Frederick W. Alt, PhD - US grants

Infection of bone marrow on fetal liver with Abelson Murine Leukemia virus (A-MuLV) transforms a small fraction of the cells into clonal continuous cell lines which derive from B-lymphoid (immunoglobulin-producing) cells. We have defined several sub-classes of these transformants which in culture undergo the molecular rearrangements associated with immunodifferentiation. The work described in this proposal is aimed at exploiting the properties of these sub-classes to study the regulation of Ig gene rearrangement and expression. Most previous studies in this area have focused on cell lines which represented the later stages of the B-cell pathway (i.e., myelomas) and which were terminally reorganized with respect to Ig gene structure. In contrast, most of our proposed studies will focus on cell lines which, in culture, undergo the rearrangement processes associated with the construction of complete heavy and light chain genes. With such lines we propose to analyze, in more detail than previously possible, the mechanism by which a given B-cell clone achieves functional rearrangement and expression of only one of its two heavy chain alleles (allelic exclusion) and one of its multiple light chain genes (allelic and isotypic exclusion). Details of the rearrangement process will be analyzed by the introduction of appropriate recombination substrates into cells which are actively undergoing heavy or light chain gene rearrangement. The hypothesis that these rearrangement processes are controlled by feedback from Ig chains will be tested by introduction of functional Ig genes into these lines -- both by cell infusion and gene transfer protocols. Finally, additional studies will focus on the mechanism by which expression of the functionally rearranged heavy chain gene is regulated over the course of B-cell differentiation. The availability of variant lines of A-MuLV tranformants with altered regulation of heavy chain gene expression should greatly facilitate these studies.

Heavy-chain class switching refers to the process in which a clone of B lymphoid cells first expresses a single heavy chain variables (V) region in association with mu heavy chains, and, subsequently, in association with another heavy chain class such as gamma or alpha. Most previous studies of this process have compared heavy-chain gene structure in unrelated tumor cell lines which secreted different classes of immunoglobulin chains. The research has developed Abelson murine leukemia virus-transformed pre-B cell lines which, in culture, undergo all of the gene reorganization events associated with the differentiation of cells of the B-lymphoid (antibody-producing) lineage including heavy-\and light-chain variable region assembly and heavy-chain class switching. The analyses of several such lines which have undergone a class switch event (mu to gamma[unreadable]2b[unreadable]) in culture, indicated that they have employed the genetic recombination/deletion mechanism thought to mediate physiological class switch events. Therefore, these lines offer an ideal model system to study the dynamic aspects of class switching. It is proposed to extend studies of this phenomenon by comparative analysis of class switch events occurring between the endogenous immunoglobulin gene segments in these lines and potential recombination events occurring within appropriate class switch recombination substrates that have been introduced into the genome of the same lines. These studies should help to answer a variety of unresolved questions concerning the class switch phenomenon including the details of the molecular mechanism, the differentiation stage at which switching occurs, and the mechanisms controlling the process. Some of the A-MuLV transformants have recently been shown to undergo a high rate of somatic mutation. Additional gene transfer studies will exploit such lines to elucidate the mechanism and control of the somatic mutation process. Finally, gene transfer technology will be used to analyze the well-established phenomenon of cellular oncogene (c-myc) translocation into the class switch region of the immunoglobulin heavy-chain genes in tumors of the B-cell lineage. These studies should help to elucidate the molecular mechanism of this translocation as well as the role of the class switch recombinase system in this aberrant translocation process. (HI)

The proposed studies are aimed at the elucidation of mechanisms that control precursor lymphocyte differentiation with a particular focus on factors that regulate antigen receptor variable region gene assembly (VDJ recombination). Cloned VDJ recombination substrates will be introduced into transgenic or chimeric animals to elucidate elements that control stage and lineage-specificity of VDJ recombinase activity within the lymphoid lineages. Other types of VDJ recombination substrates will be transfected into VDJ recombinase-positive cell lines to study molecular details of how particular elements defined in transgenic assays influence accessibility of substrate gene segments to VDJ recombinase. A particular focus of the latter studies will be to generate novel pre-B lines that provide a more physiologically representative system for these assays. Novel cellular and animals models will be generated to study factors involved with the progression of cells through the precursor stages of the B cell differentiation pathway. Animal models will include mice unable to assemble endogenous immunoglobulin heavy chain genes and mice that constitutively express VDJ recombination enzymes throughout B cell differentiation; these systems will permit elucidation of the function and control of ordered assembly of immunoglobulin heavy and light chain genes and help elucidate the function and regulation of allelic exclusion.

The N-myc gene is frequently amplified and overexpressed in a highly restricted set of related human tumors, including neuroblastomas and retinoblastomas. We have demonstrated that the N-myc gene encodes a protein very related to that encoded by the classical c-myc protooncogene. Furthermore, we have also demonstrated that this gene may have a role both in tumorgenisis and in normal development. To further characterize this unique oncogene, we will isolate both from humans and from mice, the complete N-myc gene and its flanking regions as well as complete cDNA copies of the N-myc mRNA; these reagents will be used to determine the complete structure and nucleic acid sequence of the N-myc gene(s). Comparative analyses of the coding and non-coding regions of the human and murine N-myc genes with each other and with the related c-myc should help to eludidate important regulatory or functional regions. Our previous studies have demonstrated a remarkable tissue and developmental-stage specifically of N-myc expression. To characterize this phenomenon in more detail, we will study N-myc expression in developing rats and mice by cDNA/RNA in situ hybridization techniques. To determine the molecular elements involved in tissue or stage-specific N-myc expression, we will develop model cell culture systems with which to analyze regulation of N-myc expression by gene transfer technology. In addition, we will make N-myc-specific antisera which we will use to identify the N-myc protein and to localize it within the cell. Finally, we have derived considerable evidence which suggests that there is a family of myc-related genes, possibly specific for unique cell lineages or developmental stages. We have already isolated numerous members of the myc gene family, both from humans and from mice. We will characterize the structure of these and additional members of this family and assay for their function and expression as outlined above for the N-myc gene.

The proposed studies are aimed at the elucidation of mechanisms that control precursor lymphocyte differentiation with a particular focus on factors that regulate antigen receptor variable region gene assembly (VDJ recombination). Cloned VDJ recombination substrates will be introduced into transgenic or chimeric animals to elucidate elements that control stage and lineage-specificity of VDJ recombinase activity within the lymphoid lineages. Other types of VDJ recombination substrates will be transfected into VDJ recombinase-positive cell lines to study molecular details of how particular elements defined in transgenic assays influence accessibility of substrate gene segments to VDJ recombinase. A particular focus of the latter studies will be to generate novel pre-B lines that provide a more physiologically representative system for these assays. Novel cellular and animals models will be generated to study factors involved with the progression of cells through the precursor stages of the B cell differentiation pathway. Animal models will include mice unable to assemble endogenous immunoglobulin heavy chain genes and mice that constitutively express VDJ recombination enzymes throughout B cell differentiation; these systems will permit elucidation of the function and control of ordered assembly of immunoglobulin heavy and light chain genes and help elucidate the function and regulation of allelic exclusion.

We have shown that Vav-deficiency results in defective development and activation of T lymphocytes. More recently, we found that the activation- defects in Vav-deficient murine lymphocytes are associated with defects in reorganization of the actin cytoskeleton, similar to those of human WASp- deficient lymphocytes. Members of the Rho-family of small GTPases, such as Cdc42, have been shown by others to be downstream effectors of Vav in fibroblasts and also to feed into both the cytoskeleton and stress- activated protein kinase cascades. The goal of the proposed work is to elucidate intracellular signaling pathways effected by Vav and its potential downstream effectors in lymphocyte development and activation and, in the context of these studies, to dissect the contribution of cytoskeletal versus mitogen and stress-activated protein kinase pathways. The first aim is to elucidate defects in signaling from the T and B cell antigen receptor in Vav-deficient lymphocytes. A major focus will be to employ Vav+ lymphocytes generated by RAG-2-deficient blastocyst complementation for studies aimed at elucidating potential roles of Vav in regulation of the actin-cytoskeleton, proliferation, and activation- induced cell death. We also propose to generate mice which harbor germline mutations in the Vav gene to facilitate studies of the physiologic consequences of Vav-deficiency on the immune system. The second aim is to dissect specific roles for Vav-protein domains and potential downstream effectors in the Vav-signaling pathway and will be accomplished by carrying out "rescue" experiments which involve introducing wild type or mutant cDNA expression constructs into Vav- deficient ES cells followed by assay via RAG-2-deficient blastocyst complementation. The third aim is to elucidate developmental and functional defects in lymphocytes deficient in potential downstream Vav- effectors, including Cdc42 (which has been functionally linked to both Vav and WASp), as well as particular MAP kinases (MEK-1 and SEK-1). In this aim, a major focus will be to compare and contrast potential phenotypic effects of specific mutations to those observed in the context of Vav- and WASp-deficient lympyhocytes.

[unreadable] DESCRIPTION (provided by applicant): Immunoglobulin (Ig) and T cell antigen receptor variable region genes are assembled from V, D, and J segments in developing B and T lymphocytes. These gene segments reside in large and complex chromosomal loci; yet their assembly is tightly regulated in the context of lineage and developmental stage. Also, rearrangements are feedback regulated to ensure generation of one antigen receptor gene for a given locus (allelic exclusion). Such control maintains specificity of the immune response. To effect regulation, multiple cis elements interact to modulate access of a common V(D)J recombinase to the proper substrate gene segments, a process we named "accessibility" control. Accessibility control, as related to transcription, replication, and repair, is a broadly relevant process; and V(D)J recombination control has provided a most important paradigm. Yet, specific mechanisms remain elusive. Over the past 5 years, we used gene- targeted mutation to "tailor" endogenous TCR(3 and IgH loci in mice to make amenable experimental systems. Specifically, we mutated various cis-regulatory sequences that alter normal rearrangement patterns with respect to order and, potentially, feedback. Proposed analyses of deregulated recombination events in these and other mutants will provide a basis for elucidating normal mechanisms. In addition, we developed a novel approach to measure "accessibility", which we will employ to elucidate how accessibility controls endogenous Ig gene assembly. Finally, we have collaborated with Regeneron Pharmaceuticals to use the novel VelociGene method to introduce major modifications over the 3MB endogenous Ig heavy chain locus. This approach has provided alleles that will serve as minimal locus substrates for studies of V(D)J recombination control. With the rapid accumulation of new information and technologies, we argue that the novel endogenous antigen receptor loci that we have generated leave us poised to generate important insights into variable region gene assembly that could not be readily gained by other approaches. Besides relevance to fundamental mechanisms of developmental biology, elucidation of V(D)J recombinational control has substantial implications for health and disease. Thus, the mechanisms we study are required for development of normal antibody and TCR repertoires. Correspondingly, defects in V(D)J recombination underlie immunological diseases ranging from immunodeficiency to autoimmunity. Finally, aberrant V(D)J recombination can unleash oncogenic activities via chromosomal translocations Lay Summary: We have tailored the chromosomes of mice to introduce major changes in genes that encode antibodies. Our studies of altered antibody genes will allow us to understand how the body responds to infections and how mistakes in formation of antibody genes lead to immunodeficiency, autoimmunity and cancer. These studies also may provide insights relevant to generation of antibodies as therapeutic agents for disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Immature T cell lymphomas account for a significant portion of human lymphoid malignancies and are a clinical problem as they are typically resistant to treatment. Many of these tumors harbor recurrent inter-chromosomal translocations and intra-chromosomal rearrangements (deletions) that activate proto-oncogenes, inactivate tumor suppressor genes, or create novel oncogenic fusion genes. Most oncogenic translocations of human immature T cell lymphomas likely arise through errors in the repair of specific DNA double strand breaks (DSBs) introduced at T cell receptor (TCR) loci during initiation of V(D)J recombination and/or more general DSBs at other genomic locations. In this context, mice deficient for the Ataxia Telangiectasia mutated (ATM) tumor suppressor protein, which can be inactivated in human immature T cell lymphomas, invariably develop thymic lymphoma with oncogenic TCR locus translocations. On the other hand, mice deficient or haplo-insufficient for histone H2AX and deficient for the p53 tumor suppressor rapidly and reproducibly succumb to immature T cell lymphomas with clonal translocations that, frequently, do not involve TCR loci and, instead, involve more general DSBs. Notably, H2AX is closely linked to ATM in both mice and man in a chromosomal region that in man is altered in a large number of cancers. In this application, we propose to elucidate mechanisms that lead to chromosomal translocations associated with T cell lymphomas. A major goal of this work will be to test our hypothesis that heterozygous or homozygous mutations of H2AX will function synergistically with loss of ATM to increase translocations and predisposition to tumors including thymic lymphomas. For this purpose, we will use sequential gone-targeting to generate cells and mice with combined H2AX and ATM mutations. Other experiments will test the hypothesis that ATM and H2AX prevent translocations resulting from aberrant V(D)J recombination by functioning to stabilize synaptic complexes of cleaved TCR chromosomal gone segments. Several novel approaches will be employed to test this notion, including the generation of mice which will be prone to frequent chromosomal translocations resulting from aberrant V(D)J recombination. Finally, we will also exploit thymic lymphoma-prone mouse models to test the role of TCR locus enhancer elements in the generation of oncogenic translocations. Frequent interaction with investigators in this program will greatly enhance the accomplishment of our goals as outlined. In the long term, our studies, and the mouse models that we will generate, should lead to a greater understanding of the molecular pathways which lead to thymic malignancies and which also are likely involved in the development of many other types of cancer.

Lymphoma is one of the most frequent cancers worldwide and the incidence is rising. The most commonly occurring human lymphomas represent mature B cells, of which many arise from GC B cells and are associated with characteristic chromosomal translocations that fuse immunoglobulin locus sequences with certain cellular oncogenes. Mouse models for human mature B cell lymphomas have been lacking. In work supported by this program project grant, we have now developed lymphoma models that share many characteristics with human B cell lymphomas, including the routine development of characteristic translocations. In particular, we have made a mouse model that reproducibly develops mature B cell lymphomas, termed CXP tumors, that arise when we conditionally inactivate a DNA double strand break (DSB) repair protein (XRCC4) in p53-deficient mature B cells. CXP B cell lymphomas routinely harbor a clonal translocation that fuses IgH switch (S) regions to exon 1 of c-Myc, as well as a second clonal translocation that fuses the IgX light chain locus into various chromosomal locations. Our ongoing studies wil focus on these models and related models being developed to address long-standing and fundamental questions regarding the mechanisms that generate and select for specific types of oncogenic translocations. In addition, our recent work has defined novel roles DNA DSB response factors, such as HistoneH2AX, in holding broken chromosomes together so that they can be repaired by NHEJ. Moreover, w have shown that defects in such factors can lead to broken chromosomes, translocations, and, in the appropriate backgrounds mature B cell lymphomas with clonal translocations. Our proposed studies will continue to elucidate the functions of this novel class of translocation and tumor suppressors and use conditional elimination of H2AX, as well as other approaches, to develop additional novel mature B cell lymphoma models. In the long term, our studies, and the mouse models that we will generate, should lead to a greater understanding of the molecular pathways that lead to B cell malignancies, as well as other types of cancer and facilitate development of novel treatments.

Immature T cell lymphomas comprise a significant portion of human lymphoid malignancies. Many of these tumors harbor recurrent chromosomal translocations and related aberrations that either activate proto-oncogenes, inactivate tumor suppressor genes, or create novel oncogenic fusion genes. Most oncogenic translocations of human immature T cell lymphomas are thought to occur via errors in the repair of DNA double strand breaks (DSBs) introduced at T cell receptor (TCR) loci during V(D)J recombination and/or general DSBs at other genomic locations. We propose to elucidate functions of the DSB response in suppression of translocations associated with T cell lymphomas and to generate novel mouse models for human T cell lymphoma. We also propose to elucidate molecular mechanisms that underlie recurrent translocations in T cell lymphomas, including how spatial proximity, DSB frequency and DNA repair pathway availability affect translocation patterns. Recurrent chromosome 14 translocations in the vicinity of the TCRa/d locus are found frequently in ATM-deficient mouse thymic lymphomas and similar translocations are found in human T cell lymphomas that have mutated ATM genes. In this regard, we find that a region 10 Mb upstream of the TCRa/d locus is highly amplified on chromosome 14 in most ATM-deficient mouse thymic lymphomas. We propose to fully investigate this recurrent translocation/amplification in ATM-deficient T cell lymphomas i) to elucidate mechanistic aspects, including potential roles of TCRa/d locus V(D)J recombination and TCRa/d enhancers (with Project 2, Harald von Boehmer), ii) to identify target oncogene(s) (with Project 5, Rick Young), and iii) to determine relevance to human T cell lymphomas (with Project 1 Tom Look). For translocations, participating loci on different chromosomes must be broken and must be in close proximity for joining. Thus we propose to test the hypothesis that frequent activation of certain proto-oncogenes via translocation to TCR loci in human, but not mouse, T cell lymphomas may reflect the relative frequency of DNA DSBs and the spatial proximity of target loci. To address this question, we will employ various approaches including 3D FISH and the generation of novel cell culture and mouse models in which DNA breaks are introduced into target T-cell oncogenes during T-cell development. We will also employ these models to test our hypothesis that ATM and its substrates (e.g., H2AX) prevent translocations resulting from aberrant V(D)J recombination by stabilizing TCR locus DSBs introduced during V(D)J recombination. Together, these studies should allow us to address long-standing questions regarding the mechanisms underlying chromosomal translocation targeting in T-ALL and other cancers.

DESCRIPTION (provided by applicant): Based on a mutational search of the mouse IgH locus, we have implicated the presence of two types of V(D)J recombinational control elements that depend on the integrity of the 100kb intergenic region between the germline IgH VH and DH clusters. One suppresses anti-sense transcription from the downstream DH locus, as deletion of the 100kb VH-D region leads to de novo DH anti-sense transcription that produces long non-coding transcripts in developing B and T cells. This increased transcription is associated with greatly increased D to JH joining in thymocytes implicating a positive role for anti-sense transcription in targeting V(D)J recombination. The second VH-D integenic control element, which we term "5'D4KBS", is contained with a 4kb sequence just upstream of D cluster. Remarkably, this element mediates ordered IgH variable region exon assembly in B cells by suppressing VH joining to Ds that have not joined to JHs and mediates lineage-specific joining by suppressing VH to DJH joining in thymocytes. The 5'D4KBS also enhances utilization of VHs located 2MB distant and therefore influences primary antibody repertoires. We have found that the 5'D4KBS functionally employs a CTCF looping/insulator factor binding site, at least for its lineage-specific function. We propose 3 specific aims designed to determine how these elements function in mice and in humans and, based on these studies, to identify additional regulatory elements. Our first aim is to characterize how the 5'D4KBS controls ordered and lineage specific V(D)J recombination and normalizes VH usage. These studies involve an in depth genetic analysis of the functions of individual factor binding sites within 5'D4KBS. In this regard, our preliminary studies implicate sites that bind CTCF, at least in lineage-specific control. We also will assess how assembly of a DJH intermediate during the V(D)J assembly process inactivates apparently suppressive influences of the 5'D4KBS on VH to DJH joining. Our second aim seeks to genetically scan the 100kb VH-D intergenic region to locate elements implicated in controlling transcription of the downstream D and JH segments and to test physiological roles for such transcription. Based on the apparent absence of IgH allelic exclusion control elements from the VH-D intergenic region, we also propose to search for such elements elsewhere in the mouse IgH locus. Our final aim proposes to characterize V(D)J recombination control in mice that have human VH, D, and JH segments in place of the corresponding mouse sequences. These studies will allow us to test the significance of our findings (e.g. the role of VH-D intergenic elements) in the context of whether they are evolutionarily conserved and provide a means to begin an in depth evaluation of the genetic and epigenetic mechanisms involved in generating the human antibody repertoire.) PUBLIC HEALTH RELEVANCE: Unlike most of our genes, antibody genes are assembled from gene segments to allow the generation of B lymphocytes that can produce a vast diversity of different antibodies. Our studies are aimed at discovering how this antibody gene assembly process is carried out and how it is regulated. Knowledge of antibody gene assembly mechanisms will lead to a better understanding of how the diverse sets of antibodies are generated to fight a multitude of different infections and also, of how mistakes in this gene assembly process can predispose to diseases such as immunodeficiency, autoimmunity, and cancer.)

DESCRIPTION (provided by applicant): In Hodgkin's Disease (HD), the most common lymphoma in the Western world, the malignant cells are the so-called Hodgkin and Reed-Sternberg (HRS) cells, comprising only a few percent of the lymphoma mass. HRS cells are often infected by Epstein-Barr-Virus (EBV) and express the EBV proteins LMP1 and LMP2A, partially mimicking constitutively an active CD40 co-receptor and B cell antigen receptor (BCR), respectively. Previous work, which had identified somatically mutated B cells, often bearing mutations crippling BCR expression, as HRS progenitors, led to a scenario of HD pathogenesis, in which HRS cells derive from pre-apoptotic GC B cells rescued by LMP2A and LMP1 expression. Despite their B cell origin, HRS cells have largely lost the B cell-specific gene expression program and acquired expression of genes typical for the T and/or myeloid hematopoietic lineages. We hypothesize that this lineage infidelity is dictated by the interference of certain transcription factors (TFs) known to be expressed in HRS cells, namely Id2, ABF1 and Notch1, with the B cell- specific gene expression program. This together with constitutive expression of proteins promoting cell survival and proliferation, like the TF c-Jun and TFs of the NF-kB family, may ultimately reprogram GC B cells into HRS cells. In this general scenario, EBV infection is a major initial transforming event in HD, and is indeed known to result in up-regulation of the TFs NF-kB, AP1, Id2 and ABF1 in B cells. Following this general hypothesis, we have developed a strategy to target the expression of candidate genes into GC B cells, using Cre-mediated conditional gene targeting. Using and further improving these tools, we plan to analyze whether induced expression of the various EBV-derived proteins and TFs individually and in combination in GC B cells will result in trans-differentiation and transformation of those cells as it is seen in HRS cells in HD. Our ultimate goal is to generate a mouse model of HD and to better understand the role of cellular reprogramming in lymphomagenesis. A second EBV-associated disease addressed in the present proposal is Post-Transplant Lymphoproliferative Disorder (PTLD), common in post-transplantation patients and due to a significant extent to the outgrowth of EBV-infected B cells because of immune suppression. We have found that induced expression of the EBV protein LMP1, known to be a major player in EBV-mediated B cell transformation, in developing B cells in the mouse leads to the rejection of these cells by the immune system. Depletion of T cells in the animals results in the rapid outgrowth of LMP1 expressing B cell blasts and death of the mice within a few weeks. We plan to develop this system into a first mouse model of PTLD, by targeting LMP1 expression into mature B cells, and to fully characterize disease progression and reversibility in the mutant animals, with a view of ultimately using this model for the development of new therapeutic strategies. Public Health Relevance: Hodgkin's Disease (HD) is the most common lymphoma in the Western world, and Post-Transplant Lymphoproliferative Disorder (PTLD) is an important cause of morbidity and mortality in post-transplantation patients, posing a significant clinical problem. Epstein-Barr-Virus (EBV) plays a major role in both HD and PTLD, with almost half of the HD cases and most of PTLDs being associated with it. The present proposal is based on known features of the pathogenesis of these diseases in the human and extensive own work, and aims at the generation of EBV-related mouse models of HD and PTLD, which so far do not exist, but should open the way to new therapeutic strategies and also shed new light on the role of gene expression reprogramming in lymphomagenesis.