Brent Berwin - US grants

[unreadable] DESCRIPTION (provided by applicant): Calreticulin (CRT) and gp96 (GRP94) are endoplasmic reticulum-derived molecular chaperones that elicit potent and effective immune responses that lead to the inhibition and rejection of a variety of tumors and their metastases. The efficacy of chaperones in eliciting immune responses against murine tumors has led to more than a dozen recent and ongoing human clinical trials, including current phase-Ill protocols to evaluate chaperone-induced suppression of human melanoma and renal carcinoma. Therefore, the broad goal of the proposed research is to understand molecular mechanisms of chaperone immunogenicity to enable rational and effective clinical use of these proteins. Antigen-presenting cells (APCs) are required for chaperone-mediated anti-tumor responses, which derive from: 1) stimulation of antigen-independent innate immune responses including APC maturation, activation and cytokine secretion, and 2) activation of the adaptive immune system by eliciting peptide- specific immune responses against associated antigens. At present, the mechanisms by which chaperones elicit these responses are poorly understood. The objective of the proposed studies is to define the mechanisms by which chaperones elicit immune responses from APC. We will do so by investigating: 1) How do chaperones access the APC MHC class-l antigen presentation pathway? 2) How do chaperones function as adjuvants? 3) What is the contribution of Scavenger Receptor Class-A in mediating chaperone-elicited immune responses? We recently identified a novel role for the scavenger receptors SR-A and SREC-1 as endocytic receptors of both gp96 and CRT: expression of Scavenger Receptor Class-A (SR-A) was sufficient to confer chaperone uptake, while SR-AV" macrophages and dendritic cells were impaired in this function. Moreover, SR-A ligands competed for cross-presentation of gp96-associated peptides. On the basis of these findings, we hypothesize that scavenger receptors function in mediating the immune responses stimulated by gp96 and CRT. Thus, we propose to elucidate the mechanisms by which scavenger receptors mediate the immunological effects of chaperones. We will use cellular and molecular techniques to identify the mechanisms by which chaperone complexes elicit antigen-specific responses, and genetic and immunological techniques to evaluate the contribution of scavenger receptors towards chaperone-mediated antigen presentation. Insights obtained will benefit the field of immunotherapy by elucidating the basis of a treatment that is currently undergoing clinical evaluation. These insights will facilitate the rational development and application of this anti-tumor therapy, and the understanding of chaperones as immunological molecules. [unreadable] [unreadable] [unreadable] [unreadable]

A Mouse; APC; Adjuvant; Antigen-Presenting Cells; Bacteria; Bacterial Infections; Blood leukocyte; CD8; CD8B; CD8B1; CD8B1 gene; CRISP; Cancer of the Ovary; Chaperone; Computer Retrieval of Information on Scientific Projects Database; Data; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Dendritic Cells; E coli; Escherichia coli; Family; Funding; Gram-Negative Bacteria; Grant; Immune response; Immunologic Accessory Cells; Institution; Investigators; LYT3; Leukocytes; Ligands; Malignant Ovarian Neoplasm; Malignant Ovarian Tumor; Malignant Tumor of the Ovary; Malignant neoplasm of ovary; Marrow leukocyte; Mediating; Molecular Chaperones; Monocytes / Macrophages / APC; NIH; National Institutes of Health; National Institutes of Health (U.S.); Pattern recognition receptor; Publishing; Receptor Protein; Research; Research Personnel; Research Resources; Researchers; Resources; Reticuloendothelial System, Leukocytes; Role; SR-A proteins; Source; T-Cells; T-Lymphocyte; TLR protein; Thymus-Dependent Lymphocytes; Toll-like receptors; United States National Institutes of Health; Veiled Cells; White Blood Cells; White Cell; abstracting; accessory cell; acetylated LDL receptor; bacterial disease; clinical data repository; clinical data warehouse; cytotoxic; data repository; host response; immunoresponse; in vivo; microbial; novel; ovarian cancer; receptor; receptor binding; receptor function; receptor, acetyl-LDL; relational database; response; scavenger receptor; scavenger receptors, class A; social role; thymus derived lymphocyte; trafficking; white blood cell; white blood corpuscle

Abstract The Gram-negative bacteria, which include Pseudomonas aeruginosa, cause substantial morbidity and mortality: bacterial pneumonia, septicemia and chronic disease account for ~15% of the total deaths in the USA. Therefore, it is imperative that we develop a better cellular and molecular understanding of the host interactions with bacterial pathogens, how bacteria avoid or manipulate the host's response, and to develop new strategies to prevent disease and improve patient care. Bacterial swimming motility, conferred by their flagella, has been recognized for over 25 years to influence the ability of bacteria to infect and colonize a host. Importantly, motility is required to initially infect the host, but bacteria must become non-motile to persist during clinical chronic infection. A well-described example is that the loss of P. aeruginosa motility directly correlates with increased bacterial burdens and increased disease severity in Cystic Fibrosis patients. However the underlying reasons for why and how changes in bacterial motility alter the course of infection and disease are unknown. We have recently provided the first formal demonstration that it is loss of bacterial motility, rather than loss of flagellar expression, that confers an advantage towards evasion of immune responses. Specifically, we have shown that loss of bacterial motility, in a variety of bacterial genera, results in bacterial resistance to phagocytosis in vitro and in vivo. Thus motility represents a novel and widespread mechanism by which the innate immune system recognizes and responds to bacteria ? and is a mechanism by which bacteria successfully elude immune responses during chronic infection. Therefore this proposal has the central goal of identifying the mechanisms by which immune cells respond to bacterial motility. Our recent finding that phagocyte PI3K and Akt activity are responsive to bacterial flagellar motility identifies novel regulation of an intracellular pathway that determines the phagocytic fate of Pa. We have leveraged this finding to identify two new critical molecular links in this motility-induced signal transduction pathway. Based on our preliminary data, in Specific Aim 1 our working hypothesis is that the Pa motility-stimulated phagocytic signal is transduced through a CIN85/src-family kinase pathway to PI3K/Akt. Our working hypothesis for Specific Aim 2 is that the c-Abl pathway is also responsive to, and required for, motility-induced phagocytosis. Therefore we propose to identify the c-Abl molecule(s) that contribute to motility-induced phagocytosis, and how this pathway functionally intersects with the PI3K/Akt axis. Achievement of these Aims will provide a mechanistic understanding of how loss of bacterial motility enables immune evasion and persistence of infection, and will identify molecular targets which can potentially be targeted to effect the therapeutic clearance of the non-motile, antibiotic-resistant bacteria present in chronic infections.

Abstract The Gram-negative bacteria, which include Pseudomonas aeruginosa, cause substantial morbidity and mortality: bacterial pneumonia, septicemia and chronic disease account for ~15% of the total deaths in the USA. Therefore, it is imperative that we develop a better cellular and molecular understanding of the host interactions with bacterial pathogens, how bacteria avoid or manipulate the host?s response, and to develop new strategies to prevent disease and improve patient care. For a variety of Gram-negative bacteria, flagellar swimming motility has been demonstrated to be required to initially infect the host. However, the bacteria must become non-motile to persist during clinical chronic infection. A well-described example is that the loss of P. aeruginosa motility directly correlates with bacterial persistence, increased bacterial burdens, and increased disease severity in Cystic Fibrosis patients. Our lab was the first to demonstrate that the observed loss of bacterial motility, regardless of loss of flagellar expression, enables P. aeruginosa to evade phagocytic clearance by macrophages and neutrophils both in vitro and in vivo. Additionally, sessile P. aeruginosa, especially upon formation of microcolonies or biofilms, become tolerant to standard-of-care antibiotics: this is reflected in ineffective eradication of bacteria from chronically infected patients following antibiotic treatments. These two traits ? phagocytic evasion and antibiotic tolerance ? enable non-motile P. aeruginosa to persist as chronic infections despite our best current treatments, and thus there is an obvious and pressing need for a novel intervention. Therefore the central goal of this proposal is to elucidate a novel and effective methodology that will induce the phagocytic clearance of non-motile P. aeruginosa. Our recently published studies that mechanistically demonstrate that non-motile bacteria avoid triggering the PI3K/Akt-dependent phagocytic response identify a potential therapeutic opportunity that guides the central hypothesis of this proposal: that activation of the Akt-dependent phagocytic pathway will promote the therapeutic clearance of the non- motile P. aeruginosa that persist in chronic infection. Based on our preliminary data, we propose a single, focused Specific Aim that tests the feasibility of our evidence-supported premise: that we can induce the phagocytic clearance of sessile bacteria. Achievement of this Aim will identify a molecular signaling pathway within host phagocytes, and a methodology by which to induce this pathway, which can potentially be harnessed to effect the therapeutic clearance of the non-motile, antibiotic-tolerant bacteria present in chronic infections.

Abstract/Project Summary The Gram-negative bacteria, which include Pseudomonas aeruginosa, cause substantial morbidity and mortality: bacterial pneumonia, septicemia and chronic disease account for ~15% of the total deaths in the USA, and neutropenic patients including those undergoing cancer therapies are especially susceptible to opportunistic bacterial infection. Of particular concern is the ongoing inability to eradicate chronic infections. A well- characterized example of this is P. aeruginosa for which there is no effective clinical therapy once the infection transitions from an acute infection to a chronic infection. This transition is hallmarked by the progressive loss, or down-regulation, of the bacterial flagellar swimming motility which can result in the formation of microcolonies or biofilms. Importantly, loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and to achieve antibiotic-tolerance that exceeds standard-of-care clinical treatments. Therefore, enabling the elimination of the non-motile P. aeruginosa that, despite our best current treatments, persist as chronic infections presents an obvious opportunity and need. Loss of bacterial motility confers resistance to phagocytosis due primarily to decreased bacterial association with the phagocytic cells, e.g. the non-motile bacteria avoid cell-surface interactions with the phagocytes that lead to ingestion. The proposed studies are based on the unprecedented observation that cell surface polyphosphoinositide lipids can promote a 30-fold clearance of non-motile P. aeruginosa. Moreover, this enables non-motile P. aeruginosa to be phagocytosed by a mechanism previously uniquely accessed by motile P. aeruginosa. This finding guides the central hypothesis of this proposal: that we have identified a novel mechanism by which to induce the phagocytic clearance of non-motile P. aeruginosa by human neutrophils. Thus, the proposed studies in Specific Aim 1 focus on identification of the mechanism by which addition of polyphosphoinositide lipids enable phagocytosis of non-motile P. aeruginosa. In Specific Aim 2 we provide preliminary data that support the hypothesis that P. aeruginosa interactions with phagocytic cells, in the absence of polyphosphoinositide treatment, are dependent upon endogenous cell-surface polyanions. This further reinforces the proposed mechanism by which polyphosphoinositides induce binding and, importantly, the proposed experiments will identify the cell-surface molecules that mediate bacterial interactions with host cells that result in phagocytosis. Achievement of these Aims will reveal from the host cells the mechanism by which phagocytes preferentially interact with motile bacteria and initiate their clearance while, concomitantly, non-motile P. aeruginosa evade phagocytosis. Additionally, these findings will enable and inform future studies directed at utilization of efficacious polyanions as a novel therapeutic opportunity to eradicate antibiotic-tolerant chronic infections.

Project Summary/Abstract: Career Enhancement Norris Cotton Cancer Center (NCCC) is a central provider and resource of Career Enhancement activities for its trainees, Program Members, at both Dartmouth and Dartmouth-Hitchcock Health system (D-H), and beyond. Importantly, the NCCC Career Enhancement programs link Dartmouth, and the Geisel School of Medicine, with D-H in the joint endeavor to train the next generation of cancer-focused physicians and scientists and to improve health locally and globally. In the last five years, our new initiatives have been responsive to emerging themes, needs and opportunities, and we have increased our funding base in key areas to implement these initiatives. These new programs span the breadth of stages of career development and include: 1. Successful founding of the NCI-funded Dartmouth Opportunities for Oncology Research (DOOR) Training Program for undergraduates from underrepresented minority (URM) or disadvantaged backgrounds; 2. Implementation of the Training Program in Surgical Innovation and the Quantitative Biomedical Sciences Graduate Program, both of which are recently NIH T32-funded and led by NCCC Members; 3. The establishment of two new medical residency programs in Radiation Physics and Radiation Oncology; 4. Synergistic interaction with the NIH-funded CTSA and COBRE Programs at Dartmouth to provide mentoring and grant writing support, which has directly contributed to the doubling of NIH Career Development awards (K and R00) as well as new Komen, American Cancer Society and NIH MIRA awards, to junior NCCC Members during this reporting period; 5. The expansion of training and trainee participation in Global Oncology, facilitated by active partnerships with institutions in Honduras and Rwanda; 6. NCCC has collaborated with Dartmouth, Geisel, and D-H to hire 21 cancer and oncology-focused faculty within 11 different departments during the current reporting period. Future activities and goals to facilitate Career Enhancement at NCCC have been prioritized and many are already underway. Burgeoning initiatives are described within this application and include: integration of a new Medical Masters in Science Program; the new Radiation Physics residency program underway (2018) and the Radiation Oncology residency program that will begin next summer (2019); The Translational Oncology Program Scholars (TOPS) program begins the summer of 2019, and will provide Geisel medical students a mentored research experience and exposure to clinical translational medicine in order to give an insider?s view of a career as a translational physician-scientist; and the 2019 launch of the D-H Cancer Faculty Fellows Program in which six to nine faculty members from the clinical departments will be provided with 40% protected academic time for 4-year terms to pursue cancer-focused research.