Dr. Staudts laboratory is currently focused on understanding the molecular pathogenesis of human leukemias and lymphomas. One major area of interest is in diffuse large cell lymphoma caused by the BCL-6 oncogene. Diffuse large cell lymphoma is a malignancy of mature B lymphocytes that accounts for roughly 40% of cases of non-Hodgkins lymphoma. The BCL-6 gene is translocated in approximately 32% of diffuse large cell lymphomas and 70-80% have mutations in a presumptive 5 regulatory region of the gene. BCL-6 is also frequently rearranged in other non-Hodgkins 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. To gain insight into the normal biological function of BCL-6, the gene was mutated in the mouse germ line using homologous recombination in embryonic stem cells. Mice homozygous for the mutant BCL-6 allele were born normally but display growth retardation shortly after birth and frequently die within 1 to 4 weeks. Upon immunization with a T cell-dependent antigen, the BCL-6 mutant mice failed to generate germinal centers and do not mount an IgG immune response. The second characteristic phenotype of BCL-6 mutant mice was a severe inflammatory disease consisting of myocarditis and pulmonary vasculitis. These data demonstrated that BCL-6 is required to initiate a germinal center immune response and to prevent undesired inflammatory responses and thus is a key regulator of the immune response.The inflammatory disease in BCL-6 mutant mice involved helper T cells that produce a Th2 pattern of cytokines (IL-4, IL-5, IL-13). Consistent with this pattern of lymphokine production, the inflammatory lesions displayed marked eosinophilia and the BCL-6 mutant mice produce elevated titers of IgE following immunization. Differentiation of naive T cells to Th2 cells in vitro requires IL-4 signaling through the Stat6 transcription factor. To understand the role of BCL-6 in regulating Th2 differentiation in vivo, the BCL-6 mutant mice were bred with animals deficient in IL-4 or Stat6. Surprisingly, neither IL-4 nor Stat6 were required for BCL-6 mutant mice to manifest the Th2 inflammatory phenotype. These data demonstrated that BCL-6 regulates a previously unsuspected, mechanistically distinct pathway to Th2 differentiation. These findings were confirmed and extended by the observation that BCL-6 mutant mice developed a Th2 response to the parasite Leishmania major and, consequently, the mice were unable to control infections with this parasite. Current efforts are aimed at identifying the genes which are transcriptionally repressed by BCL-6 that may account for the immune defects in BCL-6 mutant mice and in the ability of this oncogene to cause lymphomas. A second major initiative of the laboratory uses functional genomics to characterize gene expression patterns in normal and malignant lymphocytes. Recently, a new technology utilizing cDNA microarrays has emerged that permits the simultaneous quantitation of the expression of thousands of genes. In brief, this technique relies on robotic spotting of cDNAs for 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. Genomic-scale knowledge of gene expression in a malignant lymphoid cell should allow us to determine which normal lymphocyte subset gives rise to a particular subtype of lymphoid malignancy, which signaling pathways are altered in the malignant cells, and the molecular basis for responsiveness or resistance of lymphomas and leukemias to therapy.Dr. Staudts laboratory has designed a specialized cDNA 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. 50,000 sequences were obtained from this library, over 10% of which had not been observed previously in other libraries. This rich source of novel genes formed the basis of the Lymphochip microarray that currently contains over 18,000 clones.Initial experiments with the Lymphochip have focused on the three most common lymphoid malignancies: diffuse large B cell lymphoma, follicular lymphoma and chronic lymphocytic leukemia. Each malignancy could be readily distinguished from the others by gene expression profiling. Nonetheless, considerable gene expression heterogeneity was observed within each diagnostic category. Thus, each diagnosis may encompass a variety of molecularly distinct diseases that cannot be distinguished morphologically. Two subgroups of diffuse large B cell lymphoma could be discerned, each of which possessed a characteristic gene expression signature. To define the normal B cell counterparts of these two lymphoma subgroups, a comprehensive database of gene expression changes during B cell differentiation was generated. Germinal center B cells were found to have a distinct gene expression profile that was not observed in mitogenically activated peripheral blood B cells. Interestingly, Lymphochip gene expression analysis revealed that one of the diffuse large cell lymphoma subgroups strongly resembled normal germinal center B cells whereas the other subgroup resembled activated blood B cells. The two diffuse large cell lymphoma subgroups were found to have strikingly different overall and event-free survival, demonstrating that gene expression profiling can provide prognostic information that may be used eventually to guide patients to optimal therapy.
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