This proposal examines how the altered transcription of cellular genes by the E2A-Pbx1 oncoprotein blocks myeloid differentiation and causes fibroblast transformation. It tests whether E2A-Pbx1 induces transcription of known genes that regulate normal growth or of a novel population of genes that regulate differentiation and growth by an alternate mechanism. It also uses E2A-Pbx1-responsive cellular promoters as model systems to investigate transcriptional regulation by E2A-Pbx1 and by the normal cellular Pbx1 protein. E2A-Pbx1 is expressed as a consequence of the t(1;19) chromosomal translocation in pediatric pre-B ALL. It is comprised of the transactivation domain of the transcription factor E2A, and the DNA-binding domain of the new homeobox protein, Pbx1. E2A-Pbx1 is a nuclear phosphoprotein, and exhibits unique oncogenic properties. In a mouse bone marrow transplantation model, it induces acute myeloid leukemia, and infection of mouse marrow with E2A-Pbx1 virus in vitro results in the rapid outgrowth of GM-CSF-dependent myeloblasts. Therefore, E2A-Pbx1 blocks myeloid differentiation without altering growth-factor requirements. E2A-Pbx1 also transforms NIH3T3 fibroblasts, indicating that it may activate transcription of primary response genes.
Specific aims 1 -3 focus on 1) developing conditional mutants of E2A-Pbx1, 2) using them to clone E2A-Pbx1-regulated genes, 3) determining whether E2A-Pbx1-regulated genes directly control cell division or differentiation, and 4) using promoters of E2A-Pbx1-inducible genes to investigate the biochemical mechanism of transcriptional activation by E2A-Pbx1 as well as indirect transcriptional repression. The fourth specific aim proposes to identify mutations that strongly enhance fibroblast transformation by E2A-Pbx1 and to determine the biochemical property of E2A-Pbx1 that is affected. This will help establish a biochemical basis for transformation by E2A-Pbx1. The last specific aim proposes to use conditionally-transformed myeloblast cell lines to construct a model system to identify human oncogenes in AML that block myeloid differentiation. Because our major focus is the completion of aims 1-4, aim 5 will be developed as time permits. Accomplishing these aims will reveal how the pediatric leukemia protein, E2A-Pbx1, interferes with both growth and differentiation and the mechanisms through which oncogenes interfere with this regulation.
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