Mark Noble - US grants

This award from the Inorganic, Bioinorganic and Organometallic Chemistry Program will support an investigation of the chemistry of sulfur atoms which are part of metal-sulfur cluster compounds. Such compounds are useful models of the active sites of a number of important enzymes and also comprise an important class of catalysts, some of which are used in the chemical industry. The objective of the project is to investigate sulfur-nitrogen covalent interactions which result from the reactions of nitrogen substrates at sulfur as bound in sulfidometal complexes. Initial emphasis will be on dimolybdenum(V) sulfur- bridge systems, and both free radical and nucleophilic pathways to the formation of N-S bonds will be examined. Specific nitrogen substrates to be examined include diazonium salts, azo compounds, NO/NO+ compounds, sulfenamide precursors and metal dinitrogen complexes. Expected products for these substrates are, respectively, diazosulfide complexes, MSNNR; sulfenylhydrazines, MSNRNHR; thionitrite compounds, MSNO; metallosulfenamides, MSNR2; and MSNNM' complexes. Reactions will be monitored and products characterized by spectroscopic methods, including NMR (P-31, N-15, proton, C-13), EPR and IR, as well as by X-ray crystallography, as appropriate.

Single crystal x-ray crystallography is the most powerful analytical method for structure determination of solids. In synthetic inorganic, organic, bioinorganic and organmetallic chemistry, single crystal x-ray diffraction is an invaluable tool to characterize molecular structure. The information gained from the knowledge of the molecular composition and structure helps to develop new reactions of potentially general interest in catalysis or synthesis. This award from the Chemistry Research Instrumentation Program will help the Department of Chemistry at the University of Louisville acquire an upgrade of their computing facilities and the x-ray diffraction laboratory. Among the areas of chemical research that will enhance by the acquisition are the following: 1. Sulfur Chemistry within a Sulfidomolybdenum(v) complex 2. Dinuclear Iron Complexes of Polyimidazole Ligands 3. Molecular Recognition: The Electrostatic Factor 4. Bimetallic Complexes with Bridging Oxygenates 5. X-Ray Studies on Peptides and Pseudopeptides 6. Single Crystal and Powder X-Ray Diffraction Studies of Mineral Structures.

This award from the Chemistry Research Instrumentation Facilities Program will help the Department of Chemistry at the University of Louisville acquire a 300 MHz NMR spectrometer. The research activities to be supported include: (1) Sulfur-Centered Chemistry in Metal- Sulfur Complexes; (2) Cage Formation in Organophosphonates of Aluminum, Gallium, and Indium; (3) Synthesis and Confirmation of Oligosaccharides of Bacteria; (4) 1H NMR Studies on Paramagnetic FeIIFeIII and FeIIICoII Iron-Oxo Complexes of Polyimidazole Ligands; (5) Synthesis and Characterization of CO2-Bridged Bimetallic Complexes; and (6) the molecular basis of carcinogenesis, solution structures of peptide receptors, the synthesis NMR Studies on Peptides and Pseudopeptides. Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful tool available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solution. Access to state-of-the-art NMR spectrometry is essential to chemists who are carrying out frontier research. The results from these NMR studies are useful in the areas such as polymers and catalysis, and in biology.2

The Tissue Procurement and Culture Core will support investigation broadly within the Program by processing tissues and providing cultures of colon epithelium ranging from normal to fully transformed cells. Well- characterized growth protocols for colonic epithelial cells will be used to establish cultured from all biopsies obtained. These will include specimens derived from normal colon, from adenomas and from carcinomas. Of specific concern is to develop cell cultures from patients with APC, HNPC and sporadic polyps and malignancies. In addition, this Core will be responsible for analyzing antigenic markers of colon differentiation in the resulting populations, so as to provide the analytical laboratories with well-characterized populations for their research efforts.

DESCRIPTION: Optimizing repair of damaged tissue such as demyelinated CNS lesions by precursor cell transplantation entails identification of precursor cell population with extensive self-renewal capacity and of means of expanding these populations in vitro to increase cell numbers available for transplantation. The applicant has discovered (a) multiple precursor phenotypes which all generate oligodendrocytes but which differ greatly in self renewal potential and (b) multiple means of enhancing precursor cell self renewal and expansion in vitro. These discoveries provide the foundation for deeper elucidation of principles underlying oligodendrocyte precursor cell division and differentiation and will also enable significant progress in optimizing strategies for remyelinating damaged tissue by precursor cell transplantation. The differing precursor cell populations will be immunopurified and grown in conditions which enhance precursor cell self renewal. Clonal and time-lapse microcinematographic analysis of division and differentiation, together with serial passaging studies will reveal precursor cell self renewal potential and mitotic lifespan. Studies on phenotypic changes occurring over time (in vivo and in vitro) will reveal stability of precursor cell phenotype. Analysis of the timing of differentiation of purified progenitors will provide information on the role of cell intrinsic mechanisms in promoting differentiation and limiting mitotic lifespan. Growth of cells in cytokine combinations that enhance precursor cell renewal will reveal environmental conditions that override cell intrinsic limitations to populations expansion. Finally in vitro analysis of myelination and transplantation of populations of cells to the CNS of dysmyelinating mouse mutants will reveal the capacity of the different populations for oligodendrocyte replacement and myelin creation. This research will enable us to make significant advances in (1) better characterizing the phenotypically distinct oligodendrocyte precursors cells that we have discovered (ii) determining their biological relationship with each other (iii) establishing contributions of intrinsic biological clocks to modulating differentiation and limiting self renewal (iv) identifying growth conditions that override theses cell-intrinsic limitations and which enable expansion of populations without compromising ability to carry out tissue repair and (v) establishing the capacity of these primary and expanded populations to replace oligodendrocyte in vitro and in vivo models of remyelination. The combination of fundamental biological analysis with specific attention to issues of tissue repair will provide new insights into both basic issues in precursor cell biology and into aspects of oligodendrocyte precursor cell biology relevant to enhancing repair of demyelinating damage.

Many different physiological insults to the developing child result in long-lasting neurological impairment associated with a failure of myelination and/or destruction of existing myelin and a subsequent inability to repair this damage. Such impairment is associated, for example, with deficiencies in thyroid hormone, iron with inadequate nutrition, with fetal exposure to alcohol or cocaine, as a result of hypoxic episodes or in association with radiotherapy or chemotherapy. We propose that the underlying cellular basis for many childhood disorders of neurological development is disruption of specific steps in the development of the precursor cells that give rise to the differentiated cell types of the central nervous system (CNS). Consistent with this hypothesis, we have discovered that specific steps in development of the myelin-forming oligodendrocytes of the CNS are disrupted in the two specific instances of thyroid hormone and iron deficiency. Initially, we will carry out in vitro and in vivo studies on hypothyroidism, as a well-defined model of hormonal and nutritional deficiency disorders. These studies will provide a detailed map of the stages of precursor cell development vulnerable to deficiency of thyroid hormone. To determine why hormonal replacement therapy applied at too late a stage does not promote repair of CNS damage, we next will transplant defined stem cell and precursor cell populations into the CNS of animals that have been hypothyroid throughout development and examine the ability of these cells to contribute to tissue repair. These experiments will provide insight into whether the failure to reconstitute normal development is due solely to an absence of appropriate precursor cells, or also is due to the CNS becoming refractory to repair. Complementary to this cellular biological analysis, we also will determine whether intracellular redox modulation is a critical component of the mechanism by which thyroid hormone exerts its effects on all the CNS precursor cells regulated by this hormone. In addition, we will extend preliminary observations indicating that the very different disorder of iron deficiency may also work in part through alteration of intracellular redox state. By asking whether different syndromes exert their effect through overlapping mechanisms, these studies may provide important clues to potential new therapeutic approaches to the treatment of hormonal and nutritional deficiency disorders. In sum, this research program will identify both cellular and biochemical mechanisms that explain the biology of critical developmental periods and may lead to the identification of therapeutic approaches that can enhance repair in multiple deficiency syndromes.

DESCRIPTION (provided by applicant): There is an increasing recognition that cancer treatment is associated with serious neurological impairment, even in patients treated for cancers outside the central nervous system (CNS). Imaging studies are revealing a variety of abnormalities in the brain following chemotherapy and an increasing number of studies demonstrate a disturbingly high frequency of cognitive impairment in patients who have received exclusively chemotherapy. We propose that damage to oligodendrocytes and CNS precursor cells provides a cellular basis for understanding the adverse neurological consequences of treatment with chemotherapeutic agents. We have discovered that oligodendrocytes, glial precursor cells and neuronal precursor cells of the CNS are vulnerable to widely used chemotherapeutic agents, such as BCNU and cisplatin, at doses well within the range to which brain cells would normally be exposed during cancer treatment. The nature of many chemotherapeutic agents is associated with a ready penetrance into the brain, such that administration of these drugs outside, of the CNS might be expected also to be associated with neurotoxicity, a hypothesis supported by our preliminary in vivo experiments on the effects of BCNU. Our goals are to develop a detailed understanding of chemotherapy-associated neurotoxicity and to develop means of selectively protecting normal cells from the harmful effects of chemotherapy without compromising the utility of these cytotoxic agents in killing tumor cells. We will develop a detailed analysis of neurotoxicity using in vitro and in vivo approaches. We will propose two complementary protective strategies. The first paradigm involves regulation of oxidant balance, a known trigger for initiation of apoptosis. The second paradigm involves inhibition of caspases, specific components of death effector pathways. Thus, we will explore in vitro and in vivo approaches to define cellular populations at risk from being damaged or destroyed by chemotherapeutic agents. In addition, we will identify and test means of selectively protecting normal cells from such damage without simultaneously protecting cancer cells in vitro as well as in vivo.

[unreadable] DESCRIPTION (provided by applicant): This research is focused on understanding how non-catastrophic toxicant exposure disrupts normal development and function of the CNS. The continuing determination that concentrations of toxicants once thought to be safe are associated with a variety of maladies, combined with the large numbers of toxicants and potential toxicants found in our environment, make it of great importance to increase our understanding of how normal cellular function may be disrupted by such substances. A central goal of this effort is to identify general principles applicable to the understanding of large numbers of toxicants. The general principle/hypothesis that underlies this application, which emerges directly from our ongoing research, is that, regardless of its other activities, any toxicant that has pro-oxidant activity will have a highly predictable set of effects on both precursor cells and differentiated cells. These effects include inhibition of precursor cell division and enhancement of responsiveness to inducers of differentiation and cell death. This hypothesis, with its clear mechanistic predictions, allows the formulation of a general theory of developmental neurotoxicology applicable to exposure to a wide range of toxicants at concentrations that frequently occur in the environment. Moreover, the predictions of our hypothesis regarding the effects of low dose toxicant exposure on vulnerability to other potentially harmful agents may provide a new understanding of the reasons underlying the enormous variability seen in responsiveness of different individuals to putatively identical physiological stressors. Our in preliminary vitro experimentation provides strong support for the correctness of the hypothesis underlying this proposal, and has demonstrated marked effects of a variety of toxicants on neural cell function, including a striking enhancement of vulnerability to a variety of other physiological stressors. Biochemical analysis demonstrates that despite their different chemistries, all of the toxicants examined converge on Fyn and Cbl activation, leading to enhanced degradation of the PDGFRa. In sum, this research program will define the actions of sublethal concentrations of single toxicants on a variety of neural precursor cells, define the interactions of toxicants with other physiological stressors (including other toxicants) particularly in regards to synergistic toxicity reactions, will define cellular regulatory systems that are modulated by toxicant exposure, and will study clinically relevant situations in which toxicant load can enhance response to injury and in which follow on studies in human populations are both particularly important and comparatively straightforward to carry out. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): One of the disturbing findings to emerge from studies on survivors of both childhood and adult cancers is the frequency with which systemic chemotherapy is associated with adverse neurological sequelae, including leukoencephalopathy, seizures, cerebral infarctions, and cognitive impairment. In our studies designed to understand the biological foundations for these effects, we have discovered that multiple mainstream chemotherapeutic agents applied at clinically relevant exposure levels are more toxic for the progenitor cells of the CNS and for non-dividing oligodendrocytes than they are for multiple cancer cell lines. Enhancement of cell death and suppression of cell division were seen in vitro and in vivo. When administered systemically in mice, these diverse chemotherapeutic agents caused increased cell death and decreased cell division in multiple regions of the CNS, with a high degree of correlation between in vitro observations and in vivo effects. Our current efforts are focused on three questions central to increasing our understanding of the biological underpinnings of the adverse neurological effects of cancer treatment and to developing means of preventing these effects. In this proposal, Aim 1 provides the first animal model of delayed CNS damage associated with chemotherapy and tests the hypotheses that (i) transient systemic administration of chemotherapy causes delayed damage to the CNS that is more severe than damage observed at short times after treatment;(ii) a particular target of damage is the myelinated white matter tracts of the brain;(iii) early indicators of delayed damage are dysregulation of transcription factor expression in myelin-forming oligodendrocytes, followed by marked reductions in oligodendrocyte numbers and an absence of oligodendrocyte replacement;and, (iv) delayed damage is also associated with reductions in the generation of new hippocampal neurons. Aim 2 provides the first paradigm for reducing or preventing such damage, and is focused on analysis of the hypothesis that co-treatment with erythropoietin (EPO) reduces CNS damage caused by chemotherapy. Aim 3 focuses on mechanism-based discovery of protective strategies for acute and delayed adverse effects of chemotherapy, and tests the hypotheses that (i) chemically diverse chemotherapeutic agents disrupt the function of primary cells -but not cancer cells - by convergence on a newly discovered regulatory pathway (the redox/Fyn/c-Cbl pathway) that converts small increases in oxidative state into enhanced degradation of a subset of receptor tyrosine kinases important in cell division and survival, with consequent reductions in activity of signaling molecules vital in cell division and survival;and, (ii) this prevention of activation of the redox/Fyn/c-Cbl pathway provides a mechanistic strategy for protecting primary cells from the adverse effects of chemotherapy without also rescuing cancer cells in bulk or cancer stem cells in particular. PUBLIC HEALTH RELEVANCE One of the disturbing findings to emerge from studies on survivors of both childhood and adult cancers is the frequency with which systemic chemotherapy is associated with adverse neurological sequelae, including leukoencephalopathy, seizures, cerebral infarctions, and cognitive impairment. The concern of our research is to understand the biological and mechanistic foundations for these adverse effects, both to discover means of protecting against such events and to develop means of identifying individuals at increased risk for adverse events. Such protection can be achieved both by increasing the vulnerability of cancer cells to chemotherapy and by selectively protecting normal cells from the adverse effects of these therapeutic agents.

DESCRIPTION (provided by applicant): The goal of this "exploratory" application is to provide a novel mechanistic understanding of vulnerability to amyloid (A [unreadable]) protein, and of the pathways through which A[unreadable] disrupts function of those cells critical in maintenance of normal myelination. We have recently discovered a novel regulatory pathway that provides a sequential linkage between oxidative changes and control of cell signaling. In this pathway, increases in oxidative status caused by exposure of cells to chemically diverse substances with pro-oxidant activity cause activation of Fyn kinase. This leads to activation of c-Cbl, an E3 ubiquitin ligase that is a target of Fyn. Activation of c-Cbl leads to ubiquitylation of its target proteins, which include among them a subset of receptor tyrosine kinases (RTKs). As a result of their interaction with c-Cbl, degradation of these RKTs is enhanced, leading to a suppression of downstream signaling. As a consequence of this degradation, downstream activation of such signaling mediators as Erk1/2 and Akt are suppressed. As one would predict from such an effect, cell division is suppressed and cell survival may also be impaired. We propose to now test the hypothesis that activation of the Fyn/c-Cbl pathway plays an important role in amyloid (A[unreadable]) toxicity. The experiments proposed focus on the effects of A[unreadable] peptides on oligodendrocytes and their progenitor cells, due to the importance of myelin damage in AD pathology. Moreover, as the Fyn/c- Cbl hypothesis also predicts that exposure to sublethal concentrations of pro-oxidant stimuli will suppress cell division, we will further test the hypothesis that A[unreadable] peptides are cytotoxic for oligodendrocytes but also suppress division of the progenitors from which they are generated. If this prediction is correct, this would indicate that A[unreadable] both damages myelin-forming cells and suppresses the cell division required for repair. This research thus proposes a new molecular pathway by which A[unreadable] affects cell function. Several studies have previously suggested an important role of Fyn in the pathogenesis of AD. Our studies will provide novel insights into the mechanism by which Fyn activation may disrupt cellular function in AD. Aim 1 tests the hypothesis that exposure of oligodendrocytes and their progenitors to A[unreadable] causes activation of the redox/Fyn/c-Cbl pathway, degradation of RTKs that are c-Cbl targets, and selective suppression of downstream signaling events from these RTKs. This is associated with, depending on the type and concentration of A[unreadable] and the cell type examined, suppression of progenitor cell division (at sublethal doses) and induction of progenitor cell and/or oligodendrocyte death at higher concentrations. Aim 2 tests the hypothesis that activation of the Fyn/c-Cbl pathway is functionally important in A[unreadable] -mediated suppression of cell division and/or induction of cell death in the oligodendrocyte lineage. Aim 3 tests the hypothesis that anti-oxidants and trophic factors that protect against toxic effects of A[unreadable] suppress A[unreadable] -mediated activation of the redox/Fyn/c-Cbl pathway, thus providing a novel potential site of action for the protective effects of anti-oxidants in AD. PUBLIC HEALTH RELEVANCE: This research provides novel insights into the means by which amyloid [unreadable] protein causes damage to the central nervous system in Alzheimer's disease. Our studies identify a novel molecular pathway by which amyloid [unreadable] protein disrupts cell function, new insights into the pathogenesis of the extensive damage to myelinated tracts in this disease, and a new understanding of means by which anti-oxidant therapy protects from the effects of amyloid [unreadable] protein. This research will help in identifying new means of protecting against amyloid [unreadable] toxicity.

Glioblastomas (GBMs) are among the most deadly cancers known, with only limited improvements in treatment outcomes despite extensive efforts. GBMs exhibit resistance to chemotherapeutic agents, irradiation and other cell death inducers, colonize brain tissue far removed from the tumor's primary origin, and exhibit intrinsic intra-tumor heterogeneity, the presence of a robust tumor initiating cell (TIC) compartment and multiple other obstacles to treatment. Still a further significant challenge in developing effective GBM treatments is that normal CNS progenitor cells and oligodendrocytes are more vulnerable to most anticancer therapies than are cancer cells themselves. Adverse neurological side effects of cancer treatment are increasingly recognized as important problems, thus emphasizing the importance of developing treatments that are selectively toxic for transformed cells. While some new therapies offer benefit to a subset of individuals, with ongoing efforts to better identify such individuals in advance, most GBM patients remain without effective treatment. Thus, development of therapies that can overcome the multiple mechanisms of therapeutic resistance of GBM cells without causing unacceptable toxicity to normal cells of the CNS is thus a central need in this field. The central hypotheses of this research are that (i) restoring the ability to activate the c-Cbl ubiquitin ligase in GBM cells, and in particular using a non-canonical oxidation pathway to activate c-Cbl, enables targeting of multiple critical regulators of GBM cells with a single therapeutic intervention; (ii) agents that restore c-Cbl function in GBM cells can be identified by mechanism-based drug repurposing; (iii) c-Cbl restoration therapies provide a foundation for rational combinatorial treatments that are more toxic for GBM cells than for normal glial progenitors; (iv) this approach provides clinically relevant therapies that re effective in treating established human GBMs growing intra-cranially in immune-deficient NSG mice; and (v) it is possible to prospectively identify GBMs that are likely to respond to specific therapies developed in this research. Preliminary data to support each of these hypotheses is provided, To further develop this promising avenue of investigation, we now propose the following aims: Aim 1 tests the hypothesis that candidate CRAs (of which we thus far have ten) increase sensitivity to compounds relevant to GBM treatment, enable simultaneous targeting of multiple proteins and biological activities critical in GBM cell function and tumor generation and achieves these outcomes without increasing the sensitivity of normal glial progenitor cells to relevant therapeutic agents. Aim 2 tests the hypothesis that CRA-based therapies enable effective treatment of human GBMs, growing in immunodeficient mice, in a clinically relevant manner. Aim 3 tests the hypothesis that the presence of complexes between c-Cbl and Cool-1/ß-pix predicts sensitivity to our CRA-based therapies, thus potentially enabling prospective identification of tumors most likely to be responsive to these approaches.