We have divided this project into four portions. A) We have developed a transgenic mouse model of T-cell leukemia by crossing transgenic mice which overexpress SCL (also known as TAL1 or TCL5) with transgenic mice which overexpress LMO1 (also known as TTG1 or RBTN1). 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). We demonstrated that the SCL transactivation domain is not required for the oncogenic effect, supporting the hypothesis that SCL exerts its oncogenic action through a dominant negative mechanism. Transfection experiments demonstrated that SCL and LMO1 are powerful inhibitors of E2A reporter genes, suggesting that SCL and LMO1 exert their leukemogenic action via inhibition of E-proteins such as E2A and HEB. 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 are searching for additional genetic events present in the leukemic cells from SCL/LMO1 double transgenic mice. To aid in this goal, we have established a panel of leukemic cell lines from SCL/LMO1 mice. These cell lines and tumors are being analyzed by spectral karyotyping (SKY) to determine if any recurrent chromosomal abnormalities are present. We have begun experiments using high density microarrays to compare the mRNA expression profile of SCL/LMO1 leukemic cells with that of normal thymocytes. We are in the process of refining this T-cell leukemia model using cre-lox and tetracycline inducible systems to control temporal and spatial SCL expression. In addition, we have crossed the SCL and LMO1 transgenes onto a scid background, to determine if TCR gene rearrangements are required for development of these leukemias. B) Cloning and characterization of a novel t(14;21)(q11;q22). This topic involves 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. Similar to the situation seen with SCL, BHLHB1 is able to inhibit E2A function, suggesting that aberrant expression of BHLHB1 is leukemogenic due to its ability to inhibit E2A. Interestingly, we have recently detected BHLHB1 and BHLHB2 expression in several acute myeloid leukemia (AML) cell lines. As there are well-established in vitro differentiation protocols for these AML cell lines, we plan to determine if BHLHB1 or BHLHB2 expression is modulated during differentiation of these cell lines, as well as assay additional non-hematopoietic cell lines to determine if BHLHB1/2 are frequently expressed in malignant cell lines. Finally, we have recently generated transgenic mice that overexpress for BHLHB1 in the thymus, and are following a cohort of these mice to determine if they have an increased incidence of T-cell ALL. C) Chromosomal translocations involving NUP98. We have recently cloned several chromosomal translocations that generate NUP98 fusion proteins. We have taken three complementary approaches to determine how these proteins might be leukemogenic. We have begun experiments in which lethally irradiated mice are reconstituted with bone marrow that has been infected with retroviruses that express NUP98 fusion genes. In addition, we have recently generated mice that are transgenic for NUP98HOXD13 or NUP98TOP1; we plan to determine if these mice develop AML. We have also generated """"""""knock-in"""""""" ES cells that express NUP98HOXD13 but have been unable to generate chimeric mice from these ES cells; we are attempting to determine why these cells fail to contribute to the developing mouse. D) Zebrafish models. The zebrafish has recently become a useful 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.
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