Enhanced Killing of Chronic Myelogenous Leukemia Cells by Rapamycin and Imatinib Is Associated with Differential Inhibition of 4E-BP1 and eIF4E Phosphorylation and Decreased Protein Expression by Non-Overlapping Mechanisms.

The Bcr-abl tyrosine kinase is known to promote transformation by dysregulating gene transcription, but its role in dysregulating translation is less well documented. Our recent work has implicated the mammalian target of rapamycin (mTOR) signaling as a downstream target of Bcr-Abl, since we find that the mTOR effectors, 4E-BP1 and S6, are phosphorylated in a Bcr-Abl kinase-dependent manner (Ly et al., Cancer Research, 2003). Because mTOR is a central regulator of eukaryotic translation, and inhibitors of mTOR act synergistically with imatinib mesylate (imatinib) to kill CML cells, these results suggest that, like transcription, translation may be a general cellular process dysregulated by Bcr-Abl activity. If this were so, then components of the cellular apparatus co-opted by Bcr-Abl to increase translation would constitute rational therapeutic targets. These would include signaling pathways mediating increased translation, components of the cap-binding complex (eIF4E, eIF4GI, and eIF4A) that regulate cap-dependent mRNA translation, as well as proteins whose translation is increased by Bcr-Abl kinase activity. Here we identify eIF4E as well as cyclin D3 as potential therapeutic targets in CML. Since eIF4E is essential for cap-dependent translation, and increased translation parallels eIF4E phosphorylation at Ser209, we determined the status of eIF4E phosphorylation in murine hematopoietic Ba/F3 cells expressing Bcr-Abl (Ba/F3-Bcr-Abl), and its dependence on Bcr-Abl kinase activity. Using phosphospecific antibodies to eIF4E, we found that Bcr-Abl kinase activity was essential for phosphorylation of eIF4E at Ser209, but had no effects on total levels of the protein. In contrast, rapamycin had no effect on the degree of eIF4E phosphorylation, although it was able to inhibit phosphorylation of 4E-BP1 completely (unlike imatinib). By examining total mRNA and protein levels of known targets of Bcr-Abl, we determined that cyclin D3, but not cyclin D2, was post-transcriptionally regulated by Bcr-Abl. Metabolic labeling studies were also conducted in Ba/F3-Bcr-Abl cells treated with media alone, imatinib, rapamycin, or both. Our results demonstrated that translation of cyclin D3 protein is regulated by the mTOR kinase in Bcr-Abl-expressing cells, and that combined inhibition of mTOR and Bcr-Abl resulted in an additional decrease in protein levels. Together, these results demonstrate that Bcr-Abl promotes protein translation of specific genes via mTOR, and that the activity of both Bcr-Abl and mTOR kinases contribute to dysregulated protein expression via non-overlapping mechanisms in CML cells. Ongoing studies are being conducted to determine the role of both eIF4E and cyclin D3 in the pathogenesis of CML.