Abstract
In BCR/ABL positive CML, defining new, additional therapeutic targets in the pathways, activated by BCR/ABL is critical for the development of new treatment strategies, especially for patients resistant or refractory to Imatinib. While studying the involvement of PI3K/Akt/mTOR signaling pathway in the development of such resistance we have uncovered the existence of additional, Akt-independent mechanism of activation of mTOR/p70-S6 Kinase pathway. Short term treatment with Imatinib (1μM, 4 hours) of the BCR/ABL-positive cell lines LAMA84, AR320, KCL22, K562, Ba/F3-BCR/ABL caused downregulation of p70-S6K phosphorylation and of S6 ribosomal protein phosphorylation without decreasing phosphorylation levels of Akt, as detected by Western blotting using the respective phosphorylation-specific antibodies p-p70-S6K (Thr389), p-S6 (Ser240/244) and p-Akt (Ser473). Inhibition of Akt by the specific inhibitor SH-6 (10μM, 4 hours treatment) did not affect the phosphorylation of p70-S6K and S6. These results were consistent in all analyzed cell lines, and led us to consider alternative mechanism for mTOR/p70-S6K pathway activation. One such mechanism, recently described in FGF9 signaling is a Phospholipase-C-gamma (PLCγ)-controlled Calcium signaling pathway (Wing et al., JBC, 19937–47, 2005), involving Ca/Calmodulin (CaM) and Ca/Calmodulin-dependent Kinase (CaM-K). In all BCR/ABL+ cell lines analyzed we could detect strong PLCγ activation (examined by p-PLCγ-Tyr783 antibody), which was effectively suppressed by Imatinib treatment (1μM, 4 hours). Incubation of the cells for 30 minutes with 10μM U73122, a specific PLC inhibitor, in contrast to the inactive analog U73343, significantly blocked p70-S6K and S6 phosphorylation. The actual mechanism of the Akt-independent mTOR/p70-S6K activation by BCR/ABL is not yet entirely understood. In general, PLCγ activation in turn leads to the activation of different PKC isoform and increase in Ca-dependent signaling. By employing inhibitors of Calmodulin (W7), CaM-K (KN-93) and the PKC inhibitor PKC-412 we are currently studying the participation of these molecules in the pathway. The function of PLCγ/mTOR/p70-S6K pathway for the BCR/ABL driven cells is also analyzed in proliferation (MTT) and apoptosis assays where cells are treated with Imatinib alone or in combination with U73122. Knocking down of PLCγ and other involved molecules is another approach which we will utilize to better understand the necessity/sufficiency of PLCγ for full activation of mTOR/p70-S6K pathway in the BCR/ABL+ cells, as well as the (physical) interaction of PLCγ and BCR/ABL.
In summary, we demonstrate the existence of additional, Akt-independent, PLCγ-dependent mode of activation of mTOR/p70-S6K which operates in BCR/ABL-positive cells. This alternative pathway may prove novel therapeutic targets for CML treatment.
