CHRONIC MYELOGENOUS LEUKEMIA AND BCR-ABL SUBSTRATES

Chronic myelogenous leukemia (CML) is a malignancy of a pluripotent hematopoietic stem cell. The disease is characterized by the presence of a specific chromosomal translocation, t(9:22), which fuses BCR sequences on chromosome 22 with c-ABL sequences from chromosome 9. The chimeric gene, in which the first exon of c-ABL has been replaced by BCR sequences, gives rise to a 210 kDa fusion protein termed BCR-ABL. The fusion protein, p210BCR-ABL, has elevated protein tyrosine kinase activity as compared to c-ABL, has been shown to transform immature hematopoietic cells in vitro, and is capable of converting interleukin-3 dependent cells lines to growth factor independence. In bone marrow reconstitution studies using bone marrow infected with BCR-ABL, transplanted mice develop a CML-like syndrome along with other leukemias. Although the tyrosine kinase activity of BCR-ABL is essential for its biologic functions, only a few potential substrates have been identified, however their necessity for the biologic functions of BCR-ABL has not been determined. This proposal aims to continue the identification of substrates of the kinase and will attempt to determine the necessity of each of these proteins for transformation by BCR-ABL. This will be accomplished by determining the sites of BCR-ABL that interact with known substrates, mutating these sites, and determining whether these mutants have any effect on the biologic functions of BCR-ABL. The yeast two hybrid system will be used to identify new proteins that interact with BCR-ABL as well as to assist in the characterization of known substrates of the kinase. The purification and identification of a 39 kDa protein that is one of the few novel tyrosine phosphorylated proteins seen in patients with CML will be completed. This protein will similarly be analyzed for its necessity for BCR-ABL function. As proteins that are required for BCR-ABL function are identified, we will determine whether these interactions result in any specific cellular biochemical alterations. Through these studies, it is hoped to be able to design specific therapeutic agents for the treatment of this disease.