We have divided this project into four portions. 1) Evaluation of SCL as a candidate oncogene. We have developed a transgenic mouse model of T-cell leukemia by crossing transgenic mice which overexpress SCL with transgenic mice which overexpress LMO1. All of the double transgenic mice develop aggressive T-cell malignancies that are clinically, morphologically, and immunophenotypically similar to human T-cell acute lymphoblastic leukemia (T-ALL). Additionally, we have examined thymocytes from mice prior to the onset of frank malignancy, and shown that there are clear differences between the double transgenic and control littermates in terms of thymocyte number, immunophenotype, proliferative index, and clonality prior to the onset of frank leukemia. We have moved the SCL and LMO1 transgenes to a scid background, and have shown that onset of leukemia is dramatically delayed, and that all of the leukemias that develop have in-frame, clonal TCRbeta gene rearrangements. Since only rare scid thymocytes produce in frame TCRbeta gene rearrangements, this observation demonstrates that thymocytes which have developed to the TCRbeta positive stage are preferred targets for the leukemogenic effects of SCL and LMO1. We have begun experiments using cDNA microarrays to compare the mRNA expression profile of SCL/LMO1 leukemic cells with that of normal thymocytes. Cluster analysis is able to discriminate among SCL/LMO1 leukemic cells, SCL/LMO1 pre-malignant cells, and thymocytes from normal littermates. We have analyzed these tumors for the presence of cancer stem cells. Surprisingly, as few as 1 or 10 malignant cells from SCL/LMO1 mice can generate a tumor when injected into scid mice. 2) Cloning and characterization of a novel t(14;21)(q11;q22). This project began with the study of a novel t(14;21)(q11;q22) chromosome translocation in a patient with T-ALL and led to the discovery of two closely related genes named BHLHB1 and BHLHB2. These genes encode bHLH proteins whose expression is normally limited to the central nervous system. However, in leukemic cells which have undergone a t(14;21) translocation, BHLHB1 and BHLHB2 are activated and expressed at high levels. Recently, we have shown that >50% of mice transgenic for both BHLHB1 and LMO1 develop T-cell tumors. 3) Chromosomal translocations involving NUP98. We have cloned several chromosomal translocations that generate NUP98 fusion proteins. We have taken three complementary approaches to determine how these proteins might be leukemogenic. In collaboration with Dr. Keith Humphries of the Terry Fox Laboratories, we have shown that mice reconstituted with bone marrow transduced with a NUP98-HOXD13 retrovirus develop a myeloproliferative disease; a minority of these mice progress to develop frank AML. However, when reconstituted with bone marrow that overexpresses both NUP98-HOXD13 and Meis1, all of the mice rapidly develop AML. We have generated mice that are transgenic for NUP98HOXD13 or NUP98TOP1. NUP98HOXD13 mice develop myelodysplastic syndrome (MDS). This MDS is highly penetrant, and resembles the human disease in terms of peripheral blood cytopenias, dysplasia, apoptosis, and progression to frank leukemia. We have also generated """"""""knock-in"""""""" ES cells that express NUP98HOXD13 under control of endogenous NUP98 regulatory elements, but have been unable to generate chimeric mice from these ES cells. It seems likely that expression of the NUP98-HOXD13 fusion protein leads to a block in differentiation, as ES cells harboring the NUP98-HOXD13 knock-in allele are severely impaired in their ability to differentiate along hematopoietic lineages in vitro.4) Zebrafish models. The zebrafish has recently become a popular organism for developmental biologists, owing to its transparent embryo, rapid development time, and ability to develop gynogenetic adult fish, among other advantageous features. This popularity has resulted in the generation of many useful """"""""tools"""""""", ranging from genomic and cDNA sequences to large scale mutagenesis screens to techniques for developing transgenic fish. We plan to take advantage of these tools and determine whether zebrafish which overexpress SCL in the thymus will develop T-ALL, with a longer term goal of using gynogenetic fish to uncover tumor suppressor genes which might interact (genetically) with SCL during the development of T-ALL. We have generated transgenic zebrafish that express SCL under control of a fugufish lck promoter; these fish have transmitted the transgene, and a cohort of transgenic and non-transgenic fish is currenlty being evaluated.
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