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The Markey Scholars Conference: Proceedings Abstracts of Poster Sessions
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The Markey Scholars Conference: Proceedings CONFIGURATIONAL ENTROPY, BIOCHEMICAL COOPERATIVITY, AND SIGNAL TRANSDUCTION Paul H. Axelsen, M.D. Department of Pharmacology University of Pennsylvania Cooperativity is a common biochemical phenomenon in which two or more otherwise independent processes are thermodynamically coupled. Because cooperative processes are usually attended by changes in molecular conformation, thermodynamic coupling is usually attributed to an enthalpy-driven mechanism. In the family of glycopeptide antibiotics that includes vancomycin, however, cooperative phenomena occur that cannot be explained by conformational change. We have demonstrated that cooperativity in these systems can arise solely from changes in vibrational activity using molecular dynamics simulation. This result has important implications for much larger systems because ligand-induced modulation of periodic motions in a macromolecular system is an eminently plausible means of communicating the presence of bound ligand over long distances. Indeed, the function of large membrane proteins such as those involved in transmembrane signal transduction may actually require a mechanism based on configurational entropy changes because the enthalpy changes generally involved in the binding of small ligands are small compared to the magnitude of potential energy fluctuations one would expect in systems of this size. EMBRYONIC BEGINNINGS OF THE HEMATOPOIETIC AND VASCULAR SYSTEMS DURING MOUSE DEVELOPMENT Margaret H. Baron, M.D., Ph.D. Department of Medicine, Biochemistry and Molecular Biology Mount Sinai School of Medicine Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. “Blood islands” of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper (epiblast). These lineages arise from a layer of extra embryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-
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The Markey Scholars Conference: Proceedings 1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. We demonstrated previously that specification of these lineages requires a signal(s) secreted from the adjacent visceral endoderm (VE) and more recently that Indian hedgehog (Ihh) is a VE-secreted signal which alone is sufficient to induce formation of hematopoietic and endothelial cells. We have continued to investigate the mechanism by which Ihh activates these processes. As seen with VE, Ihh can also respecify prospective neural ectoderm (anterior epiblast) along hematopoietic and endothelial (posterior) lineages, as indicated by cell morphology, activation of specific transgenes (e.g., lacZ reporters controlled by embryonic globin, Flk1 and Cbfa2/Runx1/AML1 sequences), activation of endogenous markers of stem/progenitor cells (hemangioblasts) and more differentiated cells, and by immunostaining for PECAM1 and other proteins. Dispersed cells from recombinant human IHH-treated anterior epiblasts form primitive or definitive hematopoietic colonies in secondary cultures in the presence of appropriate cytokines, indicating that functional hematopoietic stem cells are produced, and endothelial cell sprouting is observed. As expected, downstream targets of the Hh signaling pathway (Ptch1, Smo, Gli1) are upregulated in anterior epiblasts cultured in the presence of Ihh protein. Blocking Ihh function in VE inhibits activation of hematopoiesis and vasculogenesis in the adjacent epiblast, suggesting that Ihh is an endogenous signal that plays a key role in the development of the earliest hemato-vascular system. The gene encoding Bone morphogenetic protein-4 (Bmp-4) is upregulated in the target epiblast in response to Ihh. Several more direct lines of evidence indicate that Ihh functions, at least in part, through activation of the Bmp signaling pathway. Hedgehog genes and protein are expressed by adult mouse and human bone marrow stromal cells and Ptch1 and Smo are expressed in hematopoietic stem/progenitor as well as endothelial cells. Therefore, these findings may have important implications for regulating hematopoiesis and vascular development for practical and therapeutic purposes.
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The Markey Scholars Conference: Proceedings FLUORESCENCE DETECTION TECHNOLOGIES AND BIOMEDICAL RESEARCH: PROTEOMICS, GENOMICS, IMAGING, ARRAYS, AND DISEASE Joseph M. Beechem, Ph.D. Molecular Probes, Inc. In 1981, when I started graduate school at The Johns Hopkins University (Baltimore, MD), fluorescence spectroscopy was a technique mainly practiced by biophysicists. Everyone else in my graduate school class was “flocking-to” laboratories in really hot-areas of research, which at that time was molecular biology/genetics. During my Ph.D. training, instead of learning the latest cloning technique or expression system, items such as: fluorescence polarization/anisotropy, fluorescence resonance energy-transfer (FRET), excited-state reactions, solvent relaxation, protein folding, fast kinetics, etc., formed the heart of my research. The relevance of these “arcane” spectroscopic methodologies for biomedical research was tenuous (at best). Fortunately, as modern biomedical research methodologies evolved, these “arcane” fluorescence spectroscopic techniques became some of the key detection technologies associated with much of the modern biomedical research “revolution.” Fluorescence polarization /anisotropy spectroscopy has become an essential component of high throughput homogenous drug screening and protein-protein interaction assays. Fluorescence energy-transfer (FRET) became the key technology behind molecular “beacons,” and high-sensitivity DNA microarray techniques. Ultra-high sensitivity protein folding dyes are becoming the core technology associated with proteomics platform imaging of 2-D gels. Fluorophores covalently coupled to chelating reagents formed the key technology associated with discovering the important role of calcium as a universal “currency” of intracellular signaling. The list goes on-and-on and, most importantly, many key applications of fluorescence technology to biomedical research have yet to be developed. In this presentation, data will be described for the “next generation” of fluorescence reagents and methodologies, which are being developed at Molecular Probes. From high resolution deconvolved intracellular imaging of organelles, to mass-spectroscopy/fluorescence-combined approaches for whole cell post-translational modification mapping will be presented. These (and additional) fluorescence technologies will continue to play an active role in helping biomedical research solve the major health problems facing the modern world.
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The Markey Scholars Conference: Proceedings IDENTIFICATION OF THE PROTEIN 4.2 GENE AS A DIRECT TARGET OF THE TAL1/CL TRANSCRIPTION FACTOR IN DIFFERENTIATING MURINE ERYTHROID CELLS Zhixiong Xu and Stephen J. Brandt, M.D. Vanderbilt University and Veterans Affairs Medical Centers The TAL1/SCL gene, originally identified through its involvement by a recurrent chromosomal translocation in leukemic T-cells, encodes a basic helix-loop-helix (bHLH) transcription factor essential for embryonic hematopoiesis and vascular remodeling. Although TAL1 is presumed to alter the transcription of a specific set of genes, no such targets have been definitively identified. Binding site selection assays using erythroid cell extracts have suggested that TAL1 contributes to a multi-protein DNA-binding complex that binds preferentially to a tandem E box (for bHLH proteins)-GATA motif and which also contains the zinc finger transcription factor GATA-1, the LIM domain protein LMO2, and the LIM domain binding protein Ldb1. We identified two such E box-GATA elements in the proximal promoter of the murine Protein 4.2 gene whose protein product plays an important role in maintaining the stability and flexibility of erythrocytes. To determine if transcription of this gene is regulated by such a complex, we analyzed the contributions of TAL1 and GATA-1 to Protein 4.2 DNA binding activity, promoter activity, and endogenous gene expression and in vivo occupancy of the Protein 4.2 promoter by TAL1. First, several TAL1-, GATA1-, LMO2-, and Ldb1-containing complexes were detected by gel mobility shift analysis of erythroid cell extracts using probes corresponding to either E box-GATA element in the Protein 4.2 promoter. An increase in these DNA-binding activities was observed with DMSO-induced differentiation of murine erythroleukemia (MEL) cells concomitant with expression of Protein 4.2 mRNA. Cold competitor studies and gel mobility shift assays with mutant probes indicated a requirement for both the E box and GATA sites in formation of these binding complexes and revealed an increased stability for the ternary complex relative to other E box- and GATA-binding complexes in MEL nuclear extracts. Using a novel modification of the gel mobility shift assay, it was shown that this TAL1- and GATA-containing complex could bridge in solution two double-stranded oligonucleotide probes corresponding to the two E box-GATA elements in the Protein 4.2 promoter. Reporter gene assays showed that DMSO-induced promoter activity was decreased by 75 percent and 90 percent, respectively, with mutation of either E box or GATA site, suggesting that both E box-GATA elements contribute to promoter activity and that both the E box and GATA sites within these elements are required for maximal induction of Protein 4.2
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The Markey Scholars Conference: Proceedings gene expression during MEL cell differentiation. In addition, a TAL1 expression vector increased Protein 4.2 promoter activity when cotransfected with vectors for its DNA-binding partner E47, GATA-1, LMO2, and Ldb1. Finally, an increase in endogenous Protein 4.2 gene expression and in E box-GATA DNA-binding activities was observed when TAL1 was overexpressed in MEL cells, a decrease in both was observed with enforced expression of a TAL1 mutant defective in DNA-binding or an Ldb1 mutant impaired in dimerization, and evidence for in vivo occupancy of the Protein 4.2 promoter by TAL1 was obtained through chromatin immunoprecipitation analysis. In sum, these data establish the Protein 4.2 gene as a direct target of a TAL1- and GATA-1-containing DNA-binding complex in differentiating erythroid cells. CELL SIGNALING NETWORKS IN C. ELEGANS MORPHOGENESIS Andrew Chisholm, Ph.D., Ian Chin-Sang, Mei Ding, Sean George, Bob Harrington, Tom Holcomb, Kris Larsen, and Sarah Moseley Department of Biology University of California The epidermis of the nematode C. elegans is a simple model for analyzing mechanisms of epithelial morphogenesis. The worm epidermis undergoes several distinct morphogenetic movements, including epiboly, cell intercalation, directed dilation, and invagination. We have focused on the epiboly movements required for epidermal enclosure of the embryo. The epidermis develops from a sheet of cells lying on the dorsal part of the embryo. Changes in epidermal cell shape cause the epidermis to expand laterally and ventrally, moving over neuronal substrate cells. This results in epidermal enclosure in the embryo at the ventral midline. Using genetics we have found that signaling between underlying neuronal precursors is important for normal enclosure. Mutations in the C. elegans Eph receptor VAB-1 and the ephrin ligand VAB-2/EFN-1 cause defects in neural and epidermal morphogenesis (George et al., 1998 Cell 92:633; Chin-Sang, et al., 1999 Cell 99:781). VAB-1 and VAB-2 are expressed in complementary sets of neurons during embryogenesis, and are required in neurons for epidermal morphogenesis. VAB-1 may have kinase-dependent and kinase-independent functions; vab-2 mutations synergise with vab-1 kinase domain mutations and not with a vab-1 extracellular domain mutation, suggesting that VAB-2 may mediate a kinase-independent function of VAB-1.
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The Markey Scholars Conference: Proceedings The almost complete C. elegans genome encodes one Eph receptor (VAB-1) and four GPI-anchored ephrins (EFN-1/VAB-2, EFN-2, EFN-3, and EFN-4). Mutations in the gene mab-26 cause morphogenetic defects related to but distinct from those seen in vab-1 and vab-2 mutants. We have shown that mab-26 corresponds to the fourth worm ephrin (EFN-4). Strikingly, mab-26 mutations display synthetic lethality with vab-1 and vab-2 mutations, suggesting that MAB-26 may function in VAB-1-independent signaling. Loss of function in the C. elegans LAR-like receptor tyrosine phosphatase PTP-1 causes morphogenetic defects. We have found that ptp-1 mutants display synthetic lethality with vab-1, vab-2, and mab-26 mutations. These results suggest that RPTP signaling may function in a parallel redundant pathway with Eph signaling. We are currently testing whether this interaction is specific to the Eph signaling mutants. PAS PROTEINS AND THE REGULATION OF DEVELOPMENT AND PHYSIOLOGY Stephen Crews, Ph.D. Department of Biochemistry and Biophysics University of North Carolina, Chapel Hill The central nervous system consists of a large variety of neuronal and glial cell types. Neural precursor cells are first specified and those precursors then generate distinct CNS cell types. We have studied the formation of the cells that lie along the midline of the Drosophila CNS. This work has lead to an understanding of how distinct regions of the CNS are generated and the discovery of a class of regulatory proteins that control a wide variety of invertebrate and vertebrate developmental and physiological processes. Single-minded and the control of CNS midline cell development. The CNS midline cells comprise a distinct set of functional neurons and glia, and also act as a signaling center that controls aspects of axon guidance, cell migration, and formation of epidermal, mesodermal, and neural cell types. We have broadly investigated CNS midline cell development using genetics and molecular techniques to identify and functionally analyze genes involved in establishing midline cell fate and function. The single-minded gene acts as a master regulator of CNS midline cell transcription and formation. Dorsal/ventral patterning proteins act in a concentration-dependent and cooperative mode in conjunction with the Notch signaling pathway to activate single-minded transcription in midline precursor cells.
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The Markey Scholars Conference: Proceedings The single-minded gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for all transcription and development of the midline cells. Single-minded protein dimerizes with the Tango bHLH-PAS protein. Together they enter the nucleus, bind to DNA control elements containing an ACGTG core sequence, and activate midline transcription in conjunction with additional unknown coregulators. Correct formation of the CNS midline cells involves activation of midline gene transcription and repression of lateral CNS transcription in the midline cells. Single-minded is required for both processes: it activates transcription of midline-expressed genes and the transcription of repressive factors. Single-minded continues to function throughout development. As midline precursor cells differentiate, the Single-minded: Tango protein complex interacts combinatorially with the Drifter and Fish-hook transcription factors to control midline glial transcription. Postembryonically, single-minded is expressed in the brain, including a group of neurons in the central complex that coordinate locomotion. Analysis of adult behavior using a single-minded temperature sensitive mutant shows defects in courtship and walking. Mutant flies only walk in circles—they can turn either left or right, but not both. Current work is focused on identifying genes that mediate CNS midline cell fate development, and understanding how Single-minded: Tango interacts with additional regulatory proteins to control their expression. PAS Proteins. Single-minded, along with Arnt and Period, constitute the founding members of the PAS protein family. PAS proteins are found in all organisms, from bacteria to humans. Prokaryotic and plant PAS proteins are commonly environmental sensors that respond to and mediate the effects of changes in light, oxygen levels, redox, and metabolic state. Most animal PAS proteins belong to the bHLH-PAS class of DNA binding proteins. The Markey Trust has an impressive heritage in bHLH-PAS protein discovery and analysis, having sponsored research in our lab and those of Drs. Tessier-Lavigne, Montell, and Semenza. Drosophila and mammalian bHLH-PAS proteins control a variety of biological processes, including toxin metabolism, circadian rhythms, vasculogenesis, tissue-specific development, cell migration, and behavior. Clinically, they are important for understanding tumor growth, sleep disorders, birth defects, and obesity. Our lab has discovered or worked on a number of Drosophila bHLH-PAS proteins, including Single-minded, Trachealess (tracheal development), Spineless (appendage formation), Similar (response to hypoxia), Cranky, and Tango (dimerization partner for all of the above). This project has allowed us to generalize about the mechanisms of action of invertebrate and vertebrate developmental bHLH-PAS proteins. Given the fundamental role that bHLH-PAS proteins play in the formation of the nervous system and respiratory system in insects, we
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The Markey Scholars Conference: Proceedings have begun to look for similar roles in other species. Drosophila trachealess is a master regulator of tracheal transcription and development. We have shown that the crustacean, Artemia, has a trachealess ortholog that is prominently expressed in the epipodal gills. Although the insect trachea and crustacean gills are divergent morphologically, these results suggest that their formation may be controlled by the same regulatory gene. Furthermore, evolutionary analysis of bHLH-PAS gene expression and function in organisms with distinct mechanisms of CNS and respiratory system development and anatomy may provide important insights into organismal evolution. STRUCTURAL BASIS OF TRANSCRIPTION Seth A. Darst, Ph.D. Laboratories of Molecular Biophysics The Rockefeller University Transcription is the major control point of gene expression. RNA in all cells is synthesized by a complex molecular machine, the RNA polymerase (RNAP). In bacteria, RNAP comprises a ~400 kDa core (subunit composition alpha2/beta/beta’/omega), conserved from bacteria to man. Promoter-specific initiation requires additional proteins. In bacteria, a single polypeptide, the sigma factor, binds core RNAP to form holoenzyme. Our goal is to understand the mechanism of transcription and its regulation. Our approach is to use a combination of structural and biophysical methods spanning resolution ranges (low-resolution: cryo-electron microscopy [EM]; medium: cross-linking analysis and X-ray crystallography; high: X-ray crystallography), complemented by the wealth of functional information already available, to determine the structure/function relationship of RNAP and its complexes with nucleic acids and regulatory factors. Our focus is on the bacterial RNAPs as a model for the cellular RNAPs in general. Low-resolution EM structures (25 -12 Å) of RNAP revealed a molecule shaped like a crab-claw, with a groove or channel for accommodating double-helical DNA.1,2 In the first step towards high-resolution structural analysis of cellular RNAPs, we determined the 3.3 Å-resolution crystal structure of the 380 kDa core RNAP3,4 from T. aquaticus (Taq), providing a basis for further structural and functional studies. For example, the path of the transcript RNA and template DNA through RNAP was tracked using crosslink mapping, resulting in a detailed model of the elongation complex.5 A co-crystal structure of RNAP with rifampicin revealed the
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The Markey Scholars Conference: Proceedings mechanism of inhibition by this important antibiotic.4 The remarkable conformational flexibility of RNAP was analysed by comparing cryo-EM and X-ray results using newly developed computational tools.2 Recent progress is highlighted by crystal structures of the 430 kDa Taq holoenzyme (alpha2/beta/beta’/omega/sigma) at 4 Å,6 and a holoenzyme/promoter DNA complex at 6 Å resolution.7 These results depended on having high-resolution structures of core RNAP3,4 and sigma factor domains8 in-hand. These structures provide fundamental insight into sigma/core RNAP interactions and conformational changes to form holoenzyme, holoenzyme recognition of promoters, and sigma’s role in melting the DNA to form the transcription bubble. The structures also provide a basis for models of holoenzyme/promoter complexes along the pathway of open complex formation. The new structural information will guide future investigations at an unprecedented level of detail. Notes 1 Darst, S. A.; Kubalek, E. W.; Kornberg, R. D. (1989) Nature 340:730-732; Darst, S. A.; Edwards, A. M., Kubalek, E. W.; Kornberg, R. D. (1991) Cell 66:121-128; Polyakov, A.; Severinova, E.; Darst, S. A. (1995) Cell 83:365-373. 2 Darst, S. A.; Opalka, N.; Chacon, P.; Polyakov, A.; Richter, C.; Zhang, G.; Wriggers, W. (2002) Proc. Natl. Acad. Sci., in press. 3 Zhang, G.; Campbell, E.; Minakhin, L.; Richter, C.; Severinov, K.; Darst, S. A. (1999) Cell 98:811-824. 4 Campbell, E. A.; Korzheva, N.; Mustaev, A.; Murakami, K.; Goldfarb, A.; Darst, S. A. (2000) Cell 104:901-912. 5 Korzheva, N.; Mustaev, A.; Kozlov, M.; Malhotra, A.; Nikiforov, V.; Goldfarb, A.; Darst, S.A. (2000) Science 289:619-625. 6 Murakami, K.; Masuda, S.; Darst, S.A. (2002) submitted. 7 Murakami, K.; Masuda, S.; Campbell, E.A.; Muzzin, O.; Darst, S. A. (2002) submitted. 8 Campbell, E. A.; Muzzin, O.; Chlenov, M.; Sun, J. L.; Olson, C. A.; Weinman, O.; Trester-Zedlitz, M. L.; Darst, S. A. (2002) submitted. HIJACKING THE RIBOSOME: STRUCTURAL BASIS FOR TRANSLATION INITIATION IN HEPATITIS C VIRUS Jennifer A. Doudna, Ph.D. Molecular Biophysics and Biochemistry Howard Hughes Medical Institute Yale University Initiation of protein synthesis is a key step in the control of gene expression in eukaryotes. In most cases, recruitment of the 40S ribosomal subunit to a messenger RNA (mRNA) involves recognition of a modified
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The Markey Scholars Conference: Proceedings IN VIVO ANALYSES OF T CELL COSTIMULATORY PATHWAYS Arlene H. Sharpe, M.D., Ph.D. Department of Pathology and Brigham and Women’s Hospital Harvard Medical School My research focuses on the in vivo biology of costimulation and its rolein regulating immune responses. Costimulation appears pivotal in determining whether T cell antigen recognition leads to T cell activation or anergy. For antigen-specific T cell activation to occur, two signals are needed. The interaction between the T cell receptor (TCR) complex and antigen-MHC is necessary, but not sufficient for T cell activation. Costimulatory signals, provided cell surface molecules are expressed on antigen-presenting cells, determine the outcome of TCR engagement, since they augment T cell proliferation and effector functions, such as lymphokine production. The interaction between CD3/TCR and antigen-MHC in the absence of costimulation not only results in a failure to induce an immune response, but often also results in functional inactivation of mature T cells, leading to a state of T cell unresponsiveness or death. Thus, T cell costimulatory signals play a critical role in determining the fate of a T cell. Several receptor-ligand pairs, which are primarily either members of the immunoglobulin supergene family or tumor necrosis factor receptor family, have costimulatory function because they induce activated T cells to proliferate after TCR signaling. The B7-CD28/CTLA4 costimulatory pathway appears to be particularly important, because of its unique capacity to prevent the induction of anergy. The main approach that my laboratory has been taking to analyze the in vivo function of costimulatory pathways is to generate and analyze the immune capabilities of mice lacking costimulatory ligands and receptors using gene targeting approaches. We have focused primarily upon the B7:CD28 superfamily. Our mouse strains have provided a genetic means for dissecting the hierarchy of costimulatory pathways in the development of an immune response. Our studies have revealed striking and unexpected functions of costimulatory pathways. Our studies of B7-1 deficient mice provided the first evidence for the existence of additional functional CD28/CTLA-4 counter-receptors in vivo. As a result of these findings, a second CD28/CTLA-4 counter-receptor, B7-2, was cloned. Our studies revealed that B7-2 is the major early activating costimulator in this pathway. Mice lacking both B7-1 and B7-2 exhibit profound immunologic deficits and have demonstrated a critical role for this pathway in IgG class switching and germinal center formation. Our CTLA-4 deficient mouse revealed a critical role for CTLA-4 in turning off activated T cells and a previously unsuspected means by which costimulation can regulate re-
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The Markey Scholars Conference: Proceedings sponses to self antigens and potentially offers new approaches for regulating Tcell activation and tolerance. There is currently great interest in manipulating costimulatory signals for therapeutic purposes. Specifically, learning how to inhibit costimulatory pathways may enable new methods for achieving tolerance for tissue transplantation and for controlling autoimmune diseases and allergies, whereas learning how to use costimulatory pathways to augment immune responses may lead to new immunization strategies for infectious agents and tumor immunity. Clinical trials using costimulatory blockade to block transplant rejection and ameliorate autoimmune diseases are in progress. LESSONS FROM ABL: IMPLICATIONS FOR THE FUTURE OF TARGETED CANCER THERAPEUTICS Richard A. Van Etten, M.D., Ph.D. Center for Blood Research and Department ofGenetics Harvard Medical School A new era of targeted cancer therapy was inaugurated in May 2001 with the FDA approval of STI-571 (Gleevec®/imatinib mesylate) for the treatment of chronic myeloid leukemia (CML). STI-571 is a phenylamino-pyrimidine compound that is a potent and selective inhibitor of the Abl, PDGFbR, and Kit tyrosine kinases. An ATP mimetic, STI-571 binds to the ATP-binding site of the Abl catalytic domain and effectively inhibits Abl kinase activity in vitro and in vivo at concentrations of 0.1-1.0 mM. In phase I trials, STI-571 was remarkably effective as a single agent in interferon-resistant CML chronic phase patients, inducing durable hematologic remissions in 90 percent and major cytogenetic responses in 55 percent of patients. This agent has already radically altered the way patients with CML are managed, and is being heralded as the paradigm for the development of anti-cancer drugs in the future. From my perspective as a hematologist studying the molecular pathophysiology of leukemia, the STI-571 story raises several important issues: (1) The importance of precise knowledge of molecular drug targets in cancer. Work from our laboratory and many others over the previous decade demonstrated that the Bcr/Abl fusion tyrosine kinase, the product of the Philadelphia chromosome, is the direct cause of CML. Definition of the fundamental genetic abnormalities in cancer cells is a prerequisite to developing targeted therapies. Our work strongly argues that accurate animal models of human cancer are critical to this effort, as results obtained in cultured cells have
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The Markey Scholars Conference: Proceedings often been misleading. (2) The remarkable activity of STI-571 in CML as a single agent, and whether this can be generalized to other cancers. Our work suggests that solitary expression of Bcr/Abl in a hematopoietic stem cell is sufficient for induction of CML, and this may account for the extreme sensitivity of this leukemia to STI-571. However, solid tumors may have additional abnormalities in addition to activated tyrosine kinases that contribute to the oncogenic phenotype. Thus, while a subset of gastrointestinal stromal cell tumors that express activated c-Kit tyrosine kinase receptors also respond to STI-571, no complete remissions are observed. Trials of EGFR inhibitors in head and neck and glial tumors are just beginning. (3) Acquired drug resistance is a central problem in cancer therapy, and targeted therapeutics will be no different. Although STI-571 is effective in CML chronic phase, in patients with advanced disease, including accelerated phase, myeloid and B-lymphoid blast crisis, and those with de novo Ph-positive B-lymphoblastic leukemia, STI-571 is less effective. Although 50-70 percent of such patients initially respond to the drug, 60 percent of myeloid blast crisis patients and all B-lymphoid leukemia patients relapse within 3 to 6 months of starting therapy. Our laboratory and others have shown that a major mechanism of resistance is point mutations in the Abl catalytic domain that directly confer drug resistance. (4) Combinations of targeted therapies may be a strategy for improving responses to STI-571 and preventing the development of resistance. By analogy to HIV infection (like CML, another acquired dominant genetic disease of the blood) where treatment targeting both viral reverse transcriptase and protease is more efficacious than monotherapy, it is plausible that targeting critical signaling pathways downstream of Bcr/Abl will synergize with kinase inhibitor therapy. Again, animal models are the best way to validate such pathways. (5) Can the success with STI-571 be repeated? This is a complex question with both scientific and economic/social aspects. The development of STI-571 is often cited as a successful example of high-throughput drug screening and/or rational drug design. Both are incorrect, as the screen that identified the basic pharmacophore of STI-571 used protein kinase C, and the structural basis for the selectivity of action of the compound against Abl is still not understood. In addition, economic forces will play a major role in dictating which drugs will be developed for which diseases. The STI-571 program was nearly cancelled by Novartis less than 18 months before FDA approval due to the small perceived market opportunity, and resurrected only after direct pressure from leukemia patients. There are other leukemias with excellent tyrosine kinase targets for which drug development may never proceed due to the comparatively small number of patients with the disease.
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The Markey Scholars Conference: Proceedings TWO FAMILIES OF BIR-CONTAINING PROTEINS: INHIBITORS OF APOPTOSIS OR REQUIRED FOR MITOSIS David L. Vaux, Ph.D. The Walter and Eliza Hall Institute ofMedical Research Melbourne, Australia Inhibitor of apoptosis (IAP) proteins all bear one or more copies of a motif termed a baculoviral IAP repeat (BIR), a novel zinc finger fold. Certain BIR bearing proteins from baculoviruses, Drosophila and mammals inhibit cell death by binding to processed caspases. The Drosophila IAPs are antagonized by small pro-apoptotic molecules Grim, HID and Reaper. Mammalian IAPs are antagonized by mitochondrial proteins Diablo/Smac and Htra2, which interact via their processed amino-termini. Survivin is a protein that bears a single, structurally distinct BIR, and is expressed in all cancer cells, but is usually undetectable in cells from normal adult tissues. We have identified and deleted surviving homologues in the yeasts S. pombe and S. cerevisiae, in C. elegans and in the mouse. The phenotypes of these mutant organisms, and the pattern of Survivin expression revealed by antibodies, indicates that Survivin, inner centromere protein (INCENP) and aurora kinase 1 and their respective homologues function in concert to coordinate chromosome segregation and cytokinesis. The requirement for Survivin for cell division explains why it is expressed in cancer cells, which are dividing, but not in most normal adult cells, which are not. A COMMON THEME IN THE PATHOGENESIS OF BACTERIAL INFECTION OF THE HUMAN RESPIRATORY TRACT Jeffrey N. Weiser, M.D. Departments of Microbiology and Pediatrics University of Pennsylvania Choline, a major component of eukaryotic membrane lipids, has been considered to be a highly unusual feature in prokaryotes. This laboratory has recently shown that choline in the form of phosphorylcholine (ChoP) decorates the oligosaccharide portion of the LPS of Haemophilus influenzae. Choline is obtained directly from the airway surface fluid or stripped off of host membrane lipids by a cell-surface glycerophosphodiesterase causing a cytotoxic effect on the ciliated epithelium. A MAb that recognizes ChoP shows that the ChoP epitope is a common feature of other major
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The Markey Scholars Conference: Proceedings pathogens that reside primarily on the mucosal surface of the human respiratory tract. In addition to Streptococcus pneumoniae, where ChoP has long been recognized as a constituent of its teichoic acids, the ChoP epitope is found on pili of Neisseria meningitidis and gonorrheae, a temperature regulated cell-envelope protein in Pseudomonas aeruginosa, the LPS of commensal Neisseria species and Actinobacillus actinomycetem-comitans, and a polar membrane lipid in various mycoplasma species. The cell-surface expression of ChoP is subject to phase variation at a frequency of 10-3/generation due to slip-stranded mispairing of multiple tandem 5′-CAAT-3′ repeats within the open reading frame of the gene expressing choline kinase, licA. This suggests that it is not always advantageous for the organism to express ChoP. Our ability to define the genetic basis of choline incorporation in H. influenzae has allowed us to compare constitutive ChoP+ and ChoP- phenotypes to demonstrate that on the mucosal surface in both animal models and in human carriage there is strong selective pressure for bacteria expressing ChoP(ChoP+). In contrast, during invasive disease there is a selection for bacteria with an out-of-frame number of 5′-CAAT-3′ repeats in licA(ChoP- phenotype). ChoP appears to have multiple effects on the ability of respiratory tract pathogens to interact with their host. ChoP may function in binding and invasion of host cells through mimicry of the natural ligand for the receptor for platelet activating factor (rPAF) expressed on the apical surface of the respiratory epithelium. In addition, ChoP renders the organism more resistant to the bactericidal effects of the antimicrobial peptide LL-37, which is present in airway surface fluid and targets structural differences between host and microbial membranes. In contrast, for some strains ChoP confers sensitivity to killing mediated by the binding of the serum acute phase reactant, C-reactive protein (CRP). Binding of CRP to ChoP causes the activation of the classical pathway of complement by ligation of C1q. In this regard, CRP seems to be a component of innate host defense that specifically targets microbes bearing cell-surface ChoP. Humans may depend on innate immunity based on CRP because the pathogens involved can quickly overwhelm their host and unlike other host species their antibody against ChoP does not appear to be protective. CRP, recently noted to be present in airway surface fluid, also inhibits binding of ChoP-expressing bacteria to rPAF. The inhibitory effect of CRP on adherence of both H. influenzae and S. pneumoniae is blocked by human surfactant, an abundant component of the lower airway that is composed largely of choline phosphate and binds to CRP. The effect of surfactant could contribute to the susceptibility of the lower airway to organisms that asymptomatically colonize the upper airway where there is no sur-
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The Markey Scholars Conference: Proceedings factant. It appears, therefore, that ChoP on the cell-surface of a variety of otherwise dissimilar pathogens may be 1) important for successful colonization of the mucosal surface, and 2) evasion of innate host defenses mechanisms. THE ROLE OF TGFSS SIGNALS DURING EMBRYOGENESIS Malcolm Whitman, Ph.D. Department of Cell Biology Harvard Medical School Our laboratory studies the mechanisms by which intercellular signals regulate the differentiation and patterning of the early vertebrate embryo. Our work is currently focused on the role of TGFβ signals during embryogenesis. TGFβ superfamily ligands have long been known to play essential roles in patterning at multiple steps in vertebrate embryogenesis, but, until recently, the signal transduction pathways by which TGFβ factors act have been obscure. Several years ago, we discovered a novel transcription factor, FAST-1, that interacts in a ligand regulated manner with the TGFβ signal transducers Smad2 and Smad4, and demonstrated that this interaction targets the Smads for specific, developmentally regulated promoters. The Smads are regulated directly by TGFβ receptors, and therefore the identification of the Smad/FAST DNA binding complex provided the first example of a direct pathway by which TGFβs regulate a specific set of transcriptional responses. We have subsequently shown that FAST-1 is an essential component of the TGFβ signaling pathway that establishes the early embryonic body plan. More recently, we have used antibodies specific for the phosphorylated, activated forms of the Smads to examine how TGFβ signaling is regulated endogenously during embryogenesis. We have found that the ability of embryonic cells to respond to specific TGFβ ligands is regulated as development progresses, and that a small extracellular protein, cripto, acts as a specific co-receptor for one subset of TGFβ ligands, the nodals, during early development. We are currently studying how cripto is regulated in the early embryo. We have also shown that nodals act via a novel mechanism, heterodimerization, to antagonize other TGFβ superfamily ligands during early embryogenesis. These studies suggest that direct interactions among distantly related TGFβ ligands may be important determinants of their activity in the embryo.
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The Markey Scholars Conference: Proceedings RO SMALL RNPS FUNCTION IN THE RECOVERY OF CELLS FROM RADIATION DAMAGE X. Chen, H. Shi, J. Smith, D. Yang,1 L. Evangelisti,1 R. Flavell1 and Sandra Wolin, M.D., Ph.D. Department of Cell Biology Yale University 1 Section of Immunobiology, HHMI, Yale University School of Medicine The Ro 60 kDa autoantigen is an RNA-binding protein that is normally bound to small cytoplasmic RNAs known as Y RNAs. Although these RNPs are components of most vertebrate cells, their function has long been mysterious. In Xenopus oocyte nuclei, the Ro protein is also complexed with a large class of variant 5S rRNA precursors. Because these variant RNAs are inefficiently processed to mature 5S rRNA and eventually degraded, the Ro protein may recognize improperly folded 5S rRNA precursors as part of a quality control pathway (O’Brien and Wolin, Genes & Dev. 8:2891-2903). Although Ro RNPs have not been detected in either S. cerevisiae or S. pombe, the genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an orthologue of the Ro protein. The Ro protein orthologue, Rsr (Ro Sixty Related) contributes to the resistance of D. radiodurans to ultraviolet irradiation. D. radiodurans cells lacking rsr are more sensitive to UV irradiation than wild-type cells. During recovery from irradiation, the levels of Rsr increase approximately fourfold. Rsr binds several small RNAs, encoded upstream of rsr, that also accumulate during recovery from UV irradiation. Remarkably, one of these RNAs resembles the Y RNAs bound by the Ro autoantigen in higher eukaryotes (Chen et al., Genes & Dev. 14:777-82). We have been examining the role of Ro RNPs in the recovery of higher cells following UV irradiation. Using gene knockout technology, we generated mouse embryonic stem cells that lack the Ro protein. Mouse cells lacking Ro have drastically reduced levels of Y RNAs, suggesting that Ro protein binding stabilizes these RNAs from degradation. Most interestingly, cells lacking the Ro protein are more sensitive to ultraviolet light than wild-type cells. Thus, in both mouse and bacterial cells, Ro RNPs contribute to survival following radiation damage. Although the mechanism is under investigation, one possibility is that the Ro protein binds to misfolded, mutant RNAs that are transcribed from DNA molecules containing radiation-induced mutations.
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The Markey Scholars Conference: Proceedings ENVIRONMENTAL RESPONSIVENESS OF THE DIMORPHIC FUNGAL PATHOGEN HISTOPLASMA CAPSULATUM Jon P. Woods, M.D., Ph.D. Department of Medical Microbiology and Immunology University of Wisconsin, Madison Histoplasma capsulatum (Hc) is a thermally dimorphic fungus that is a significant cause of respiratory and systemic disease in humans and other mammals. Its clinical importance has increased along with the growing immunodeficiency of the human population associated with HIV/AIDS, cancer and its treatments, immunosuppressive therapy for transplants and inflammatory syndromes, aging, and hospitalization. Hc lives saprobically in the soil as a mold, which is a successful member of a competitive polymicrobial ecosystem. The host-adapted parasitic morphotype is a budding yeast which is a facultative intracellular pathogen of macrophages. This microbe faces a variety of different environments and must survive under harsh conditions or modulate its microenvironment to achieve success as a pathogen in a professionally antimicrobial host cell. We have used several molecular techniques to identify fungal genes that are differentially expressed during infection of host macrophages and/or mice, when Hc is subjected to a complex range of environmental conditions. These methods have included in vivo expression technology (IVET), differential display, and cDNA representational difference analysis (RDA). Such approaches do not provide exhaustive genomic surveys in this eukaryotic microorganism, but we have identified several interesting genes. One differentially expressed gene encodes a small transcript in antisense orientation to a homolog of a negative regulatory protein kinase gene from another fungus, which is important in mating and starvation responses. We are examining both upstream and downstream aspects of this potential regulatory system in Hc, such as the specific environmental stimuli influencing expression of the antisense transcript, whether expression of the antisense transcript affects sense transcript expression, whether the sense transcript encodes a protein kinase functional in Hc, what the downstream targets of the putative kinase are, and the role of this locus in Hc biology and pathogenesis. A second target gene is expressed specifically in the yeast morphotype and not in mold, and the predicted encoded protein displays significant sequence homology with epidermal growth factor (EGF) domains found in a variety of proteins from other organisms, that typically function in attachment or intercellular signaling. Finally, we have preliminarily identified homologs of genes in other organisms that are involved in iron uptake. This finding interfaces with our separate interest in iron acquistion and fungal responses to
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The Markey Scholars Conference: Proceedings the specific environmental stress of iron limitation. The essential nutrient iron lies at the competitive interface between the mammalian host and nearly all microbial pathogens, including Hc. The host displays both constitutive and inducible iron sequestration mechanisms. Iron limitation acts as an important host defense mechanism against Hc in human and mouse macrophage cell culture infection models. As a successful pathogen, Hc must express iron acquisition mechanisms to obtain this nutrient in the competitive host environment in which it resides during infection. Hc previously has been shown to produce hydroxamate siderophores, which typically act as iron-scavenging compounds. We have demonstrated ferric reduction by Hc via at least three moieties—an extracellular ferric reductase enzyme, extracellular ferric reductant(s), and cell-surface ferric reducing agent(s). Reduction of ferric to ferrous iron causes removal from both host (e.g., transferrin) and fungal (siderophore) iron-binding compounds. Siderophore-mediated and reductive processes may provide important alternate, complementary, or interactive mechanisms for acquiring iron in the soil and/or the host. MERGING BIOLOGY WITH DRUG DISCOVERY IN OBESITY, INFLAMMATION, ANGIOGENESIS, MUSCLE DISEASE AND OTHER SETTINGS George D. Yancopoulos M.D., Ph.D. President, Regeneron Laboratories Chief Scientific Officer, Regeneron Pharmaceuticals, Inc. Growth factor and cytokines, released by one cell and acting via cell surface receptors on a second cell, mediate intercellular communications required for the initiation and/or regulation of all biologic processes. A major focus for us at Regeneron has been to identify new growth factors and/or their receptor systems, with the notion that identification of such critical master regulatory systems would present new therapeutic opportunities. We have particularly focused on growth factor/receptor systems that specifically act on a single or limited number of cell types, so that manipulation of these systems could be attempted so as to benefit diseases involving those cell types, without having widespread side effects. Over the last decade, our efforts have led to the discovery and characterization of multiple growth factor/receptor systems (e.g., neurotrophins and their Trk receptors; CNTF/IL6 family and their gp130-related receptors; agrin and its MuSK receptor; collagens and their DDR receptors; ephrins and their Eph receptors; angiopoietins and their Tie receptors;
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The Markey Scholars Conference: Proceedings cartilage-specific ROR receptors), as well as to novel approaches for blocking these and other growth factor systems using engineered versions of soluble receptors we term Traps. In many cases, the growth factors or their blockers are in, or are approaching, clinical testing. I will discuss the progress of a second-generation version of CNTF, termed Axokine, which is in Phase III clinical testing for obesity, as well as Traps that we are using to block IL-1 in rheumatoid arthritis, VEGF in cancer, and IL-4 and IL-13 in asthma and allergy. I will also discuss our new high-throughput knockout and transgenic technology, termed Velocigene, which we use to rapidly assign function to genes, with examples involving genes we have realized play key roles in the biology and pathology of muscle, cartilage, blood vessels, and lymphatic vessels. STUDIES OF 53BP1 MAY REVEAL AN UNEXPECTED LINK BETWEEN DNA DAMAGE AND MITOSIS S.T. Liu, Y. Adachi,1 and Tim J. Yen, Ph.D. Fox Chase Cancer Center 1 University of Edinburgh, Edinburgh, U.K. 53BP1 was identified as a yeast two-hybrid interactor of the p53 tumor suppressor but the functional significance of this interaction remains unclear. 53BP1 contains two copies of a BRCT motif that is found in a large number of proteins that are involved with various aspects of DNA replication, repair, and recombination. Furthermore, 53BP1 has been shown to accumulate at dozens of foci within nuclei that contain damaged DNA. Although the functionality of DNA damage induced foci remain to be clarified, it is generally believed to represent a macromolecular assembly of multiple proteins at or near the site of broken DNA. These findings strongly suggest that 53BP1 is a component of the DNA damage response pathway. We have recently discovered that 53BP1 may have a different function besides DNA damage. 53BP1 accumulates in several large aggregates in nuclei of normal cycling cells. In mitotic cells, 53BP1 is dispersed from these aggregates and becomes concentrated at kinetochores, a structure that links chromosomes to the mitotic spindle. We determined that the earliest time that 53BP1 can be detected at kinetochores is shortly after nuclear envelope breakdown. 53BP1 assembles onto kinetochores after several other proteins that assemble onto kinetochore just prior to nuclear envelope breakdown. The temporal pattern of kinetochore binding exhib-
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The Markey Scholars Conference: Proceedings ited by 53BP1 suggests that it is likely to provide functions important for the final steps of kinetochore assembly. To begin to understand the importance of 53BP1 to kinetochore function, we have localized the kinetochore targeting domain to lie within the region that contains the BRCT repeats. Given that 53BP1 form foci of similar size at sites of DNA damage and kinetochores, we speculate that 53BP1 provides functions that are shared by these two different cellular functions. Thus, our studies of the mitotic functions of 53BP1 may provide novel insights into the mechanism by which cells monitor and respond to double stranded DNA breaks.
Representative terms from entire chapter: