David Ross - US grants

Chronic exposure to benzene, an extensively used industrial chemical and widespread environmental contaminant, results in progressive deterioration of haematopoietic function, acute myelogenous leukemia and lymphoma in man. The mechanisms underlying benzene-induced myelotoxicity are unknown but metabolism of benzene is required for it to induce toxic effects. The major metabolite of benzene is phenol but myelotoxicity has been correlated with bone marrow concentrations of the secondary benzene metabolites - catechol and hydroquinone. No information exists concerning activation of catechol or hydroquinone to reactive species by bone marrow cells or by enzymes present in high concentration in bone marrow such as myeloperoxidase (MPO). We therefore propose to examine the metabolism of benzene and its phenolic metabolites by rat and human MPO and by various rat bone marrow cell populations. Use of both MPO and bone marrow cells will allow us to compare metabolite profiles produced by the isolated enzyme and by the cellular systems and to observe the biochemical changes associated with cytotoxicity in bone marrow cells. We also propose to examine whether MPO-catalyzed oxidation of the phenolic metabolites of benzene results in the production of species which can bind to cellular macromolecules (DNA, protein, glutathione) and if so to characterize any DNA or glutathione adducts that are formed. The contribution of autoxidation reactions of phenolic metabolites of benzene and more specifically, the production of active oxygen species to cytotoxocity in cellular systems will be examined, as will the role of other potential toxifying and detoxifying enzymes in bone marrow cells. As one of the most sensitive cell populations to benzene-induced toxicity is peripheral leukocytes, we propose to examine the activation of benzene and its metabolites to binding species when incubated with leukocytes stimulated to perform an oxidative burst. This work should greatly increase our understanding of the metabolic mechanisms underlying benzene-induced myelotoxicity.

DT-diaphorase, although generally considered to be a protective enzyme against quinone-induced toxicity, has been suggested to play a role in the bioactivation of antitumor quinones. The precise role of DT-diaphorase in bioreductive activation of quinones remains controversial. We propose to utilize DT-diaphorase purified from HT-29 human colon carcinoma cells to examine the role of this enzyme in bioactive of AZQ* and Mitomycin C. Whether AZQ, Mitomycin C and their analogs are substrates for HT-29 DT- diaphorase and whether metabolism by this enzyme results in the production of metabolites which can induce damage to cellular macromolecules (DNA, proteins, soluble thiols) will be examined. Studies in cellular systems will also be performed to identify intracellular targets of antitumor quinones. These studies will allow unequivocal confirmation of the role of DT- diaphorase in bioreductive activation of AZQ and Mitomycin C in human tumor cells. * AZQ - Diaziquone-(2,5-bis(1-aziridinyl)-3,0-bis-(carboethoxyamino)1,4- benzoquinone.

DT-Diaphorose or NAD(P)H:quinone oxidoreductase (NQO1) plays a key role in the deactivation of many environmental quinones and xenobiotics and has been proposed to be important in both chemoprotection and chemoprevention. NQO1 has also attracted considerable attention because of its ability to activate certain bioreductive antitumor quinones and its elevated activity in tumors such as non small cell lung cancer (NSCLC). In an effort to develop new agents for the therapy of NSCLC, we will examine the ability of novel antitumor quinones to serve as substrates for human NQO1 and test the hypothesis that compounds which are efficiently bioactivated by NQO1 are selectively cytotoxic to NSCLC cells with elevated NQO1 activity. It is also critical to understand the mechanisms underlying the elevated expression of NQO1 in NSCLC relative to SCLC. We therefore propose to characterize the specific cis-acting DNA sequences and trans-acting nuclear protein-DNA interactions that mediate NSCLC specific expression of NQO1. We have characterized a polymorphism in NQO1, a homozygous mutation which leads to a total loss of NQO1 protein and enzyme activity, although NQO1 mRNA can readily be detected. We will determine if regulation of the mutant hNQO1 is at the level of translation or protein instability and define the alterations in protein structure which occur as a result of the mutation. Whether NQO1 plays any physiological role or acts on endogenous substrates is as yet unknown. Our data suggests that NQO1 regenerates antioxidant forms of alpha-tocopherol after free radical attack and this observation will be pursued in this application. The potential role of NQO1 as an antioxidant enzyme may also contribute to the chemopreventive role of NQO1. The characterization of a polymorphism in NQO1 which leads to a total loss of NQO1 protein and activity gives us a unique opportunity to examine the association between lack of NQO1 and susceptibility to cancer in a human population. We will examine the prevalence of the NQO1 polymorphism in patients with colon ademomas, an intermediate stage in the development of colon cancer, and matched disease-free individuals. The proposed studies represent an integrated chemical, biochemical and molecular approach to determining the significance of NQO1 for bioreductive drug activation and chemoprevention.

The behavior of an organism is a set of characters that is just as critical to the understanding of biology as the morphological and physiological features of the individual. Statistically robust description of behavioral processes is, unfortunately, more difficult to obtain than in the more traditional fields of biology. Especially when the task is to indicate the occurrence of subtle behavioral changes, merely counting the number or duration of a behavior pattern is insufficient. Recent applications of Continuous Time Markov Chain (CTMC) models to Ethology have shown great promise as a descriptive and hypothesis-testing tool. Researchers have indicated otherwise undetectable changes of sub-threshold doses of amphetamines and electrical stimulation of the brain. Unfortunately, many of the statistical techniques available are poorly suited for fitting CTMC models to behavior for a variety of reasons such as high alpha error rates, inability to deal with normally encountered sample sizes, number of ties and censored observations, and the need to have computationally simple solutions that can be used as standard PC-based routines. This project will consider the design and implementation of generalizations of the CTMC model. In particular, while the transition times in such models usually follow simple exponential distributions, this project will examine the possibility that these times follow a distribution with nonconstant, piecewise-constant hazard rates. The goal is to create computationally efficient, unbiased, robust statistical techniques to fit such models, paying special attention to estimation of, and inference on, the time or times when the hazard rate changes. These techniques will then be evaluated by testing them on both actual and simulated data sets.

This B/START application proposes to test hypothesis about the optokinetic and smooth pursuit eye movement systems in schizophrenic patients. Background: Despite decades of research on abnormal smooth pursuit eye movements in schizophrenia, the potential of this area of study remains unfulfilled. Current theory suggests that the abnormal pursuit is caused by dysfunction of cerebral, not brainstem, ocular motor regions. However, this theory remains unproven. An important test of it would be to examine an oculomotor system which shares brainstem regions with the pursuit system, but which bypasses cerebral regions. This system should function normally in schizophrenia. One such system is the optokinetic system, which is used to stabilize a steadily moving environment on the fovea. Only rarely has this system been studied in schizophrenia. The most recent study concluded that the optokinetic system of functions normally in schizophrenia (Latham et al., 1981), and this may be the predominant view in the field (Holzman, 1987). However, by today's standards, all of the previous studies of the optokinetic system in schizophrenia were much too limited in their methodology to support this conclusion. Therefore, the important hypothesis that the optokinetic system functions normally in schizophrenia remains to be adequately tested. Specific aims: To test the hypothesis that schizophrenic patients will have an abnormal pattern of responses to pursuit and optokinetic stimuli, indicating normal function of the optokinetic system and abnormal functions of the pursuit system. Preliminary studies : We have shown that patient shave abnormalities in the initiation and maintenance of smooth pursuit eye movements. Optokinetic testing will complement such ongoing studies in our laboratory. Methods: Pursuit and optokinetic eye movements will be tested in 20 normal controls, 20 schizophrenic patient with eye tracking disorder (ETD) and 20 non-ETD patient. The optokinetic stimulus will be a full-field, rotating environment, which will invoke optokinetic nystagmus (OKN). Measures of the slow phase in response to both pursuit and optokinetic stimuli will include initial acceleration and steady- state gain. In addition, the optokinetic stimulus will completely darkened during certain periods of time in order to test buildup and discharge of velocity storage, which is related to optokinetic after- nystagmus (OKAN). In addition, monocular testing in response to the optokinetic stimulus will test temporal-nasal preference, a characteristic of the optokinetic system.

DESCRIPTION: (Adapted from the Investigator's Abstract) NAD(P)H:quinone oxidoreductase (NQO1) is known to protect against benzene-induced hematopoietic toxicity. The proposed mechanism is protection against reactive benzene-derived quinones and oxygen radicals formed in bone marrow. The major problem with this mechanism is that NQO1 levels are non-detectable in human bone marrow aspirates. This observation is inconsistent with a proposed protective role for NQO1 in human bone marrow and the increased benzene-induced hematotoxicity observed in individuals carrying a homozygous NQO1 mutation which results in the absence of NQO1 activity. The investigator will test the following two hypotheses in this proposal which could provide an adequate explanation for the protective effects of NQO1 against benzene toxicity. Hypothesis 1. Stromal microenvironments exist in-situ in human bone marrow which have significant NQO1 activity and are not removed by bone marrow aspiration. They propose that individuals carrying the homozygous NQO1 mutation would not have stromal NQO1 leading to impaired detoxification by NQO1. Hypothesis 2. The mechanism underlying the protective effect of NQO1 against benzene-induced hematotoxicity is that NQO1 can be induced in bone marrow by benzene metabolites. In individuals carrying the homozygous NQO1 mutation, however, NQO1 activity is not induced after exposure to benzene metabolites leading to impaired detoxification and chemoprotection. This hypothesis will be tested by characterizing induction of NQO1 by benzene metabolites in the human hematopoietic cell line KG-1a, in human bone marrow and potential target cells such as stroma and CD34+ progenitor cells. The molecular mechanism underlying induction of NQO1 by benzene metabolites will be examined and the investigators will confirm that the toxicity of benzene metabolites is ameliorated by NQO1 induction in human bone marrow. These experiments will provide a mechanistic explanation for the protective effects of NQO1 against benzene-induced hematopoietic toxicity.

[unreadable] DESCRIPTION (provided by applicant): We have shown that quinones can be bioactivated by NQO1 (DT-diaphorase) and that human solid tumors contain markedly elevated NQO1 levels making NQO1 an attractive target for the development of antitumor quinones. We will focus on two groups of NQO1 directed antitumor quinones a) aziridinylbenzoquinones with RH1 as the lead compound and b) the benzoquinone ansamycin Hsp90 inhibitors typified by 17-AAG. RH1 is as an excellent substrate for NQO1 and is currently in phase 1 clinical trials. NQO1 markedly potentiates RH1-induced DNA crosslinking, cell cycle arrest, apoptosis and toxicity. However, although NQO1 activates RH1, a cytotoxic effect could still be observed in NQO1 null cell lines and xenografts. The mechanisms of RH1 toxicity in NQO1-null cells are undefined but have been suggested to depend on the one electron reductases, P450R and b5R. Using isogenic cell systems differing only in NQO1, P450R or b5R levels, we will define NQO1-dependent and NQO1-independent mechanisms of RH1 toxicity both in-vitro and in xenograft systems in-vivo. Elucidation of these mechanisms will allow predictions of the efficacy of RH1 in different types of tumors. Pancreatic tumors contain high levels of NQO1 and we will therefore define the efficacy of RH1 in pancreatic tumor systems both in-vitro and in both pancreatic xenograft and orthotopic models in-vivo. A second class of quinone antitumor agents in clinical trial are the benzoquinone ansamycin Hsp90 inhibitors. Inhibition of Hsp90 results in the aberrant folding of a number of oncogenic client proteins making Hsp90 an attractive target. Our data using both recombinant Hsp90 and cellular systems has shown that the quinone form of 17-AAG does not appear to be the active Hsp90 inhibitor and that generation of the hydroquinone via NQO1 metabolism results in more potent Hsp90 inhibition and cytotoxicity. Molecular modeling studies have confirmed more favorable binding energies of the hydroquinone ansamycins in the active ATPase site of the Hsp90 protein. We will therefore test the hypothesis both in-vitro and in-vivo that the hydroquinone forms of the ansamycins are more active Hsp90 inhibitors than their parent quinones. We will also examine the stability and redox properties of the hydroquinone ansamycins and test the hypothesis that novel prodrug forms of the hydroquinones lead to effective Hsp90 inhibition and antitumor activity. Our studies will generate data that can be applied to ongoing and future clinical studies of RH1 and both benzoquinone and hydroquinone ansamycins. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Increased oxidative stress in dopaminergic neurons has been implicated as a causative factor in Parkinson's disease (PD). The catechols DOPA dopamine and DOPAC generated in these cells can undergo oxidation to produce aggressive oxygen species and reactive arylating quinones which are capable of cellular damage. We have characterized a null polymorphism in the NQO1 gene (NQO1 *2) and individuals carrying the variant NQO1 *2 allele have been found to be at a markedly increased risk of PD relative to control individuals emphasizing the role of NQO1 as a risk factor for PD. PD has also been associated with inhibition of the ubiquitin-proteasomal system (UPS). Inhibition of the UPS by aggregated proteins in neurodegenerative disease leads to increased oxidative stress. Since oxidative stress can result in the misfolding and aggregation of proteins resulting in further frustration of the UPS, a picture is beginning to emerge where both oxidative stress and UPS inhibition are part of a vicious cycle and are important factors in the etiology of PD.In this proposal, we will test the following hypotheses; 1) that NQO1 protects cells against reactive arylating quinones and oxidizing species generated during metabolism of DOPA, dopamine and DOPAC. We will use dopaminergic cells, mechanism based inhibitors of NQO1 and an isogenic cellular model developed in our lab to explore the role of NQOl in these studies; 2) that DOPA, dopamine or DOPAC derived o-quinones contribute to UPS inhibition and that NQO1 protects against this inhibitory effect; 3) that NQO1 protects against oxidative stress generated as a result of inhibition of the UPS. UPS inhibition in cells will be achieved by the use of both chemical proteasome inhibitors and by transfection of mutant forms of alpha-synuclein which have been associated with PD and inhibit the UPS by mechanisms involving protein aggregation and 4) that mutant NQO1*2 protein, which is normally rapidly degraded by the UPS, generates oxygen radicals leading to increased oxidative stress. The ability of mutant NQO1 *2 protein to generate oxygen radicals becomes particularly important under conditions where the UPS is inhibited and the protein accumulates.

Unconscious transference occurs when an eyewitness misidentifies a familiar but innocent person from a police lineup. This phenomenon has been studied with young adult participants and with children ranging from 5-12 years of age. This research project expands the work on this important topic by replicating the aforementioned studies using middle-aged and elderly participants. The intellectual merit of the proposal is addressed in the following ways. First, these studies represent one of the first attempts to examine unconscious transference in the elderly. The results from the elderly and adult samples are compared with those obtained with the child and young adult samples. This permits a unique developmental examination of unconscious transference, as well as other memory phenomena, across the life span because the same methodology has been used in each study. According to recent U.S. Census data, the elderly population in the United States is steadily increasing. Moreover, based on U.S. Bureau of Crime Statistics reports, the elderly are likely to be victims of crimes where they would be called to provide eyewitness testimony. Specifically, the elderly are very likely to be victims of crimes in which they see and/or interact with the perpetrator. Thus, it is extremely important to determine the factors that affect the accuracy of eyewitness judgments in older adults. Second, these experiments are theoretically motivated to determine not only if unconscious transference occurs, but also to identify the mechanism producing the effect and whether it varies by the age of the witness. Third, the researchers examine how to improve eyewitness identification accuracy in general, and reduce unconscious transference, in particular, by providing the witness with a lineup instruction that would help him or her identify the source of the memory for the assailant. Fourth, the researchers examine whether the participants demonstrate a "Pop Out" effect and reach their lineup decisions faster (within a range of 10-12 seconds) and use an automatic process as compared to inaccurate witnesses who use a slower, more deliberate process of elimination procedure when making a lineup identification. Fifth, because the researchers have included an array of recognition and meta-cognition measures, they can explore other important memory issues that are related to unconscious transference but which span beyond that topic as well. Consequently, the proposed research addresses the broader impact criteria as it has theoretical and applied importance. The results of this research provide a major contribution to the literatures in human development, memory, and the law because it investigates a variety of memory processes, as well as unconscious transference, and provide empirically-derived suggestions on how to improve the accuracy of eyewitness identifications.

[unreadable] DESCRIPTION (provided by applicant): The homozygous NQO1*2 polymorphism results in a total lack of NQO1 activity due to accelerated degradation of the mutant NQO1*2 protein by the ubiquitin/proteasomal pathway. The NQO1*2 polymorphism has been found to be a risk factor for benzene-induced myeloid toxicity but also for childhood and adult de-novo leukemias and secondary leukemias arising as a result of chemotherapy. The mechanisms underlying the protective effects of NQO1 against benzene-induced myelotoxicity and both de-novo and secondary leukemias were puzzling since NQO1 was not expressed in aspirated human bone marrow cells or human bone marrow CD34+ progenitor cells, the target cell for induction of both aplastic anemia and leukemia. However, we observed that NQO1 was present in human bone marrow endothelial cells (HBMEC), which are not harvested by bone marrow aspiration. In the present application, we wish to explore the potential role of NQO1 in HBMEC in protection against benzene induced aplastic anemia and have established HBMEC cultures in our lab for this purpose. We propose a mechanism whereby HBMEC exposed to benzene metabolites produce increasing amounts of endothelial IL8 (elL8) which results in apoptosis of neighboring hematopoietic cells and myeloid progenitor cells resulting in aplastic anemia. We will also examine the mechanism underlying the lack of expression of NQO1 in human myeloid cells at the transcriptional level by characterizing cis acting DNA sequences and trans acting nuclear protein-DNA interactions that modulate NQO1 expression. One of the major tumor suppressor genes characterized in mammalian systems is p53 and a high percentage of leukemias contain mutations or allelic losses of p53. In preliminary data, we demonstrate that NQO1 forms a protein complex with wild type p53. We propose to examine whether the interaction of NQO1 and p53 is specific for wild type p53 and whether it has consequences for p53 stability and p53-dependent transcriptional activation of downstream genes. If NQO1 stabilizes p53 and the interaction has functional consequences, this would provide a mechanism for the increased incidence of leukemia of diverse origin that has been associated with a lack of NQO1 protein due to the NQO1*2 polymorphism.

DESCRIPTION (provided by applicant): It is clear that toxic oxidative stress in nigrostriatal dopaminergic neurons plays an important role in the pathogenesis of Parkinson's disease (PD). Since scientific evidence support mitochondria (mito) as a critical site for the induction of oxidative stress and cell death in PD, an attractive neuroprotective strategy would be to enrich nigrostriatal mito with the antioxidant, d-alpha-tocopherol (T). Accordingly, we contend that ineffective protection observed with vitamin E treatments in previous clinical and experimental investigations on PD resulted from the inability of these vitamin E treatments to enrich nigrostriatal mito with protective levels of T. In support of the critical role of mito T in the protection of cells against toxic oxidative stress, we have recently discovered that a single injection of d-alpha-tocopheryl succinate (TS) [but not T or d-alpha-tocopheryl acetate (TA)] as well as 7 days of dietary vitamin E (1400 IU TA/kg diet) result in dramatic T enrichment of hepatic mito membranes which completely prevented rotenone- and ferrous iron-induced necrotic cell death in rat hepatocytes. It is our working hypothesis that protection against toxic oxidative stress in experimental PD is dependent on the nigrostriatal enrichment of mito membranes with protective levels of T that attenuates mito reactive oxygen species (ROS) production and protects mito from ROS-mediated mito lipid, protein and DNA damage and these dopaminergic cells from death. This hypothesis will be tested both in vitro and in vivo using the oxidative insults: MPTP (or MPP+), rotenone or ferrous iron by: (1) determining the effect of T enrichment of mito membranes in dopaminergic cells on oxidative stress-induced mito ROS production, mito lipid, protein and DNA damage and apoptotic and necrotic cell death and (2) determining the ability of in vivo parenteral TS administration and chronic dietary vitamin E administration to enrich striatal mito membranes with T and protect against experimental PD. We believe that these novel studies will result in breakthrough discoveries that will clearly define the role of mito T in experimental PD as well as identify effective T treatment strategies for PD.

DESCRIPTION (provided by applicant): Inhibition of the enzyme NQO1 in pancreatic tumor cells using the non-specific inhibitor dicoumarol resulted in increased intracellular superoxide, inhibition of cell growth and inhibition of the in-vitro malignant phenotype of cells. The hypothesis proposed was that inhibition of NQO1 led to increased superoxide levels which inhibited pancreatic cancer cell growth and the in-vitro malignant phenotype. We have recently shown that NQO1 can directly scavenge superoxide providing both a potential approach and a mechanism of action for the use of NQO1 inhibitors in the therapy of pancreatic cancer. In this proposal, we will test the hypothesis that specific mechanism-based inhibitors of NQO1 are effective compounds for the therapy of pancreatic tumors both in-vitro and in-vivo. One of these inhibitors, the indolequinone ES936, is a potent inhibitor of pancreatic cancer cell growth and the in-vitro malignant phenotype. We will therefore test the hypothesis that ES936 can be employed as an effective therapeutic agent in pancreatic cancer and extend in-vitro data to in-vivo xenograft and orthotopic pancreatic tumor models. We will define the mechanism of action of ES936 and will attempt to dissociate NQO1 inhibition from effects on pancreatic cancer cell growth and the in-vitro malignant phenotype using both chemical/pharmacological and genetic approaches. Whether the effect of ES936 is due to increased levels of superoxide as a result of NQO1 inhibition will be characterized and downstream effects of ES936 on modulation of the cell cycle and apoptosis will be determined both in-vitro and in-vivo. This approach represents a novel therapeutic strategy for the treatment of pancreatic cancer, a disease where therapeutic options are very limited and where chemotherapy has made minimal impact.

Project Abstract
Rumor Propagation: Modeling & Testing Dynamic Social Influence Mechanisms

Rumors are a powerful, pervasive, and persistent force affecting people and groups. They play an important role in a variety of human attitudes and actions, yet we know little about how they propagate over time and in the context of the complex social networks that exist in our world. Like infectious diseases, many rumors engender mistrust, suspicion, and conflict between people groups; such rumors "survive"-even thrive-and are believed as fact despite well-meaning attempts to dispel them. How does this happen? That is, what are the mechanisms involved in rumor propagation over time and across social spaces? This project will address that question in a couple of new ways, using a dynamical systems framework, and in the context of an unusual collaboration between mathematicians and psychologists. First, rumor selection (choosing whether or not to share a particular rumor) and belief in that rumor will be computationally modeled using insights and data gained from empirical research and using a dynamic framework that accounts for different ways in which social networks are configured. Second, a series of laboratory experiments will be conducted in which groups of networked participants will select and discuss rumors via email. Both the mathematical modeling and the computer assisted laboratory experiments will investigate how social space (how a person's social network is configured) and network homogeneity (the extent of same group membership in a network) affect group-level rumor outcomes over time. Finally, an exploratory arm of the study will pilot a web-site for collection of field data related to propagation mechanisms and will search for archival repositories of rumor. In summary, the project will study how social space configuration and network homogeneity affect temporal and spatial patterns of rumor selection and belief, resulting in a dynamical understanding of rumor activity and increased knowledge about how motivational processes associated with group membership affect rumor activity over time and space (e.g., how desiring to build up one's self or one's ingroup may lead to the popularity of and belief in rumors derogating a different group). Such knowledge is vital for the effective prevention of and response to harmful rumors, especially those that foster intergroup distrust, discord, and hostility.

DESCRIPTION (provided by applicant): Imatinib was developed to target specifically the deregulated tyrosine kinase activity of the oncoprotein BCR-ABL in chronic myeloid leukemia (CML) and it has high specificity against its major kinase target. In a surprising and unexpected finding in 2007, NQO2 was identified as the first non-kinase target of imatinib in two independent studies. Imatinib was found to bind potently and specifically to NQO2 resulting in inhibition of catalytic activity of the enzyme. One of the conclusions of these two studies was that inhibition of NQO2 may contribute to the therapeutic activity of imatinib in CML cells. In this proposal, we will define the outcome of specifically targeting NQO2 in CML. Levels of NQO2 have not been examined in human CML and in aim 1 we will characterize NQO2 levels in a panel of imatinib-naive and imatinib resistant archived CML patient samples. In aim 2, we will examine whether specific targeting of NQO2 using novel mechanism-based (suicide) inhibitors developed in our laboratory results in antiproliferative activity in human CML cell lines. We will also validate the role of NQO2 in the proliferative activity of CML cells by employing anti-NQO2 shRNA and characterize the effect of stable knockdown of NQO2 on CML growth. BCR-ABL stability is known to be influenced by interactions with other proteins. In aim 3, we will therefore examine whether there is a protein-protein interaction between NQO2 and BCR-ABL, whether this interaction is inhibited by imatinib, whether it leads to increased stability of BCR-ABL and whether it depends on the catalytic activity of NQO2. The proposed studies will characterize the potential role of NQO2 in the therapeutic activity of imatinib and define the outcome of specifically targeting NQO2 as a therapeutic approach in CML. PUBLIC HEALTH RELEVANCE: Imatinib has been found to bind potently and specifically to NQO2 resulting in inhibition of catalytic activity of the enzyme. The implication of this work is that inhibition of NQO2 may contribute to the therapeutic activity of imatinib in chronic myeloid leukemia (CML). The proposed studies will define the outcome of specifically targeting NQO2 in CML. The development of novel small molecule suicide inhibitors of NQO2 may provide a novel therapeutic approach in CML.

ABSTRACT FOR THE NSF PROPOSAL GK-12: SUPER-M
PI: MONIQUE CHYBA
DEPARTMENT OF MATHEMATICS, UNIVERSITY OF HAWAII

The School and University Partnership for Educational Renewal in Mathematics project (SUPER-M) will bring the knowledge and expertise of research mathematicians into K?12 classrooms, making an important contribution to improving school mathematics in Hawaii. The project will provide K?12 students with an enriching learning environment where mathematics is interesting and dynamic. The project will also contribute to the formation of a cadre of highly qualified teachers, bringing new mathematics expertise to schools throughout the State of Hawaii and helping to sustain the program. Over five years, 38 Fellows will be selected to partner with K?12 schools on Oahu, the Big Island and Maui. Fellows will take a semester long course on ?Issues in K?12 Mathematics Education,? learning about best practices in the design of professional development courses for teachers. Fellows will design and lead workshops for K?12 teachers arising from their areas of research. Upon completion of the course, Fellows will partner with a cooperating teacher, creating and leading mathematics activities for K?12 students. In this way, SUPER-M will provide K?12 students with a solid grounding in mathematics, increasing their opportunities to pursue careers in STEM disciplines. SUPER-M will serve under-represented populations by placing a special emphasis on recruiting Native Hawaiian and women Fellows. SUPER-M expects to profoundly impact the community at large through special events such as summer camps, public outreach events, and family math days.

DESCRIPTION (provided by applicant): Both benzoquinones and naphthoquinones have been found to perturb protein handling and degradation in a variety of cellular systems. Protein handling and degradation is not restricted to the proteasome and also involves protein chaperones, the unfolded protein response (UPR)/endoplasmic reticulum (ER) stress response, formation of aggresomes and lysosomal autophagy. Quinones have been found to affect each of these systems and altered protein handling is emerging as a potentially key mechanism of quinone induced toxicity. Our studies will focus on model 1,4-benzo- and naphtho-quinones as well as the dopamine derived 1,2-quinone, aminochrome which have all been shown to induce changes in protein handling. In aim 1, we will characterize changes in all major protein handling systems induced by model benzo- and naphtho- quinones and by aminochrome. These experiments will characterize altered mechanisms of protein handling as a result of treatment of cells with reactive quinones and the relevance of such changes for toxicity in cellular systems. In aim 2, we will define the respective roles of arylation and quinone-induced oxidative stress in inhibited protein handling using quinones capable only of either redox cycling or of both redox cycling and arylation . We will also examine quinone induced changes in protein handling in cells stably transfected with the one electron reductases cytochrome P450 reductase or cytochrome b5 reductase which cause increased quinone one electron redox cycling and increased reactive oxygen generation. The major mammalian quinone reductases NQO1 and NQO2 are highly polymorphic with a high prevalence of variant alleles resulting in marked phenotypic changes. A lack or variation in activity of these enzymes may therefore represent susceptibility factors for quinone induced toxicity. In aim 3, we will examine the role of NQO1 and NQO2 in modulating quinone induced protein handling changes and toxicity. These experiments will be performed in isogenic pancreatic, breast and neural cellular systems specifically designed to explore the roles of NQO1 and NQO2 in the same genetic background Overall, these experiments will characterize novel mechanisms of quinone-induced toxicity at the level of protein handling, define the inter-relationships and the respective roles of protein handling changes in toxicity and define the role of NQO1 and NQO2 as susceptibility factors for these changes. The studies will have broad mechanistic applicability to a variety of organ systems.

This project builds upon the successful Phase 1 project, STEM Real World Applications Modules. New modules are developed which are closely tied to classroom concepts with mathematics used in industry. The team of investigators includes leaders in the mathematical research and educational communities, and mathematicians with years of industry experience. The overarching goal of this team is to show undergraduate students that strong mathematical skills lead to more employment options and greater chances of success.

The dissemination plan has three main components. The project impacts students and faculty members nationwide through distribution of effective teaching materials. A key part of the dissemination starts with 12 faculty members at 7 additional institutions beta testing the developed modules. After beta-testing, the modules are made available on the project website. Finally presentations about the modules and their effect on student learning are given at conferences, and articles about the modules are submitted to educational and research journals.

The last major advance in the treatment of metastatic bladder cancer (BC) took place in 1997 with the advent of gemcitabine. Despite this advance, visceral metastases are usually fatal. The overall goal of the proposed studies is to develop small molecule inhibitors that block a critical node in the metastatic process. We found that Rai GTPases serve as the molecular switches of a therapeutically tractable signaling pathway that allows UC cells to grow in the lung, the most common visceral metastatic site. The clinical significance of this pathway and validity of Rai as a therapeutic target is supported by finding that high Rai expression in tumors places patients at higher risk for metastasis and the requirement of Rai expression for lung metastasis to occur in animal models of UC. Our Guiding Hypothesis for this application is that small molecules targeting Rai provide effective therapy for metastatic UC. With support from the MD Anderson Bladder SPORE Developmental Research Program (DRP), we evaluated >500K compounds for their ability to bind RalA or RalB in computational and combinatorial screens and selected 99 hits. These were evaluated in a series of secondary assays allowing us to select Rai Binding Compound (RUC)8 and 10 to be pursued in this application. RUC8 and 10 were selected because they: 1) inhibit RalA to RalBPI binding in human UC cells and RalA induced spreading in murine embryo fibroblasts; 2) inhibit in vitro monolayer growth (IC50 0.5-1.9 pM) of human UC cells; 3) bind RalB directly by nuclear magnetic resonance (NMR) spectroscopy; and 4) have good pharmacokinetic (PK) properties in mice (Cmax 1.3-23 pM, T1/2 3.7-4.6 hrs). To develop this novel class of agents we propose the following Specific Aims: Aim 1: Characterize higher potency 2^* generation compounds based on RUC8 and 10 using medicinal chemistry, computational fragment-based design, and similarity search of chemical databases. In the unlikely situation that higher potency compounds are not found in Aim 1, we will pursue Aim 2 and 3 using RUC8 and 10, given their adequate IC50 and in vivo PK. Aim 2: Evaluate 2 generation compounds for their in vivo therapeutic efficacy in novel human UC models of visceral metastasis. Aim 3: Develop predictive biomarkers of response to antlRal therapeutics in human tissues that will position us for Phase 1 trials by end of this project. Documented interest by Astra Zeneca in our work improves overall chances for success in translating our novel Rai inhibitors into the clinical setting as anticancer therapeutics.

DESCRIPTION (provided by applicant): The benzoquinone ansamycin Hsp90 inhibitors 17-AAG and 17-DMAG are in Phase II trials but their use is limited by hepatotoxicity. In the last grant period we found that the active forms of the BQA Hsp90 inhibitors were their hydroquinone metabolites generated by the quinone reductase NQO1 rather than the parent quinones. We defined the molecular determinants of hepatotoxicity of BQA Hsp90 inhibitors and have focused on the development of entirely novel 19-substituted BQAs (19BQAs) which are metabolized by NQO1 to active hydroquinone ansamycin Hsp90 inhibitors. 19BQAs were rationally designed in our labs (DR and CJM) specifically to prevent off-target interactions with glutathione and protein thiols and to be markedly less toxic than the current BQA derivatives in clinical trials. Preliminary data validates the underlying rationale and shows that 19BQAs do not interact with cellular thiols and are not toxic in mouse hepatocyte systems. In this proposal, we will focus on testing the hypothesis that 19BQAs are potent Hsp90 inhibitors that have markedly less toxicity than their parent quinones using both in-vitro and in-vivo approaches. We will finalize the structure-activity relationship for 19BQAs in the geldanamycin, 17-AAG and 17-DMAG series and characterize their structure and binding to purified Hsp90 using X-ray and NMR analysis. Cellular studies will define a) the Hsp90 and growth inhibitory effects of 19BQAs in both human breast and pancreatic tumor cells b) metabolism of 19BQAs to their hydroquinones by NQO1 using isogenic breast and pancreatic tumor cell lines differing only in NQO1 and c) the effects of 19 BQAs on association of Hsp90 with co-chaperones which modulate Hsp90 function and deliver key client proteins essential for growth to Hsp90. The hepatotoxicity of 19BQAs in both mouse and human hepatocyte systems will be characterized and together with data from studies in tumor cell systems will allow us to advance compounds with the greatest therapeutic index to in-vivo testing in xenograft and explant systems where the use of an integrated PK-PD model will address the in-vivo therapeutic selectivity of 19BQAs. Using a mechanistic approach, these studies will validate the potential of an entirely novel class of Hsp90 inhibitors designed to be markedly less hepatotoxic than the BQA Hsp90 inhibitors currently in clinical trials.

CBET - 1604712
PI: Das, Moumita

The human eye is filled with a vitreous gel that becomes progressively more fluid-like with age. The gel is a composite material composed of a network of stiff collagen fibers and flexible polysaccharide chains in an aqueous solution. This project will explore the relationships between the structure of the gel and its mechanical and thermodynamic properties in order to understand how changes in those properties are correlated with vision impairment. The project will address these relationships by studying a reconstituted gel made of the primary components of the vitreous material. The team of investigators will use a variety of experimental and theoretical methods to develop a quantitative understanding of the mechanical properties of the network and the thermodynamics of the reconstituted gel. The results of the study will provide insight into the mechanical origin of vitreous related pathologies and may provide guidance into effective therapeutic strategies to treat vision disorders. Results from the project will be used in courses for university students in biophysics and related fields. The team of investigators will participate in several outreach activities that encourage the participation of students, especially those from underrepresented groups, in science and engineering subjects. In addition, the team will participate with high school teachers in summer workshops to emphasize the importance of high-school mathematics as a foundation for success in college studies and professional careers.

The investigators will characterize experimentally the microrheology of the primary components of vitreous gel using purified extracts of hyaluronic acid, type II heterotypic collagen network, and reconstituted composites made of the collagen network and hyaluronic acid. They will use a combination of experimental and theoretical approaches to construct a mechanical phase diagram of reconstituted gel that can be used to relate material properties to structure, micromechanics, and concentrations of hyaluronic acid and collagen. They will construct a thermodynamic phase diagram of the reconstituted gel using experimental and analytical methods to investigate the sol-gel, gel collapse, and complex coacervation transitions in mixtures of hyaluronic acid and collagen. Finally, they will establish connections between changes in mechanical and thermodynamic properties of the reconstituted vitreous gel and pathological changes that lead to vision problems. The project will fill a gap in our understanding of how material properties of vitreous gel are quantitatively related to its macromolecular organization and to different types of environmental cues. It will provide important insights into changes in the vitreous gel with age or with onset of ocular pathologies. Results of the project may apply broadly to other materials such as soft biological tissues and biomimetic hydrogels.

Metabolic syndrome comprises a group of cardiovascular risk factors including obesity, high blood pressure, glucose intolerance and dyslipidemia whose underlying pathology is related to insulin resistance. We have shown that the xenobiotic-metabolizing enzyme NQO1 is critical to insulin sensitivity and protection from diet-induced morbidities. Increasing NQO1 expression using NQO1 transgenic mice protects against the negative biochemical and physiological effects of a high fat diet and confers increased insulin sensitivity. Pharmacological inhibition of NQO1 using selective mechanism-based inhibitors led to a severe impairment in insulin sensitivity in mice. NQO1 knockout animals are insulin-insensitive resulting in a diabetes-like phenotype and a null NQO1 polymorphism is associated with metabolic syndrome phenotypes in humans. Our data shows that NQO1 is required for activation of AKT by enabling its insulin-inducible interaction with the protein complex TORC 2 allowing AKT serine phosphorylation and full glucose utilization. Insulin administration led to a rapid and marked increase in NQO1 tyrosine phosphorylation by the insulin receptor and mutational analysis suggests that phosphorylation leads to major changes in NQO1 functionality. Conformation-dependent antibodies and electrophoresis also indicated marked changes in NQO1 conformation as a result of either altered pyridine nucleotide redox ratios or addition of insulin. Based on the cellular functions of NQO1, we will test 3 biologically plausible mechanisms underlying the observed role of NQO1 in insulin signaling and protection against metabolic syndrome phenotypes. 1) NQO1 modulates insulin-dependent signaling by protein scaffolding. We will test the hypothesis that the observed effects of NQO1 on insulin sensitivity are modulated by a change in NQO1 conformation induced either by alterations in pyridine nucleotide levels or by phosphorylation of NQO1 by the insulin receptor, facilitating optimal association of AKT and Rictor and downstream insulin signaling; 2) NQO1 generates NAD+ for optimal SIRT activity. Deacetylation via SIRTs is critical in insulin signaling and NQO1 can rapidly generate high levels of NAD+ for optimal sirtuin activity; 3) Stabilization of critical metabolic regulators. The key metabolic regulator PGC1? is protected against proteasomal degradation by NQO1. Both PGC1? and AMPK influence mitochondrial oxidative phosphorylation and biogenesis and protect against metabolic syndrome. We will therefore define whether NQO1 influences metabolic syndrome by modulation of PGC1? and AMPK levels. Our working hypothesis is that NQO1 plays a critical role in insulin sensitivity and protection against metabolic syndrome phenotypes but the critical question that remains to be answered is the mechanism(s) underlying the beneficial effects of NQO1.

Project Summary Declines in the numbers of physicians pursuing careers in patient-oriented research have raised national concerns about an impending crisis in public health care. While numerous medical fields are affected, the problem in psychiatry is particularly acute, with projected shortages prompting an analysis of the problem by the Institute of Medicine. Ironically, shortages of psychiatrists pursuing patient-oriented research careers occur at a time when discoveries in the fields of genetics and neuroscience are having an unprecedented impact on our understanding of behavior. Translating such basic science insights into discoveries in the patient-care realm will ne crucial for improving out understanding and treatment of several mental illnesses. The current R25 renewal is intended to further address the current crisis in mental heath research education through several specific aims, aims that build upon prior success of the past 10 years. These include 1) continuing to educate psychiatry residents under established (?traditional?) model of Intergrated Mentored Patient-Oriented Research Training (IMPORT), 2) building upon this model through the addition of a modified (?early immersion?) track that is designed to better accommodate trainees with greater research experience and respond to increasingly complex translational research methods, and 3) augmenting both models by the addition of early (PG1) elective time, mentoring committees, educational experiences in grant writing (including opportunities for securing pilot funding), and enhanced recruitment. If successfully renewed, the program will continue to benefit the nation, through the increased identification, recruitment and retention of a highly skilled cadre of patient-oriented psychiatric physician scientists, and by piloting and implementing a flexible, individualized model of research education for psychiatry residency programs nationally.