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Aim 1. Mimicking human Burkitt lymphoma t(8;14)(q24;q32) and mouse plasmacytoma T(12;15)(F2;D3) translocations by gene insertion in mice
Mouse T(12;15) and human t(8;14) translocations are widely considered as crucial initiating transforming events in mouse plasmacytoma (PCT) and human Burkitt lymphoma (BL) development. To evaluate the biological and oncogenic consequences of these translocations in greater depth, including the mechanisms that activate Myc and drive Myc-dependent tumor progression, we used gene targeting to reproduce the T(12;15)/t(8;14) in mice. Previous research has empirically associated different states (fine structures) of the T(12;15)/t(8;14) with different types of human BL (e.g., endemic versus HIV/AIDs-related BL), mouse PCT (spontaneous versus inflammation-induced tumors), and mouse PCT progression states (tumor precursors versus frank tumor cells). To accurately recreate these different states of translocation, we inserted a His6-tagged Myc cDNA, MycHis, into three different sites of the immunoglobulin heavy-chain gene cluster, Igh, just 5’ of the following loci: Eμ, Cμ (thereby deleting Eμ, and Cα). The gene-insertion strains were designated iMycEμ, iMycCμ and iMycCα, respectively (Fig. 1).
Common to all three mouse strains is the potential of MycHis to interact with the correctly spaced Igh enhancers, Eμ and Eα, designed to create the same complex interplay of promoter and enhancer interactions that govern the expression of translocated Myc in mouse PCT and human BL. Another advantage of the present “iMyc” mice over previously developed Myc-transgenics is that the insertion of MycHis in the Igh chromatin domain subjects the gene to the same higher-order regulatory influences that affect Myc in naturally occurring T(12;15)/t(8;14) exchanges. Influences of this sort include positional effects in the interphase nucleus and chromatin remodeling processes.
We are now in the process of studying the incidence, onset, and spectrum of tumors that arise in the iMyc mice. Evaluating neoplasms of different morphologies by gene expression profiling on microarrays may permit us to define the differentiation stages and putative precursors of the various tumor types. Acceleration of iMyc-driven tumor development by retroviral insertional mutagenesis may lead to the identification of tumor progressor genes, a notoriously difficult area in human cancer genetics. Analyzing the mouse tumors cytogenetically using SKY and CGH may further facilitate these tumor progression studies. Comparing the various characteristics of the mouse tumors with published data in human B-cell neoplasms may uncover new mouse-human counterparts.
Aim 2. Elucidating the genetics, molecular mechanisms, and epidemiology of Myc-activating chromosomal translocations
We have generated gene-targeted mice in which the gene encoding an enhanced version of the green fluorescence protein (GFP) has been inserted in the vicinity of Myc. In these mice, the GFP is designed to function as the reporter of T(12;15)(Igh-Myc) translocation according to the scheme presented in Fig. 2. In normal B cells, the GFP reporter (located on Chr 15) is silent because it is driven by the VH promoter that cannot be activated in trans by the Igh enhancers (residing on Chr 12). However, in cells undergoing T(12;15), GFP becomes activated due to the juxtaposition of the VH promoter in cis to an Igh enhancer. Two possibilities exist, based on the location of the GFP-encoding gene relative to Myc: GFP residing 3’ of Myc is activated by Eα on the Myc-deregulating (and Myc protein encoding) product of translocation, der(12), whereas GFP located 5’ of Myc is activated by Eμ on the reciprocal product of translocation, der(15).
Our most recent studies have shown that T(12;15)-harboring PCT induced in GFP mice express the reporter gene, as expected. GFP expression was determined using FACS, fluorescence microscopy, molecular methods (RT-PCR, Western blotting), and immunohistochemistry. The underlying T(12;15) translocation was confirmed cytogenetically (FISH, SKY) and by PCR analysis of genomic Igh-Myc junction fragments. In future studies we will be concerned with fundamental outstanding questions on the biology, genetics, and mechanism of T(12;15). We will use flow cytometry to estimate the frequency of GFP-expressing cells in vivo and pinpoint the stage of B-cell differentiation at which translocations take place. This approach may yield highly purified, pre-malignant, T(12;15)-harboring cells for studies on the biology of early tumor precursors, a rare opportunity in cancer research. We shall also employ fluorescence microscopy on tissue sections of transgenic mice to assess the anatomical location, distribution pattern, and trafficking of T(12;15)+ cells in vivo. To further our understanding of the mechanisms of translocation, we will cross the GFP reporter gene onto various transgenic backgrounds.
Related collaborative studies on the epidemiology of t(8;14) translocations involve the determination of this exchange in peripheral blood lymphocytes (PBL) and tumor tissues of patients, case-control and cohort analyses to associate the occurrence of t(8;14) in PBL with lymphoma risk, and the re-detection of the clonotypic translocation breakpoints of serial pre-cancer PBL samples in clonally related subsequent lymphomas. This may yield important information on tumor latency in humans and identify the significance of circulating lymphocyte translocations for the etiology of B cell lymphomas in the general population.
Aim 3. Modeling human plasma cell neoplasms in transgenic mice
Since the over-expression of human MYC by (poorly defined) trans-activating mechanisms is an important feature of human osseous and extraosseous plasma cell tumors, such as multiple myeloma (MM) and plasmacytoma, respectively; the acquisition of cytogenetic rearrangements at the MYC locus is commonly observed during MM progression (~15% of early-stage MM, ~50% of advanced MM, and ~90% of MM cell lines have been reported to contain such rearrangements); a good mouse model for elucidating the mechanism by which MYC promotes human plasma cell tumor development is currently lacking. It may be promising to use the above-described iMyc mouse strains as platform for improved modeling of human plasma cell tumors in mice. Preliminary findings in the iMyc mice suggest that this may be a reasonable strategy (Fig. 3). Possible approaches include double transgenic mice that combine the iMyc with the transgenic expression of MM progressor genes; e.g., IL6 or the BCL2 family gene MCL1, or genes involved in MM bone disease (e.g., the WNT inhibitor DKK1). Considering that human MM is a disease of elderly patients, it may also become necessary to delay transgene expression to aging mice; i.e., generating mice with inducible transgenes, such as our newly developed Tet-off/Myc strain. Delaying expression of certain transgenes during B-cell development to the plasma cell stage with the assistance of plasma cell-restricted promoters and enhancers may be equally important.
