Numerous animal studies have demonstrated that the fusion protein p210 Bcr-Abl is the causative agent of Chronic Myelogenous Leukemia (CML), and pharmacological inhibitors (such as imatinib) that target this molecule produce effective, durable responses in many patients. Imatinib targets a tyrosine kinase activity contained within the Abl sequences that is considered to be the principle driving force behind CML. Although the success of imatinib has validated rational drug design in cancer therapy, two clinical issues are emerging in the treatment of CML. First, patients are developing resistance to imatinib by acquiring mutations within the kinase domain (acquired resistance). Second, and probably more problematic, over 95% of patients still have p210 Bcr-Abl positive cells, even after 12 months of imatinib treatment (minimal residual disease). Whether or not acquired resistance and minimal residual disease are mechanistically related is unclear, but both problems highlight the need to develop complementary inhibitors, with different modalities, that can target additional functions of p210 Bcr-Abl. Although the tumorigenic properties of p210 Bcr-Abl can be primarily attributed to the Abl-encoded kinase activity, numerous studies have determined that there are also domains present within the Bcr sequences that are required to support oncogenic activity. Although poorly characterized, these domains are intriguing since they represent unexploited targets for pharmacological intervention. In the previous funding period, our laboratory as been exploring the hypothesis that the characterization of Bcr- encoded activities will identify viable new targets for p210 Bcr-Abl inhibition. Three such activities have now been identified. First, it has been determined that the RhoGEF domain, which is autoinhibited in Bcr, is constitutively activated in p210 Bcr-Abl. Mutations that eliminate this activity in p210 Bcr-Abl exhibit diminished transforming activity in cell- and animal-based models suggesting that this domain may be required for CML. Second, it has been determined that Bcr is a regulatory component of the endosomal sorting pathway, and that this pathway is disrupted by overexpression of p210 Bcr-Abl in hematopoietic cells. This represents a novel, and completely unexplored aspect of p210 Bcr-Abl biology, which undoubtedly will be relevant to CML. Finally, it has been determined that Bcr has a nuclear function through which it can regulate levels of protein expression. This includes the c-Myc oncoprotein which is known to contribute to CML. Bcr-Abl regulates c- Myc through binding to XPB, and disruption of this interaction is also associated with diminished transforming activity in cell- and animal-based models. In this continuation application these activities will be characterized in the context of hematopoietic cells, and it will be determined whether they are viable targets for p210 Bcr-Abl inhibition. For this analysis, p210 Bcr-Abl mutants have been generated that are impaired in these activities, and which can be evaluated in cell- and animal-based models for CML.
Imatinib is a pharmacological inhibitor that causes clinical remission in many patients with Chronic Myelogenous Leukemia. However, imatinib controls rather than cures the disease, and many patients develop resistance to the treatment. Thus, the focus of this proposal is the development of novel approaches and inhibitors that can be used to complement imatinib treatment.
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