Increased eIF4E Expression and Phosphorylation in Late Phase Chronic Myelogenous Leukemia Occurs in a Bcr-Abl-Dependent Manner, and Can Be Targeted by a Novel Mnk Kinase Inhibitor, CGP57380, To Overcome Imatinib-Resistance.

The molecular mechanisms which mediate progression of chronic phase (CP) CML to accelerated and blast phase (BP) disease remain unclear, although one feature that correlates with progression is increased expression of the Bcr-Abl protein itself (Barnes et al., Can. Res. 2005). Increased Bcr-Abl expression is likely to contribute to the more aggressive behavior of BP disease, but the downstream factors that are dysregulated by the increased amounts of Bcr-Abl protein remain to be determined. In these studies we turned our attention to eIF4E since forced expression of eIF4E is transforming, and because increased levels of eIF4E have been found in BP but not CP CML (Topisirovic et al., Mol. Cell. Bio. 2003). eIF4E plays a critical role in cap-dependent translation and allows recruitment of the translation machinery to mRNA. eIF4E is phosphorylated at Ser209, and phosphorylation correlates with exposure to growth factors and increased cap-dependent translation. Using a panel of primary CML cells representing patients at various stages of disease, we confirmed that both Bcr-Abl and eIF4E protein levels were elevated in BP samples compared to those in CP, and furthermore that phosphorylation at Ser209 was dependent on Bcr-Abl kinase activity in BP but not CP samples. We next went on to explore the role of eIF4E phosphorylation in BP CML. Because eIF4E is exclusively phosphorylated at Ser209 by the MAPK signal-integrating kinases (Mnk1/2), we used a small molecule inhibitor of Mnk1/2, CGP57380, to inhibit eIF4E phosphorylation (kind gift of Dr. H. Gram, Novartis). Using MTS assays, we found that CGP57380 exhibited synergistic activity with imatinib mesyalte (IM) against Ba/F3-Bcr-Abl and K562 cells, and that this was associated with increased caspase-3 activation. Consistent with a role for eIF4E phosphorylation in cap-dependent translation, we found that CGP57380 augmented the IM-mediated inhibition of cap-binding complex (eIF4F) formation, as well as loading of mRNA onto polysomes. Interestingly, we also uncovered the existence of a novel negative-feedback loop regulating Mnk kinase. Here, treatment with CGP57380 resulted in increased phosphorylation of Mnk1 as well as its upstream activator, ERK, in a time- and dose-dependent manner. Because activation of the MEK/ERK pathway is essential to Bcr-Abl-mediated transformation, this finding suggested that the full activity of CGP57380 might be obscured by this feedback loop. In support of this, the addition of the MEK inhibitor, U0126, to the IM/CGP57380combination resulted in increased activity against CML cells. The triple combination was also effective against Ba/F3-Bcr-Abl cells harboring the E255K and T315I mutations, but not parental Ba/F3 cells (reduced by 50, 23, and 15% respectively of DMSO-treated controls by MTS assay). Colony forming assays also demonstrated the activity of the IM/CGP57380 combination against CML progenitor cells. In conclusion, our data demonstrate that: eIF4E protein expression and phosphorylation are upregulated in a Bcr-Abl-dependent manner in BP CML; Inhibition of eIF4E phosphorylation by the novel Mnk kinase inhibitor, CGP57380, synergizes with IM in killing CML cells, as well as overcomes certain forms of IM-resistance; The addition of CGP57380 to IM results in inhibition of key steps in cap-dependent mRNA translation, and may provide a mechanistic explanation for the activity of this agent in CML.