Linda M. Boxer - US grants

DESCRIPTION (provided by applicant): A number of lymphoid malignancies are associated with specific chromosomal translocations. Many of these translocations result in the activation of an oncogene. The majority of low-grade follicular lymphomas have a t(14; 18) with translocation of the bcl-2 gene to the immunoglobulin heavy chain locus. Bcl-2 expression is deregulated in these lymphomas, presumably due to the influence of the immunoglobulin enhancers, although the molecular mechanisms of the deregulation are not understood.We propose to examine the molecular mechanisms involved in the deregulation of the translocated bcl-2 gene using several model systems. We postulate that the IgH enhancers maintain the bcl-2 promoter in an open transcriptional conformation through the recruitment of histone acetylases allowing the binding of transcription factors to the promoter. Interference with the function of the most active transcription factor(s) should therefore decrease bcl-2 expression. Studies will be performed to test these assumptions. The most active elements of the immunogiobulin heavy chain enhancers will be characterized, and the elements of both the 5' and 3' bcl-2 promoters that interact with them will be defined. The regulation of the normal bcl-2 gene in B cells will also be studied and compared to that of the translocated and untranslocated bcl-2 genes. A mouse model of the translocation will be made to gain further insight into the mechanisms involved in the deregulation of the bcl-2 gene. Strategies to interfere with transcription factor function will be developed to decrease bcl-2 expression in cells with deregulated expression. 1. Elucidation of the role of the CRE and CDX sites in the regulation of bcl-2 expression in B cells without the t(14;18). We have determined that these sites bind several transcription factors that are critical for bcl-2 regulation. 2. Mechanisms involved in deregulation of the translocated bcl-2 allele. a. Elucidation of the molecular mechanisms involved in deregulation of the bcl-2 promoter in t(14; 18) lymphomas: role of the CRE and CDX sites. b. Determination of the role of the IgH enhancers in the deregulation of expression of bcl-2. The most active site in each enhancer will be studied. 3. Construction of a mouse model to study the deregulation of bcl-2 by the 3' immunoglobulin heavy chain enhancers. Molecular studies will be performed to determine the mechanisms involved in deregulation of bcl-2, and methods will be developed to interfere with bcl-2 activation. We will attempt to create a mouse model of the t(14;18) lymphoma.These studies should provide the groundwork for the development of novel therapies for future therapeutic trials in lymphomas with deregulated bcl-2 expression.

DESCRIPTION: (adapted from the investigator's abstract) The applicant proposes to study the mechanism of activation of c-myc at a molecular level in both Burkitt's lymphoma and transformed lymphomas with the t(8;14) translocation. The goal is to reach a better understanding of the mechanisms of malignant transformation of B cells. A. Identification of the molecular mechanisms of transcriptional control of the c-myc gene in B cells. In vivo footprinting and in vitro protein-DNA binding studies will be used to locate regulatory regions important for c-myc expression in normal B cells and in B cell lines that do not contain the t(8;14) translocation. The identity of the proteins that bind in vivo will be determined by a technique of UV crosslinking and immunoprecipitation followed by hybridization analysis. Functional assessment of the sites will be performed with transient transfection experiments in B cell lines. B. Identification of the molecular mechanisms of transcriptional control of the c-myc gene in Burkitt's lymphomas. Conditions have been optimized for the separation of the translocation from the normal c-myc gene in several Burkitt's cell lines. Differences in the binding of transcription factors between the two alleles have been found and these studies will be continued. The identification and characterization of the proteins that bind in vivo will be determined as described in A. The methylation status of each allele will be compared. C. Identification of the molecular mechanisms of transcriptional control of the c-myc and bcl-2 genes in transformed lymphomas and their low grade counterparts. The deregulation of both c-myc and bcl-2 by the immunoglobulin locus will be studied as outlined in B. D. Effect on c-myc expression of mutations identified in the regulatory regions. The effects of any mutations on the binding of regulatory proteins will be investigated by in vivo and in vitro studies. E. Role of the immunoglobulin locus in the activation of c-myc expression. Model constructs which reproduce the c-myc translocations will be constructed and used to define the important regions of the immunoglobulin locus. The successful completion of this proposal will represent the first instance where the molecular mechanisms involved in the transcriptional activation of an oncogene by translocation are known. The transcription factors that are required for the activation of c-myc will be identified and the regions of the immunoglobulin locus which are responsible for this will be defined.

DESCRIPTION: (adapted from the investigator's abstract) The c-myb oncogene is expressed at high levels in immature hematopoietic cells and in many leukemias. Leukemic cells appear to be more sensitive to antisense inhibition of c-myb expression than normal hematopoietic cells are. Although it is clear that leukemic cells require increased c-myb expression for proliferation, the molecular mechanisms that lead to this increased expression are not known. The goal of this proposal is to determine the molecular mechanisms responsible for the transcriptional control of the c-myb gene in both normal and malignant hematopoietic cells. 1. Identification of the molecular mechanisms of transcriptional control of c-myb in normal T cells. Activation of T cells causes increased expression of c-myb mRNA. They will perform in vivo footprinting, in vitro protein-DNA binding studies, and transient transfection experiments to study the transcriptional regulation of the c-myb gene. 2. Identification of the molecular mechanism of transcriptional control of c-myb in leukemic cells. Studies as described in Aim 1 will be performed on leukemic samples that express high levels of c-myb mRNA and cell lines will be used for the transient transfection experiments. The regulatory proteins identified in leukemic cells will be compared to those identified in normal T cells. 3. Role of the first intron in the transcriptional control of c-myb. They have shown that a positive regulatory region exists in the first intron. They will continue to study this and also investigate the role it plays in leukemic cells. They will also determine whether the block to transcription elongation is in the first intron or at the transcription start site. 4. Determination of the methylation status of c-myb. Methylation of CpG dinucleotides is thought to be involved in a gene silencing mechanism. They will determine the methylation status of c-myb in both quiescent and activated T cells, in HeLa cells which do not express c-myb, and in leukemic cells. With this information they hope to discover how c-myb expression is deregulated in leukemias and to begin to understand how this contributes to malignant transformation.

DESCRIPTION: Specific chromosomal translocations are associated with a number of hematologic malignancies. Several of these have been demonstrated to result in the activation of an oncogene. How an oncogene becomes activated by a translocation that does not affect its coding sequence is not known and is a central question in cancer research. This proposal will attempt to answer that question for the BCL-2 oncogene. In lymphomas with the t(14;18) translocation, the BCL-2 gene is translocated to the immunoglobulin locus. Because the immunoglobulin gene is active in B cells, a reasonable hypothesis is that the translocation somehow activates BCL-2, this is one step along the way to development of the malignant phenotype. The applicant proposes to study the mechanism of activation at a molecular level in human lymphoma tissue. The activation of the translocated BCL-2 gene will be compared to the transcriptional regulation of the normal BCL-2 gene during B-cell development and during B-cell activation. The goal is to reach a better understanding of the mechanisms of malignant transformation. The specific aims are: A) determination of the role of CREB in the regulation of BCL-2 in normal B cells; B) determination of the role of other transcription factors in modifying the activity of CREB on the BCL-2 promoter in normal B cells; C) determination of the role of CREB, WT1, pi1, p53, the URE factor, Myb and the immunoglobulin enhancers in the deregulation of the translocated BCL-2 gene in t(14;18) lymphomas; D) creation of a model system of the t(14;18) translocation to test the hypotheses for the deregulation of BCL-2. The successful completion of this proposal will represent the first instance where the molecular mechanisms involved in the transcriptional activation of an oncogene by translocation are known. The transcription factors that are required for the activation of expression of BCL-2 will be identified, and the region of the immunoglobulin locus that are responsible for this will be characterized.

DESCRIPTION: (Adapted from the applicant's abstract) We proposed to study the mechanism of activation of c-myc at a molecular level in human lymphoma tissue and in mouse model of the translocation. The goal is to reach a better understanding of the mechanisms of malignant transformation. 1. Completion of the characterization of the regulatory elements in the murine and human immunoglobulin heavy chain (IgH) enhancers that deregulate c-myc transcription. We will continue to identify the regulatory elements that are required for activation of c-myc, including increased transcription and P2 to P1 promoter shift. 2. Identification of the regions of the c-myc promoter that are required for the interaction with the IgH enhancers for maximun transcriptional activity and the promoter shift. Study of the mechanisms involved. 3. Determination of the mechanisms of transcriptional silencing of the normal c-myc allele in Burkitt's lymphoma. 4. Construction of a mouse model of the c-myc-IgH translocation. We will target the active sites of the IgH enhancers to the murine c-myc gene to recreate the Burkitt's translocation. 5.Development of strategies to interfere with c-myc transcription that is driven by the IgH enhancers. We have shown that several NF-kB sites are critical for deregulated c-myc expression, and we will first target the function of NF-kB.

DESCRIPTION (provided by applicant): Molecular cytogenetic studies have provided new information for the determination of prognosis in pediatric ALL. The genes involved in the major chromosomal translocations have been identified, and a number of studies have characterized the resulting fusion proteins. It is not known how the fusion proteins contribute to the process of leukemogenesis, however. Although knowledge of the different translocations in ALL is useful for predicting response to treatment, heterogeneity of response exists within the cytogenetic subgroups of ALL. We hypothesize that gene expression analysis will be able to distinguish among the different cytogenetic subgroups of pediatric B cell ALL. In addition, we will determine whether subgroups can be identified within several of the translocation groups of ALL and whether further information on prognosis can be obtained. We will examine whether a "relapse signature" can be identified by gene expression analysis of relapsed ALL samples within a translocation subtype. Evidence suggests that in vitro drug sensitivity correlates with in vivo response to the drug as measured by event-free survival. Microarray analysis will be used to follow changes in gene expression in response to a chemotherapeutic agent in both sensitive and resistant ALL cells. Further studies will be designed to identify genes that are directly correlated with expression of the fusion gene and important in the process of leukemogenesis. 1. Analysis and comparison of the gene expression profiles in pediatric ALL samples with t(9;22), t(1;19), t(4;ll), t(11;19), and t(12;21). 2. Correlation of gene expression patterns within the t(1;19) and t(12;21) ALL groups with prognosis and with relapse. 3. Determination of changes in gene expression in ALL cells, both sensitive and resistant, in response to chemotherapeutic agents. 4. Identification of fusion protein-specific events and studies of important genes and pathways in cell line

DESCRIPTION: (Adapted from the applicant's abstract) We proposed to study the mechanism of activation of c-myc at a molecular level in human lymphoma tissue and in mouse model of the translocation. The goal is to reach a better understanding of the mechanisms of malignant transformation. 1. Completion of the characterization of the regulatory elements in the murine and human immunoglobulin heavy chain (IgH) enhancers that deregulate c-myc transcription. We will continue to identify the regulatory elements that are required for activation of c-myc, including increased transcription and P2 to P1 promoter shift. 2. Identification of the regions of the c-myc promoter that are required for the interaction with the IgH enhancers for maximun transcriptional activity and the promoter shift. Study of the mechanisms involved. 3. Determination of the mechanisms of transcriptional silencing of the normal c-myc allele in Burkitt's lymphoma. 4. Construction of a mouse model of the c-myc-IgH translocation. We will target the active sites of the IgH enhancers to the murine c-myc gene to recreate the Burkitt's translocation. 5.Development of strategies to interfere with c-myc transcription that is driven by the IgH enhancers. We have shown that several NF-kB sites are critical for deregulated c-myc expression, and we will first target the function of NF-kB.

Follicular lymphoma is characterized by the t(14;18) translocation, which links the bcl-2 gene with the immunoglobulin heavy chain gene (IgH). The translocated bcl-2 allele is expressed at high levels, and the normal allele is silent. The increased levels of Bcl-2 contribute to resistance to chemotherapeutic agents, and follicular lymphoma is rarely cured. We have investigated how the translocated bcl-2 gene is deregulated by the IgH locus and have identified regulatory sites in the bcl-2 promoter and in the 3' IgH enhancers. The involvement of these sites has been confirmed in an episomal model of the translocation. In this proposal, we will build upon our previous results using a murine model of deregulated bcl-2 expression induced by the IgH 3' enhancers (lgH-bcl-2 mice). Our studies have provided insight into the mechanisms involved in the deregulation of bcl-2 expression and have also provided evidence that the activation of bcl-2 expression is more complex than previously thought. Based on these results, we propose to more closely examine the regulation of the two bcl-2 promoters in normal B cells and study how one promoter influences the other. In addition, our data demonstrate that while the IgH enhancers are required for the deregulation of bcl-2 expression, they are not sufficient, and that additional changes occur during the transformation process of the B cell. Our investigations will be performed on the bcl-2 gene in the genomic context so that we can study the chromosomal structure at the bcl-2 promoter and define the interactions with the IgH enhancers. Additionally, we will characterize the lymphomas that develop in lgH-bcl-2 mice and compare them to human follicular lymphomas. 1. Characterization of the mechanisms involved in the lack of transcription from the bcl-2 P2 promoter in the absence of the IgH enhancers. 2. Characterization of the molecular mechanisms of bcl-2 deregulation in B cells from lgH-bcl-2 mice to further clarify the mechanisms of bcl-2 deregulation in t(14;18) lymphomas. 3. Development of a preclinical model for t(14;18) lymphomas utilizing the lgH-bcl-2 mice. Relevance: We are studying a malignancy of B lymphocytes to understand the role of a genetic abnormality involving the bcl-2 gene, which codes for a protein that extends the survival of the malignant cells. Our investigations are designed to determine how the bcl-2 gene is expressed at high levels and to develop a mouse model for the human malignancy (follicular lymphoma). These studies will provide new information that can be used to treat the disease in humans.