Dr. Staudt's laboratory is currently focused on understanding the molecular pathogenesis of human leukemias and lymphomas. Many of the abnormalities in lymphomas, leukemias and multiple myeloma involve genes that also regulate normal lymphoid development. In particular, the laboratory is interested in the normal development of germinal center B cells since many of the non-Hodgkin's lymphomas are derived from cells that have passed through this developmental stage. A major current tool in the lab is genome-wide DNA microarray analysis of gene expression which is capable of quantitating the expression of tens of thousands of genes in parallel. This technology is used to view the gene expression profiles of distinct stages of lymphoid development and compare these to the gene expression profiles of human lymphoid malignancies. The goal of this approach is to provide a new molecular classification of these cancers. A complementary and parallel effort in the laboratory is aimed at understanding how individual oncogenes, tumor suppressors and signaling pathways cause lymphomas. The Staudt laboratory creates DNA microarrays by robotically spotting cDNAs representing defined genes in an ordered microscopic array on a glass slide. Fluorescently labeled cDNA probes are then prepared from total cellular mRNA derived from the cell of interest and hybridized at high concentration to this microarray. The extent of hybridization of the probes to each cDNA on the microarray is then quantitated using a modified confocal microscope. Two different cell types can be directly compared with each other on the same microarray by labeling the cDNA from each cell with a different fluorochrome. To study gene expression in lymphoid malignancies, Dr. Staudt's laboratory constructed a specialized DNA microarray, termed the """"""""Lymphochip"""""""", that is enriched in genes which are selectively expressed in lymphocytes and genes which regulate lymphocyte function. Since the majority of human lymphomas appear to represent malignant transformation of the germinal center B lymphocyte, a cDNA library was created from germinal center B lymphocytes that were purified by flow sorting from human tonsils. Through the Cancer Genome Anatomy Project, 50,898 sequences were obtained from this library, over 10% of which had not been observed previously in other libraries. In addition, 14,645 EST sequences have been generated from cDNA libraries of a variety of B cell malignancies, with a similarly high rate of gene discovery. This rich source of novel genes formed the basis of the Lymphochip microarray, which currently contains over 18,000 clones. Initial experiments with the Lymphochip have focused on three common B cell malignancies: diffuse large cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia. These malignancies were chosen for study since they may encompass a variety of molecularly distinct diseases that cannot be distinguished morphologically. Disease-specific sets of genes were identified that were characteristically expressed in all cases of one malignancy and not the others. Nonetheless, substantial variation in gene expression was observed between patients within a given diagnostic group. Lymphochip microarray analysis of gene expression in diffuse large B-cell lymphoma samples revealed that this single diagnosis actually contains two different diseases that differ in the expression of hundreds of genes. The two types of diffuse large B-cell lymphoma that were discovered each resemble a different type of normal B lymphocyte, suggesting that these cancers have distinct cellular origins. Clinically, patients with these two types of diffuse large B-cell lymphoma had strikingly different responses to chemotherapy. Patients with one lymphoma subtype, termed germinal center B-like diffuse large B-cell lymphoma, had a favorable prognosis: 75% of these patients were cured by chemotherapy. Patients with the other lymphoma subtype, termed activated B-like diffuse large B-cell lymphoma, had a poor response to chemotherapy with less than one quarter of these patients achieving a long term remission. This study provided a clear demonstration that genomic-scale gene expression analysis can define clinically important subtypes of human cancer. Recently, the question has been raised whether the diagnosis of CLL comprises two distinct diseases, one in which the immunoglobulin genes of the leukemic cells are mutated (Ig-mutated CLL) and another one in which the immunoglobulin genes are unmutated (Ig-unmutated CLL). Patients with Ig-unmutated CLL have a much more aggressive disease requiring earlier treatment. The Staudt Laboroatory used gene expression profiling to test the hypothesis that CLL is two distinct diseases. These studies revealed that all CLL patients share a common gene expression signature demonstrating that CLL should be thought of as a single disease. Nonetheless, highly significant gene expression differences were found between Ig-mutated CLL and Ig-unmutated CLL. The CLL subtype distinction genes were enriched in genes that are modulated during mitogenic signaling of B lymphocytes through the antigen receptor, raising the intriguing possibility that antigen stimulation may contribute to the pathogenesis of CLL. Furthermore, the Staudt laboratory showed that the CLL subtype distinction genes could be used to make a predictive test that distinguished Ig-mutated and Ig-unmutated CLL with 100% accuracy, a test which could be used clinically to guide treatment decisions for these patients. The Staudt laboratory is currently studying the expression profiles of hundreds of lymphoma, leukemia and multiple myeloma specimens provided by a consortium of cooperating institutions from around the world in an effort called the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). The goal of the LLMPP is to provide a molecular classification of all lymphoid malignancies that is useful clinically. Ultimately, this effort will guide patients towards therapies that are tailored for their particular diseases and will identify new molecular targets for therapeutic development. Another major area of interest in the Staudt laboratory is how dysregulation of the oncogene BCL-6 causes non-Hodgkin's lymphomas. The BCL-6 gene is translocated in approximately 32% of diffuse large cell lymphomas and is also frequently rearranged in other non-Hodgkin's lymphoma sub-types and in AIDS-associated diffuse lymphomas. The coding region of BCL-6 remains unmutated in the lymphomas suggesting that dysregulation of gene expression underlies the lymphomagenesis. The laboratory has previously shown that the BCL-6 protein is a zinc finger transcriptional repressor protein which is expressed at highest levels in germinal center B cells and a subset of T cells. By disrupting the BCL-6 gene in the mouse germ line, the laboratory demonstrated that BCL-6 is required to initiate a germinal center immune response and to prevent undesired inflammatory responses. Since BCL-6 is a potent transcriptional repressor, Lymphochip microarrays were used to screen for genes that were downregulated after introduction of wild type BCL-6 into B cell lines that lack endogenous BCL-6 expression. In addition, a dominant negative form of BCL-6 consisting only of its DNA binding domain was introduced into B cell lines that naturally express BCL-6 and Lymphochip microarrays were used to detect genes that were """"""""de-repressed"""""""". Only genes that were affected by both of these manipulations were deemed presumptive BCL-6 target genes. The BCL-6 target genes provided rich insights into the known BCL-6-regulated phenotypes. One set of BCL-6 target genes consists of many B cell activation genes such as CD69, CD44, Id2 and cyclin D2. These genes are induced by BCR stimulation of resting B cells and BCL-6 was able to block this induction. In contrast, none of these genes is expressed in germinal center B cells, which have high levels of BCL-6 protein. A second critical BCL-6 target gene was blimp-1, a transcriptional repressor that is critical for plasmacytic differentiation. The ability of BCL-6 to repress blimp-1 suggests that BCL-6 blocks plasmacytic differentiation. Indeed, dominant negative BCL-6 was able to cause partial plasmacytic differentiation in a Burkitt's lymphoma cell line. These target genes may explain how BCL-6 controls the fate of a B cell following antigen exposure. A naive splenic B cell encountering antigen can either become activated and differentiate rapidly into plasmacytic cells in the periarteriolar lymphoid sheath or differentiate into a germinal center B cell in the follicular region. By blocking expression of B cell activation genes and blimp-1, BCL-6 may skew the fate of the B cell towards the germinal center program. The inflammatory phenotype of BCL-6 mutant mice may be explained, in part, by the fact that BCL-6 inhibits expression of the chemokines, MIP-1 alpha and IP-10. These chemokines attract monocytes and activated T cells to sites of inflammation and their derepression in BCL-6 mutant mice could contribute to the observed myocarditis and pulmonary vasculitis. Finally, BCL-6 target genes provide a plausible mechanism by which BCL-6 causes lymphomas. Differentiation of germinal center B cells into plasma cells is accompanied by loss of BCL-6 expression, thus allowing blimp-1 to be expressed. Translocation of BCL-6 in non-Hodgkin's lymphomas prevents this physiological down-regulation of BCL-6 expression, thus blocking blimp-1 expression and trapping the cell at the germinal center stage of differentiation. A target gene of blimp-1 repression is c-myc and therefore BCL-6 translocations would indirectly maintain progression through the cell cycle by elevating c-myc expression. These findings place BCL-6 at the top of a regulatory cascade of transcription factors. Cell cycle progression is also promoted by the ability of BCL-6 to repress p27kip1, a cyclin-dependent kinase inhibitor. Thus, BCL-6 translocations co-opt the normal regulatory functions of BCL-6, thereby promoting proliferation, preventing terminal plasmacytic differentiation and possibly allowing secondary oncogenic hits to further transform the cells.

Agency
National Institute of Health (NIH)
Institute
Division of Clinical Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01SC004024-15
Application #
6756240
Study Section
Metabolism Study Section (MET)
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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