Abstract
Abstract 1672
The reciprocal chromosomal translocation of Abl and Bcr locus [t(9: 22)] is present in 95% of chronic myeloid leukemia (CML) patients. The resulting Bcr-Abl oncoprotein contains a persistently activated tyrosine kinase activity that activates Jak2/Stat5 signaling pathways. Little is known about the molecular mechanism of Jak2 activation in Bcr-Abl positive CML cells, except that the IL-3 receptor is required (Tao et al., Oncogene 2008). We found that the Jak2 activity (measured by pY1007/1008) in 32D mouse hematopoietic cells steadily diminished immediately upon IL-3 withdrawal. However, expression of kinase-inactive form of Bcr-Abl (p210K1172R) in 32D cells maintained Jak2 activity for up to 8 hrs after IL-3 withdrawal. Our previous studies have shown that the C-terminal region (CT-4) of c-Abl binds to Jak2 as does the kinase domain of c-Abl (Xie et al. Oncogene, 2001). We found that Jak2 activation depends on its binding to the CT-4 region of c-Abl using Bimolecular fluorescence complementation assays. In order to examine the role of c-Abl in Jak2 activation, we expressed c-Abl in both 32D cells (32D-Abl) and 32D cells expressing p210K1172R (32D-p210K1172R+Abl). We found that unlike 32D-Abl cells which remained cytokine-dependent, a minor population (∼7%) of 32D- p210K1172R+Abl cells gained growth independency of IL-3. Compared to 32D-Abl cells in which the level of Jak2 activity was barely detected by pY1007/1008 antibody, 32D-p210K1172R+Abl cells showed a dramatic elevation of Jak2 activation, indicating that c-Abl alone is unable to induce Jak2 activation in hematopoietic cells. Phosphorylation on p210Y177 in 32D-p210K1172R+Abl cells was also strongly increased, indicative of activated Jak2 activity (Samanta et al., Leukemia 2011). We found that 32D-p210K1172R+Abl cells were sensitive to Imatinib Mesylate (IM), as 80% of 32D-p210K1172R+Abl cells were apoptotic after treatment with 5μM IM for 24hrs, indicating that the cell survival depends on the activated c-Abl kinase. The apoptosis induced by IM in 32D-p210K1172R+Abl cells could be effectively rescued by addition of IL-3, indicating the importance of Jak2 activation through IL-3 pathway in maintaining cell survival. The above results suggest that a higher level of c-Abl enables cells expressing a Bcr-Abl kinase defective protein to acquire cytokine-independent growth. The elevation of Jak2 activity in 32D-p210K1172R+ABL cells correlated with the increased c-Abl kinase activity. We propose that the c-Abl kinase plays two crucial roles in these Bcr-Abl kinase mutant cells: 1) making cells cytokine-independent for growth, and 2) promoting persistent Jak2 activation. These results lead us to propose that the Abl kinase domain within Bcr-Abl promotes Jak2 activation by binding to the Jak2 kinase. As our recent findings indicate that Jak2 is a dominant player in CML (Samanta et al., Leukemia 2011) and particularly in later stages of Bcr-Abl positive CML, we propose that the inhibition of both Jak2 and Bcr-Abl kinase activities will result in a near complete elimination of leukemia cells including CD34+CML progenitor cells.
Disclosures:
No relevant conflicts of interest to declare.
Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis
