Abstract
Abstract The interferon-inducible, dsRNA-dependent protein kinase, PKR, is activated by diverse cellular stresses. Loss of PKR expression or activity has been found in tumors including various types of leukemia, suggesting that loss of PKR may contribute to malignancy. However, no convincing conclusion has been reached regarding the relationship between PKR and leukemia progression. We analyzed 511 AML primary bone marrow samples by reverse phase protein array (RPPA) and discovered that PKR protein expression is > 50% reduced in CD34+ blasts from AML patients compared to CD34+ cells from healthy donors. This finding prompted us to further study how PKR affected progression of leukemia. PKR expression was either knocked down (by 82%) by specific SiRNA or directly inhibited by specific PKR inhibitor in leukemia cell lines. Significantly, inhibition of PKR decreased the rate of apoptosis following treatment with hydrogen peroxide (H2O2) or doxorubicin for 48 hours. In addition, bone marrow mononuclear cells form wild type, PKR null (-/-) or PKR transgenic mice were harvested and subjected to growth factor withdrawal for up to 120 hours. Cells isolated from PKR null mice had decreased apoptosis while cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. Further investigation showed that PKR inhibition led to reduction of PP2A activity, and a 2.4 fold increase in Bcl-2 phosphorylation. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with a PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to H2O2. Furthermore, our study showed that following PKR inhibition, phosphorylation of Bcl-2 stabilized Bcl-2/Bax complex following H2O2 treatment, preventing Bax from being released, anchoring and perforating the mitochondrial outer membrane, which would initiate apoptotic process. To confirm our in vitro findings, a leukemia xenograft model was established in NSG mice using REH cells expressing luciferase. Mice receiving REH SiPKR cells displayed significantly faster tumor progression compared to mice receiving Sicontrol cells. Furthermore, tumors from REH SiPKR cells were more resistant to doxorubicin treatment compared to tumors from REH sicontrol cells. Taken together our results demonstrate that loss of PKR function inhibits PP2A-dependent Bcl-2 dephosphorylation resulting in increased leukemia progression and survival. In addition, since our studies indicate that PKR activation enhances the effectiveness of chemotherapy, PKR level/function status may serve as a clinical predictor of prognosis, disease relapse and resistance to chemotherapy. Citation Format: Xiaodong Cheng, Richard L. Bennett, Xiangfei Liu, Stratford May. Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4606. doi:10.1158/1538-7445.AM2013-4606
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