Pilot Project Grant Program Awardees

2007-2008 Pilot and Feasibility Grant Winners:

Richard Flavell
Professor & Chairman of Immunobiology

Role of c-Cbl in the regulation of hematopoietic stem cell self renewal and function

Michael Hodsdon
Associate Professor of Laboratory Medicine and Pharmacology

Cytokine-Glycosaminoglycan Interactions in Hematopoiesis

Zhong Yun
Assistant Professor of Therapeutic Radiology

Hypoxia and Hematopoietic Stem Cells

2006-2007 Pilot Project Recipients

Peter Glazer, MD, PhD
Professor of Therapeutic Radiology and Genetics
Chairman of Therapeutic Radiology

Targeted Correction of the Human b-Globin Gene
The ability to genetically manipulate human stem cells has the potential to enhance the utility of stem cell therapy for human disease. One emerging approach to targeted genome modification is the use of triplex-forming oligonucleotides (TFOs). These molecules bind to duplex DNA in a sequence-specific manner, and this binding can be used to stimulate recombination in mammalian cells. The overall hypothesis driving this application is that high-affinity, site-specific DNA binding by TFOs can be used as a tool to accomplish gene targeting in stem cells. Our long-range goal is to develop such molecules as reagents for targeted genome modification of disease-related genes in human stem cells. In Aim 1, we will develop TFOs to bind to the human b-globin gene. We have identified two sites in intron 2 of the b-globin gene (IVS2-24 and IVS2-64) that are amenable to triplex formation. We have developed and tested a series of TFO chemical modifications to optimize binding to these sequences under physiologic conditions. Modifications to be tested include 2’-O-aminoethyl (2’AE), 8-oxoadenine (8oxoA), and 7-deaza-8-azaguanine (7c8nG) substitutions. We will also develop peptide nucleic acids (PNAs) specifically targeted to b-globin sequences. In Aim 2, we will test b-globin-directed TFOs and PNAs to target b-globin gene sequences in specially designed reporter gene assays in cell lines as well as in primary cells.

Melissa Kacena, Ph.D.
Assistant Professor of Orthopaedics

Translational study of megakaryocyte GATA-1 missense mutations and bone density
A powerful new regulatory pathway between megakaryocytes and bone cells identified in mice also exists in the human correlate, according to translational research. Mice deficient in GATA-1, a transcription factor required for megakaryocyte development, have increased numbers of immature megakaryocytes (10 to 100-fold increase), a drastic reduction of platelets (15% of control level), and an unanticipated three-fold increase in bone volume. Recently, eight families with GATA-1 missense mutations have been identified, having hematological phenotypes similar to those seen in GATA-1 deficient mice. It is our hypothesis that humans with mutations in GATA-1 develop a high bone mass phenotype similar to that in GATA-1 deficient mice. We intend to evaluate the bone density in GATA-1 patients and characterize their biochemical markers of bone turnover. Next, we will investigate the osteoclastogenic potential of peripheral blood mononuclear cells from these patients. These studies are relevant to megakaryocyte associated diseases such as thrombocytopenia and idiopathic myelofibrosis, new pathways of bone mass regulation, and bone diseases such as osteoporosis and osteopetrosis.

Peining Li, Ph.D.
Assistant Professor of Genetics

Lineage-oriented molecular cytogenetic approach and clone-based genomic analysis for hematologic disorders
This pilot study will develop new FISH (fluorescence in situ hybridization) probes and validate an antibody-captured cell array for lineage-oriented in situ cell culture and FISH testing.

There are more than 250 known chromosome rearrangements in various leukemias and only 40-50 probes are available from commercial sources. With the completion of the Human Genome Project, gene-specific BAC (bacterial artificial chromosome) clones are readily available to cover all major recurrent multi-partner translocations (MPTs) and chromosome deletions. These probes can be organized into test panels for the screening and detection of lineage-specific chromosome rearrangements and clonal evolution pathways.

Recent reports have demonstrated that microarrayed antibodies can be used to capture cells expressing surface antigens. To test the technical feasibility and clinical validity of this cell array technology in in situ karyotyping, FISH screening, and genomic analysis, antibody array prototypes will be constructed in capture lymphoid cells (T and B cells) and myeloid cells. The affinity of antibody-cell binding, specificity of cell capture, and feasibility of in situ cell karyotyping and direct FISH testing will be evaluated.

Successful completion of this pilot study will lead to further development of a lineage-oriented molecular cytogenetic approach and a clone-based genomic analysis for patients with hematological disorders.

E. Scott Swenson, M.D., Ph.D.
Instructor in the Department of Internal Medicine, Section of Digestive Diseases

Liver injury and repopulation by bone marrow stem cells
Cell therapy may be an alternative to liver transplant. Surprisingly, adult bone marrow contains cells capable of engrafting the liver and treating disease. The studies proposed here are designed to distinguish between mechanisms of engraftment and differentiation of bone marrow cells into functional liver cells. Cells from normal bone marrow can replace up to half of the diseased liver in the FAH knockout mouse model of hereditary tyrosinemia. The repair mechanism involves fusion events between marrow-derived cells and diseased liver cells. However, evidence from other models indicates that liver cells can arise from bone marrow cells in the absence of fusion with host cells.

A Cre-Lox reporter system will be used to test the hypothesis that marrow-derived hepatocytes can arise through cell-cell fusion or through direct differentiation, using FAH knockout mice or wild type mice subjected to subsequent injury with a liver toxin or focal liver irradiation. Using this binary reporter system, marrow-derived cells ubiquitously express β-galactosidase. However, fusion of a transplanted, marrow-derived cell with a host (Cre+) cell results in excision of the lacZ cassette and activation of GFP expression. Thus cells arising through direct differentiation will be lacZ+, while cells arising through cell-cell fusion will be GFP+.

2005-2006 Recipients

Erica Herzog, MD, PhD
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 phentotype 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, MD
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 Eµ-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, PhD
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).