MOLECULAR INHIBITION OF BCR-ABL TYROSINE KINASE BY BCR SEQUENCES

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The Bcr-Abl oncoprotein is the primary causative factor in Philadelphia chromosome (Ph) associated leukemias. The activated tyrosine kinase of the Bcr-Abl oncoprotein is the primary driving force behind its oncogenic activity. We have shown that a deleted form of Bcr [Bcr(64-413)], encompassing the Abl SH2 binding domains of Bcr, reduced the phosphotyrosine content of c-Abl and Bcr-Abl within cells, and inhibited Bcr-A autophosphorylation activity in vitro. Similarly, a Bcr peptide phosphorylated on Ser 354 blocked the c-Abl and Bcr-Abl kinases in vitro whereas the unphosphorylated peptide was not inhibitory. Bcr(64-413) was also resistant to tyrosine phosphorylation by either activated c-Abl or Bcr-Abl. Importantly, Bcr(64-413) interfered with the growth of BCR-Abl expressing cell liners. Our findings indicate that the Abl SH2 binding domain of Bcr in the phospho-serine form inhibits the Bcr-Abl oncoprotein, but that tyrosine phosphorylation of this domain of Bcr reverse its inhibitory effects of Bcr-Abl. The results of these findings form the basis of a new strategy to treat chronic phase CML patients. The specific aims are: 1) Determine the optimum form of Bcr(64-413) will be tested in human hemopoietic cells expressing Bcr-Abl for their ability to reverse growth/apoptosis effects induced by Bcr-Abl. These constructs will be compared to vector only, BCR(1-413), and S354A BCR(64-413) transfected cells. These studies will be done using transfection and positive selection with the tetracycline repressor promoter system. 2) Construct a BCR(64-413) recombinant, replication-defective adenovirus for insertion of the BCR inhibitor sequence into Bcr-Abl positive and negative cell lines. 3) Test the effectiveness of a recombinant defective Bcr adenovirus in SCID/NOD SCID mouse systems programmed with cell lines derived from CML patients. We plan to test the effects of Bcr(64-413) on patient cell lines growing in a SCID mouse model. We plan to infect K562 cells and KBM-5 cells with Ad5 CMV BCR(64-413) to determine its effects on the growth properties of these patient cell lines in the appropriate SCID model compared to an adenovirus preparation encoding a unrelated gene (anti- sense C-CAM). Recent findings indicate that Ad5 CMV BCR(64-413) induced cell death in 75% in K562 cells infected for 2 days. The cell killing effects was specific to BCR sequences because Ad5 CMV anti-sense C CAM did not affect cell growth or induce cell death. 4) Test the effects of recombinant, replication defective BCR(64-413) adenovirus infection in human bone marrow preparations from CML chronic phase patients, Ph- positive ALL, and normal bone marrow. These effects will be compared to negative control adenoviruses. Cell death (50%) has been induced in a low density marrow preparation from an untreated CML patient. We anticipate that Bcr(64-413) will inhibit growth of Ph-positive colonies. 5) Investigate the inhibitory effects of synthetic peptides derived from Bcr(64-413) for their utility as inhibitors of Bcr-Abl expressing cell lines and marrow samples. Positive results from either virus-mediated introduction of Bcr(64-413) or soluble peptides from this region of Bcr will be useful for inclusion in therapeutic protocols for treatment of chronic phase CML patients.