PKR negatively regulates leukemia progression in association with PP2A activation, Bcl-2 inhibition and increased apoptosis

Abstract

Reduced expression and activity of the proapoptotic, double-stranded RNA-dependent protein kinase, PKR (protein kinase R) is observed in breast, lung and various leukemias, suggesting that loss of PKR potentiates transformation. Now we report that decreased PKR activity inhibits chemotherapy-induced apoptosis of leukemia cells both in vitro and in vivo. Inhibition of PKR expression or activity reduces protein phosphatase 2A (PP2A) activity, a B-cell lymphoma 2 (Bcl-2) phosphatase, resulting in enhanced Bcl-2 phosphorylation. Thus, inhibition of PKR activity leads to hyperphosphorylation of Bcl-2, stabilization of Bcl-2/Bax interaction and decreased Bax insertion into the outer mitochondrial membrane. Treatment with the PP2A activator, FTY720, restores Bcl-2 dephosphorylation and apoptosis in cells with reduced PKR expression following stress. Significantly, xenografts of REH leukemic cells with reduced PKR display significantly increased tumor volume, increased resistance to doxorubicin treatment and shorter survival. Importantly, FTY720 treatment restores sensitivity to chemotherapy and prolongs overall survival of these mice. Collectively, these findings suggest that PP2A activation is a downstream target of PKR and the PKR/PP2A signaling axis is required for rapid and potent stress-induced apoptosis. Importantly, loss of PKR promotes leukemia progression and may serve as a biomarker for predicting chemosensitivity.

PKR inhibits the DNA damage response, and is associated with poor survival in AML and accelerated leukemia in NHD13 mice

“…11 Generation of cell lines expressing short hairpin RNA (shRNA) is described in supplemental Methods. PKRI compound, okadaic acid (OA), and antiphospho(serine 1981)-ataxia-telangiectasia mutated (p-ATM) antibody, clone 10H11.E12, were purchased from EMD Millipore (Darmstadt, Germany).…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting was performed as previously described. 11 For IP, 500 mg of protein lysate was precleared with nonspecific mouse immunoglobulin (Ig)G and protein G, then incubated overnight with the indicated antibody (1:100 dilution) and protein G agarose (Life Technologies, Grand Island, NY) at 4°C. Agaroseconjugated complexes were washed with PBS and detected by immunoblotting as described.…”

“…Because we previously demonstrated that cells from PKRKO mice or treated with a pharmacologic PKRI are resistant to genotoxic stress, 3,11 we tested whether PKR may regulate DDR signaling in primary …”

“…[1][2][3][4][5][6][7][8][9][10][11] Due to proapoptotic functions, PKR has been considered to have tumor suppressor properties. However, PKR knockout mice (PKRKO) do not display any increased tumor incidence.…”

The impact of PKR activation: from neurodegeneration to cancer

“…In addition, it has been reported that PKR is suppressed or inactivated in some malignancies, and increased PKR expression has been shown to correlate with better prognosis in head and neck cancer, melanoma, and lung and colon cancer (71)(72)(73)(74)(75)(76). Furthermore, some evidence shows that PKR may act as a tumor suppressor in leukemia (77) and could play an important role in hematologic disorders (78,79).…”