Abstract
Abstract 396
Acute myeloid leukemia (AML) is still highly fatal so a better understanding of the mechanisms controlling chemoresistance is critical for the development of more effective therapies. The Protein Kinase B (AKT) regulates diverse cellular functions including protein translation, transcription, cell proliferation and apoptosis and is emerging as an important target for AML therapy. We have reported that activation of AKT predicts poor clinical outcome for patients with AML. Mutations of upstream activators of AKT such as Phosphatidylinositol 3 Kinase (PI3K) and RAS are well characterized and have been implicated in many cancers including AML. However, signal transduction is a dynamic process and the possibility arises that loss of protein phosphatase function can promote kinase activation in the absence of constitutive activation of the kinase by upstream activators. AKT has been shown to be regulated by Protein Phosphatase 2A (PP2A). PP2A is a family of hetero-trimeric serine/threonine phosphatases. While family members share a fairly common catalytic core (i.e. A and C subunits), the functional component of each PP2A isoform lies in its regulatory subunit (i.e. B subunit). Recent data suggests that suppression of PP2A can promote activation of survival signaling including the activation of AKT in AML. We recently identified that low expression of the PP2A isoform that acts as the AKT phosphatase (i.e. the one containing the B55α subunit) in AML negatively correlates with phosphorylation of AKT at threonine 308 and with AKT activation in a cohort of 511 AML patients (Ruvolo et al Leukemia, 2011). Patients with low B55α expression had shorter complete remission duration. In the current study, we now found by Reverse Phase Protein Analysis (RPPA) that there is a negative correlation between B55α expression and expression and phosphorylation of a number of survival kinases including Protein Kinase C a (PKCα) and SRC in AML patients. PP2A has been shown to negatively regulate the PKC family of kinases but this is the first report to implicate B55α in that process. While levels of phosphorylated PKCα negatively correlated with B55α, total levels of the kinase also were negatively correlated with the B subunit suggesting that phosphatase likely does not dephosphorylates PKCα but rather suppresses expression of the kinase. While SRC has not been linked to B55α, the kinase has been shown to negatively regulate the PP2A catalytic subunit (PP2A/C). Since PP2A is an obligate hetero-trimer, inactivation of PP2A/C via SRC could result in proteolytic cleavage of the B subunit. Consistent with this notion, treatment of OCI-AML3 cells with the tyrosine kinase inhibitor Dasatinib resulted in reduction of PP2A/C levels. Consistent with a mechanism whereby SRC regulated B55α rather than vice-versa, OCI-AML3 cells expressing B55α shRNA did not display changes in SRC phosphorylation status though there was a >67% loss of the B subunit in these cells. To determine the role of the AKT Phosphatase in AML cell survival, the OCI-AML3 cells expressing B55α shRNA were used to determine if reduction of levels of the phosphatase would promote AKT activation and promote chemoresistance. Cells with B55α shRNA exhibited higher levels of AKT S473 phosphorylation suggesting activation of the kinase. Furthermore, OCI-AML3 cells expressing B55α shRNA were more resistant to killing by the conventional chemotherapy agent AraC. One surprising finding was that OCI-AML3 B55α shRNA transductant cells were more resistant to okadaic acid, a PP2A inhibitor that is highly toxic to AML cells. Despite effects on AKT activation and survival, the reduction of the B subunit had no effect on total PP2A activity as determined by molybdate dye assay. Reduction of B55α did promote expression of at least one PP2A B regulatory subunit (i.e. B56α levels increased nearly 2 fold). Interestingly, B56α is positively regulated by AKT suggesting a possible feedback mechanism may exist between AKT and the various PP2A isoforms. An intriguing possibility arises that while PP2A is generally considered a tumor suppressor, inhibition of the AKT Phosphatase may activate an okadaic acid resistant PP2A isoform with survival function. Taken together, these results suggest that B55α may suppress survival signaling by kinases such as AKT and loss of function of the B subunit may promote resistance to chemotherapy in AML.
Disclosures:
No relevant conflicts of interest to declare.
Faculty Opinions recommendation of Striatin-1 is a B subunit of protein phosphatase PP2A that regulates dendritic arborization and spine development in striatal neurons.
