Pilot Project Grant Program Awardees

2005-2006 Pilot and Feasibility Grant Winners

• Erica Herzog, M.D., Ph.D.
  Assistant Professor of Internal Medicine (Pulmonary and Critical Care)
  Bone Marrow Derived Stem Cells Contributing to Alveolar Repair
• Gary Vanasse, M.D.
  Assistant Professor of Internal Medicine (Hematology)
  Role of SOCS3 in Lymphopoiesis
• Arati Khanna-Gupta, Ph.D.
  Research Scientist (Internal Medicine-Hematology)
  Role of Gfi-1 in Specific Granule Deficiency and Myelodysplasia
• Richard Flavell, Ph.D.
  Professor and Chairman of Immunobiology
  HHMI Investigator
  Role of c-Cbl in the regulation of hematopoietic stem cell self renewal and function

2005-2006 WINNERS' ABSTRACTS

Erica Herzog, M.D., Ph.D.
Assistant Professor of Internal Medicine (Pulmonary and Critical Care)

Bone Marrow Derived Stem Cells Contributing to Alveolar Repair
Until recently, local stem cells were thought to be the sole sources of epithelial repopulation following lung injury. However, an increasing body of data indicates a role for bone marrow derived cells (BMDCs) in this process. Despite evidence for a functional phenotype of these marrow derived cells, to date most of the studies done in this area have used whole bone marrow cells (WBMCs) as the donor population. While appraising the ability of marrow transplantation to ameliorate the functional characteristics of a number of disease states, these studies raise questions regarding eh specific marrow cell population with the ability to adopt the phenotype of mature pneumocytes.

This grant will present studies designed to evaluate the cell surface phenotype of BM derived subpopulations that can give rise to functional type II alveolar cells. This will be performed by transplanting wild type marrow that has been separated by FACS on the basis of surface markers, into mice that lack a functional gene for the pathognomic T2 cell gene product surfactant protein C (SPC). The mechanism of this change will also be addressed by subsequent experiments designed to evaluate for direct differentiation of heterokaryon transgenic mouse models, single cell analysis of pneumocytes using flow cytometry and immunohistochemical techniques, assessment of marrow origin using in situ hybridization, and contribution of these marrow derived cells to lung homeostasis on a number of biochemical, molecular, and functional levels.

Gary Vanasse, M.D.
Assistant Professor of Internal Medicine (Hematology)

Role of SOCS3 in Lymphopoiesis
Apoptosis or programmed cell death is a physiologic process required to maintain cellular homeostasis. Impaired apoptosis has protean consequences and has been implicated as a central event in a wide range of hematologic disorders, including neoplasia, autoimmunity, and ineffective hematopoiesis. The Bcl-2 family of pro- and anti-apoptotic proteins is the primary mediator of caspase activation involved in the regulation of apoptosis, with deregulated expression of Bcl-2 resulting in abrogation of the majority of apoptotic pathways in B and T cells. My preliminary investigations into genes that serve as downstream effectors of Bcl-2 reveal that overexpression of Bcl-2 is associated with induction of the suppressor of cytokine signaling 3 (SOCS3) gene in both murine CD19+ polyclonal B cells and human follicular lymphoma (FL) B cells from patients with de novo FL. The factors which regulate SOCS3 expression in B cells as well as its possible role in B cell development and signaling remain to be clearly defined. This pilot proposal coordinates the efforts of interdisciplinary faculty members and aims to utilize innovative and centralized core facilities to test the broad hypothesis that Bcl-2-associated induction of SOCS3 promotes aberrancy in B cell signaling and/or development and leads to B cell proliferation. The Cell Preparation and Analysis Core will be utilized to investigate the role of SOCS3 overexpression in the presence or absence of Bcl-2 on the proliferative capacity of B cells. Specifically, bone marrow derived hematopoietic stem cells retrovirally transduced with SOCS3 will be transplanted into recipient mice and effects on B cell development and cytokine-induced B cell proliferation analyzed. In addition, to investigate the role of SOCS3 in IL-6-mediated B cell signaling and to determine whether some affects of Bcl-2 overexpression are mediated via SOCS3, SOCS3-specific small interfering RNA will be employed to downregulate SOCS3 expression in B cells from E µ-Bcl-2 transgenic mice that have been stimulated with IL-6. Finally, the Genomics and Bioinformatics Core facilities will be used to further investigate differential gene expression pathways important for Bcl-2 mediated induction of SOCS3 in B cells. Characterization of Bcl-2 and SOCS3-associated pathways may provide important insight into B lymphopoiesis and may identify novel cellular targets for future therapeutic intervention.

Arati Khanna-Gupta, Ph.D.
Research Scientist, Internal Medicine (Hematology)

Role of Gfi-1 in Specific Granule Deficiency and Myelodysplasia
Neutrophil secondary granule deficiency (SGD) is an extremely rare congenital disorder characterized by frequent bacterial infections. Previous studies from our laboratory demonstrated defects in mRNA expression of the secondary granule protein (SGP) genes and defensins in one of these patients. Interestingly, SGP expression is also uniformly absent in induced leukemic cell lines, in primary leukemic cells and in myelodysplasia (MDS). We have therefore sought to elucidate the molecular basis for SGD both to provide insight into the regulation of neutrophil specific gene expression and as an entry point for the study of transcriptional dysregulation in premalignant and malignant myeloid disorders. Extensive studies from our laboratory and others have demonstrated that the positive regulation of SGP depends in part on members of the C/EBP family of transcription factors, and that C/EBPe in particular is a critical regulator of SGP transcription. In support of this hypothesis, neutrophils from C/EBPe-/- mice have morphological and biochemical features very similar to those observed in patients with SGD. Noting this similarity, sequence analysis of genomic DNA from two SGD patients revealed mutations within the C/EBPe gene, resulting in a mutant protein that lacked transcriptional activity. We conducted a similar genomic DNA analysis at the C/EBPe locus of two SGD patients. However, no mutations in the C/EBPe genes were found and lowered protein levels of the zinc-finger transcription factor Gfi-1 were found. Our data suggest that SGD can be classified into two categories: C/EBPe negative and C/EBPe positive. We hypothesize that C/EBPe positive SGD involves mechanisms that likely involve abrogation of functional C/EBPe activity. In order to gain a understanding of the mechanisms underlying SGD we propose the following: i) to generate myeloid cell lines from the bone marrow of Gfi-1+/- and Gfi-1-/- as well as C/EBPe+/- and C/EBPe-/- mice to recapitulate the two SGD types in cell line models for comparative analysis and ii) to identify the contribution of Gfi-1 to the neutropenia associated with myelodysplasia (MDS).

Richard Flavell, Ph.D.
Professor and Chairman of Immunobiology
HHMI Investigator

Role of c-Cbl in the regulation of hematopoietic stem cell self renewal and function
Hematopietic stem cells (HSCs) are multipotent progenitors that give rise to all types of blood cells. HSCs have the ability to strike a balance between self-renewal and lineage commitment. Even though the phenotypic and functional properties of HSCs have been extensively characterized, molecular machinery that governs self-renewal of HSCs remains largely unknown. In the proposed project, we aim to dissect the role of an E3 ubiquitin ligase- c-Cbl in controlling the self renewal of HSCs.

Our initial studies will be focused on characterizing the HSC compartment in the bone marrow of c-Cbl deficient mice. Next, we will assessth e function of c-Cbl deficient HSCs through competitive repopulation and serial transplantation experiments. In addltiono the capacity of c-Cbl deficient HSCs to differentiate into various hematopoietic lineages will be evaluated through in vitro and in vivo differentiation experiments. Finally, we would focus on understanding the molecular mechanisms through which c-Cbl might regulate the development and function of HSCs.

In summary the proposed project might provide valuable insights into the ubiquitin mediated control of HSC self renewal. A better understanding on the roles played by c-Cbl in cytokine signaling might be useful for manipulating stem cells for tissue engineering and cell based therapies.