of viral DNA, setting the stage for immune signal initiation. Second, we will use molecular biology, biochemistry, optogenetics, and live cell imaging to define the IFI16 properties required for initiating nuclear immune signals. We hypothesize that signal initiation requires IFI16 oligomerization via its pyrin domain at PML-NBs. Third, we will determine how the nucleus-derived immune signal is transmitted to the cytoplasm. We discovered that IFI16 interacts with interferon-inducible IFIT proteins, which shuttle to the nucleus upon infection. We propose mechanistic studies to test our hypothesis that IFI16-dependent immune signaling is relayed by such interactions to the STING hub. Collectively, our results will characterize a newly discovered aspect of immunity, nuclear sensing, by defining the fundamental mechanisms required for immune signal propagation.

PROJECT SUMMARY/ABSTRACT We request partial funding support for the Conference of the United States Human Proteome Organization (US HUPO) to be held at the Hilton Washington DC/Rockville Hotel & Executive Meeting Center in Bethesda/Rockville, MD on March 3-6, 2019. This meeting has established itself as a highly valued contribution to the interdisciplinary scientific field of proteomics, with a particular focus on current technologies in the field and their application to solving biological and clinical questions relevant to human health. The topics covered at the US HUPO conference reflect the multiple disciplines, strategies, and technologies encompassing the proteomics field, and their use to address complex biomedical problems and enable new discoveries aimed at advancing diagnosis, prognosis and treatment of human disease. The title of the conference, ?Proteomics at the Frontiers of Biology and Medicine, encapsulates this interplay between technology (e.g. mass spectrometry, bioinformatics, clinical/cellular assays) and biological discovery inherent to the proteomics field. The 2019 conference co-organizers are Ileana Cristea (Princeton University), Steve Carr (Broad Institute), and David Fenyo (New York University). The topics of the oral sessions have been carefully selected to provide attendees exposure to the latest proteomic technologies and their application to biomedical research questions ? in particular in the fields of cancer, infectious disease, immunity, microbiome, aging, and clinical diagnostics and assays. Oral presentation sessions with biological and clinical focus include: Glycoproteomics in Biology and Medicine, Infectious Diseases, Immunity and the Microbiome, Cancer Early Detection and Prevention, Metabolism and Disease, Protein Proteoforms in Health and Disease, Aging and Neurological Diseases, and Posttranslational Regulation and Signaling. Additionally, sessions dedicated to technology developments include Multi-Omics, Proteome Organization in Space and Time, Advances in Technology, Structural/Chemical Proteomics, and Informatics: Emerging/ New Approaches. Poster sessions will offer a less formal format for presentation and discussion of scientific topics. Organized social activities will provide opportunities for further discussion and collaborative networking between attendees. A Business Meeting will provide an open forum to hear about the mission of US HUPO and give feedback. Attendance is expected to be 350-400, including 100- 150 students/postdocs. A special effort will be made to recruit minority and industrial participants, and also provide a venue for young investigators to present their work ? through mechanisms such as ?lightning? talks. Funding requested through this R13 mechanism will help support minority and young investigator participation in the conference. Short courses and workshops will also be offered to either train attendees in advanced proteomic technologies or provide a forum for discussing career options and other timely scientific topics.

The Genetics and Molecular Biology (GMB) Training Program at Princeton University educates carefully selected individuals for the research, teaching, and advocacy needs of the nation. The GMB Training Program brings together the multi-disciplinary life science community at Princeton, which has a 57-member training faculty who are currently mentoring 126 graduate students, 103 postdoctoral fellows, and 109 undergraduate majors. Our program is highly collaborative and we train our students with the skills to become leaders in any field they choose. The 36 training faculty from the Department of Molecular Biology include 6 with joint appointments in the Lewis Sigler Institute for Integrative Genomics (LSI) and 5 with joint appointments in the Princeton Neuroscience Institute (PNI). The GMB training program also includes 5 faculty from Chemistry, 6 from Chemical & Biological Engineering, 3 from Ecology & Evolutionary Biology, 2 from Computer Science, 2 from Physics, 1 from Mechanical & Aerospace Engineering and 2 Institute professors (1 from LSI and 1 from PNI). The faculty provides expertise in diverse biological systems and offers training in genetics, genomics, biochemistry, biophysics, structural biology, cancer, cell biology, development, microbiology, immunology, and neuroscience. All of the faculty focus on model systems appropriate for training on a Genetics and Molecular Biology training grant. We receive approximately 350 applications to our graduate program per year and we attract some of the best students in the country. Research is performed in well-equipped laboratories with support from state-of-the-art core facilities. The training program consists of formal course work, laboratory rotations, a general exam, thesis research, thesis committee meetings, Individual Development Plans, and an array of special activities that enhance the formal education and provide our students with expert speaking and writing skills and exposure to the full breadth of career possibilities. In addition, trainees gain teaching experience and receive broad training in responsible conduct of research. We have developed a Diversity Program that is highly successful in identifying and recruiting students from groups that are under-represented in the life sciences. Indeed, our gender and racial demographics match that of the nation and we have sustained those demographics for nearly a decade. As part of our Mentorship Program, we have developed activities that benefit all students. Routine evaluation of the GMB Training Program involves both faculty and students and enables us to identify needs for new courses, policies, and activities that keep the program fresh. Its success is best judged by the productivity and success of our graduates, more than 90% of whom are actively engaged in science-related careers. Our GMB program thus nucleates the vibrant multi-disciplinary research community at Princeton and delivers scientists who have been broadly trained and provided with the interdisciplinary perspective and cross-disciplinary skills they need to solve tomorrow's problems.