Idenfificafion of new targets and novel treatment strategies for chronic mvelold leukemia. Ralph B. Arilnahaus. Ph.D.- My laboratory has published new information on several key proteins in chronic myelogenous leukemia that are regulated by the Bcr-Abl oncoprotein. These proteins are lipocalin 2 (NGAL/24p3), Jak2 and Bcr. We were first to discover the role of lipocalin 2 in Bcr-Abl induced leukemia (Lin et al. Oncogene 2005). Our recent studies with 24p3 null mice showed that 24p3 secretion by Bcr-Abl+ cells is a requirement for leukemia inducfion by Bcr-Abl in a mouse model (Leng et al.. Oncogene 2008). Regarding Jak2, we show that it is part of a large signaling network (Samanta et al., Can Res., 2006). Inhibifion of Jak2 induces apoptosis in imatinib-sensitive/resistant Bcr-Abl + cells, in CML cell lines and in cells from blast crisis CML patients. Concerning Bcr, wild-type Bcr protein inhibits the oncogenic function of Bcr-Abl but serine/threonine kinase-defective Bcr enhances the oncogenic effects of Bcr-Abl (Perazzona et al.. Oncogene, 2008). Aim #1, Develop a monoclonal anfibody that blocks lipocalin 2 (NGAL/24p3) activities induced by Bcr-Abl, and investigate the effects in mouse leukemia models. Our goal is to interfere with NGAL function for treatment of CML in combination with other therapeutic regimens such as Gleevec therapy. Aim #2, Investigate the mechanistic effects of new Jak2 inhibitors in imatinib-sensitive and resistant CML cell lines, patient cells, and in patient cells from advanced stages of CML with the long-term goal of doing clinical trials in imafinib-resistant CML and late stage CML (with Dr. Cortes). We hypothesize that a potent Jak2 inhibitor will be useful in the treatment of drug-resistant CML as well as in all stages of CML because of the dominant role of Jak2 in oncogenic signaling in CML cells. We have identified a new Jak2 inhibitor (WP1193) that disrupts the Bcr-Abl/Jak2/HSP90 signaling network complex leading to apoptosis of drug-resistant CML cells and blast crisis CML cells. Surprisingly, preliminary results indicate that the Jak2 kinase phosphorylates Tyr 177 of Bcr-Abl, which upon Jak2 inhibifion drasfically reduced Grb2 binding to the network complex and caused the disruption of the Ras and PI-3 kinase signaling pathways. Importantly, Jak2 inhibition also drastically reduced Bcr-Abl protein levels, causing down-regulafion of STAT5 and STAT3 signaling. Thus, Jak2 inhibition disrupts many If not all oncogenic effects in CML. Aim #3, Invesfigate the role ofthe Bcr Ser/Thr kinase in regulafing Bcr-Abl oncogenic activity. We will search for mutations in the Bcr kinase domain, as we hypothesize that such mutations play a role in CML disease progression.