The objective of this proposal is to understand how expression of an altered form of PBX1, the prototype of a new homebox gene family, alters hematopoiesis and induces leukemia in man. I have shown that expression of a fusion transcript between the transcription factor gene, E2A, and the homebox gene, PBX1, is the genetic consequence of the t(1;19) translocation of childhood pre-B cell acute lymphocytic leukemia, and that the presumptive chimeric transcription factor encoded by this transcript produces myeloid leukemia in mice. Discovery of this new human oncogene prompts three distinct questions: first, what biochemical activities are required to make E2A-Pbx1 a transforming gene (Aims 1 and 2); second, what specific cellular events does E2A-Pbx1 alter to produce leukemia (Aim 3); and third, do other PBX-related genes control elements of hematopoetic differentiation (Aim 4).
Aim 1 : Determine the structural domains of E2A-Pbx1 required for transformation. Whereas E2A-pbx1 cDNA's are in hand, a full-length pbx1 cDNA must be isolated. The leukemic potential of normal Pbx1, an aminoterminal truncation of Pbx1 at the E2A- Pbx1 junction, and Pbx1 fusion proteins with other transactivation domains will then be examined in mice. Analysis of a shorter spliced variant of E2A-Pbx1 that lacks 7 Kda's of Pbx1 will permit further resolution of the functional requirements of Pbx1.
Aim 2 : Determine the transcriptional activity of Pbx1 and E2A-Pbx1. Recombinant Pbx1 proteins will be expressed in bacteria, purified, and then used to select a Pbx1 DNA-binding sequence from degenerate oligonucleotides or genomic DNA fragments. Transcriptional activities of Pbx1 and E2A-Pbx1 will then be assessed by cotransfecting a reporter construct (CAT), transcriptionally regulated by the Pbx1-responsive DNA sequence, with an expression plasmid, producing forms of Pbx1 or E2A-Pbx1. Transcriptional properties will then be correlated with transforming ability to determine a biochemical basis for transformation.
Aim 3 : Examine the cellular effects of E2A-Pbx1. The ability of E2A-Pbx1 to alter the growth and/or differentiation of granulocyte and granulocyte-macrophage, colony- forming cells from mouse marrow will be examined in vitro. The ability of E2A-Pbx1 to inhibit inducible differentiation of cell lines in vitro will also be tested. These results will reveal whether E2A-Pbx1 alone can mediate, the factor-dependent, differentiation-inhibited, in vivo phenotype of E2A-Pbx1-induced leukemias.
Aim 4 : Initiate studies to determine whether other PBX1 homologues are expressed during hematopoesis. The possibility that other Pbx1 homologs are expressed during hematopoetic differentiation and bind the Pbx recognition sequences will be addressed by cloning PBX homologues, using the known sequence identity between PBX1 and PBX2 to develop identification strategies. New cloned PBX CDNA'S will provide the basis to initiate studies on the role of PBX gene expression during hematopoeisis, as well as provide a basis for determining whether other PBX genes are involved in other types of human malignancy.
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