Abstract
Cancer cells with activating kinase mutations are singularly hypersensitive to drugs that target these kinases, a phenomenon referred to as kinase or oncogene addiction. Although the AKT kinase is not known to be mutated in multiple myeloma (MM), it is frequently hyperactivated due to mutations in upstream signaling proteins. Previous work indicated that MM cells with heightened AKT activity were hypersensitive to drugs targeted to mTOR, a downstream substrate of AKT, suggesting an addiction to this pathway. “Kinase addiction” would theortically render the cell dependent on the kinase for growth. This dependency implies that other cascades of viability and/or proliferation may become downregulated subsequent to kinase activation. To test for AKT kinase addiction in MM we stably transfected the U266 MM cell line that is known to be protected against apoptosis by an autocrine Il-6/IL6R pathway (Caitlett-Falcone et al., Immunity 10, 105–115, 1999) with a constitutively active AKT construct (U266-AKT). AKT-transfected MM cells were protected against fas-induced apoptosis even when the autocrine IL-6 protective pathway was paralyzed by anti-IL6 and anti-IL6 receptor antibodies. However, as expected for an AKT addicted cell line, the U266-AKT cell line was more sensitive to agents that target the PI3/AKT/mTOR pathway, including two AKT inhibitors and the PI3K inhibitors LY294002 and wortmanin in terms of increased level of apoptosis and/or growth inhibition in comparison to the control U266 cell line. This “kinase-addicted” state was associated with down regulation of several components of the autocrine anti-apoptotic pathway of U266 cells. Expression of IL6, the IL6 receptor subunits gp80 and gp130 and STAT3 were all markedly silenced following AKT transfection. This down regulation of the survival pathway was relatively specific as expression of the fas receptor and VEGF receptors were relatively unaffected. The decrease in protein expression was also reflected in the decrease of mRNA for the receptor subunits, suggesting a transcriptional mechanism for down regulation. As expected from the decrease in autocrine IL-6 signaling, AKT-transfected cells also demonstrated a marked inhibition of Jak1 and MAPK 42/44 phosphorylation and enzymatic activity. In summary, over expression of activated AKT results in an AKT-addicted state whereby the MM cell becomes dependent on AKT for survival as other growth-promoting and anti-apoptotic cascades become paralyzed. These data support AKT as an important target for therapeutic intervention of MM and suggest such therapy would be most successful in patients whose MM cells contain the highest amount of activated AKT.
Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis
