Interferon (IFNa) is used extensively in the treatment of malignancies, but the mechanisms by which it exhibits its anti-tumor effects remain largely unknown. The applicants have identified a novel IFNa-signaling pathway involving engagement of the cbl proto-oncogene product and Crk-proteins (CrkL and CrkII). The working hypothesis is that this pathway plays a critical role in IFNa-signaling by providing a link between the functional Type I IFN receptor complex and downstream cascades that mediate the anti-tumor effects of IFNa. The current proposal is a systematic approach that will define the molecular functions of the Cbl-Crk pathway in IFNa-signaling, establish its biological relevance and determine its specific role in chronic myelogenous leukemia.
Specific aim A is to identify the signaling functions of Cbl and Crk-proteins in IFNa sensitive cells. The hypothesis will be examined that Cbl, CrkL and CrkII play the role of adapter proteins, providing a link between the Type I IFN receptor and downstream pathways. This will be achieved by identifying the precise mechanisms by which IFNa-dependent tyrosine kinases regulate phosphorylation of these signaling molecules, and by identifying the elements activated downstream of them.
Specific aim B is to determine the biological consequences of activation of this pathway. Two different methodologies will be used to achieve this goal. One approach will involve the generation of mutant, dominant-negative Cbl- and Crk-proteins and the expression of them in IFNa-responsive cell lines. The second approach will involve studies to determine the effect of antisense mRNA and antisense oligonucleotides against Cbl and Crk-proteins on the biological effects of Type I IFNs on normal and malignant cells.
Specific aim C is to examine the potential role of Crk-proteins in mediating the growth inhibitory effects of IFNa in chronic myelogenous leukemia. The effect of IFNa on the interaction of BCR-ABL with CrkL and on the BCR-ABL-regulated activation of mitogenic signaing pathways. Further experiments determine the sensitivity of leukemic progenitors from CML-patients to the antiproliferative effect of IFNa, and will examine whether this sensitivity correlates with specific IFNa-induced signaling events. Altogether, these studies should provide valuable information the mechanisms by which signals generated at the Type I IFN receptor mediate inhibition of malignant cell proliferation.
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