The objective of the proposed research program is to examine molecular interactions between BCR/ABL oncogenic protein tyrosine kinase and phosphatidylinositol-3 kinase (PI-3K), and to investigate if the simultaneous inhibition of BCR/ABL and PI-3K has synergistic antitumor effect against the leukemia. Oncogenic protein tyrosine kinases (OPTKs) appear to initiate and maintain the neoplastic phenotype of tumor cells. Because of their cytoplasmic or membrane localization it is likely that OPTKs exert their oncogenic potential in cooperation with several cytoplasmic and nuclear molecules. Previous studies (Skorski, et al., Blood, 86:726, 1995) have indicated that PI-3K is a downstream target of cytoplasmic BCR/ABL oncogenic protein tyrosine kinase. The first and second aim of this proposal is to further investigate the role that the interaction(s) between BCR/ABL and PI-3K have in initiation and maintenance of the leukemic phenotype. For this purpose we will employ BCR/ABL function-less mutants, dominant negative mutants, and dominant active mutants of Pl-3K. Bone marrow retroviral infections, in vitro and in vivo (SCID mice) leukemia development assays, transfections of the cell lines, site-directed mutagenesis and subcloning, enzymatic assays (RAS, RAF, PI-3K, MAPK, JNK), immunoprecipitation, Western blotting, protein phosphorylation assay, will be used in these aims.
The third aim i s to investigate if simultaneous inhibition of BCR/ABL and PI-3K exerts synergistic antileukemia effect. Our previous studies revealed that downregulation of BCR/ABL expression by the antisense strategy or inhibition of PI-3K by its specific inhibitor wortmannin (WT), strongly inhibited proliferation of chronic myelogenous leukemia cells without affecting normal hematopoiesis [Skorski, et al., Blood, 86:726, 1995]. Our preliminary data indicate that simultaneous inhibition of both BCR/ABL and PI-3K exerts synergistic antileukemia effect. The antisense strategy will he employed to downregulate BCR/ABL expression, and PI-3K activity will be inhibited by WT. Bone marrow purging experiments will be performed to test the antitumor effectiveness of the simultaneous inhibition of BCR/ABL and PI-3K in conditions mimicking those of bone marrow purging as described [Skorski et al., J.Clin.Invest., 92:194,1993]. Finally, we will test the effects of a systemic therapy consisting of simultaneous inhibition of BCR/ABL and Pl-3K using our in vivo models of CML growth in SCID mice [Skorski et al., Proc. Natl. Acad. Sci. USA, 88:2351,1994].
