PKR Inhibits the DNA Damage Response and Correlates with Poor Clinical Outcomes in AML

Introduction:

Dysregulation of the double-stranded RNA-activated protein kinase (PKR) disturbs cellular signaling pathways that influence proliferation, differentiation, inflammation and apoptosis. Given the multifaceted functions of PKR in cellular stress signaling and inflammation, how PKR may play a role in tumorigenesis is a significant but underexplored area. Using a proteomic approach, we stratified AML patients according to PKR expression level in CD34+ leukemic cells and find that patients with high PKR expression have inferior clinical outcomes. Significantly, results from both acute leukemia derived cell lines and primary AML BM samples reveal that increased PKR expression leads to inhibition of DNA damage response (DDR) and DNA double strand break (DSB) repair. Thus, high PKR expression/activity in leukemic cells may retard the DNA damage response, enhance genomic instability and facilitate leukemia progression.

Method:

Peripheral blood and bone marrow specimens were collected from 414 patients with newly diagnosed AML and evaluated for outcomes at The University of Texas M.D. Anderson Cancer Center (MDACC) between September 1999 and March 2007. Samples were acquired during routine diagnostic assessments in accordance with the regulations and protocols (Lab 01-473) approved by the Investigational Review Board (IRB) of MDACC and analyzed under IRB-approved laboratory protocol (Lab 05-0654). Transgenic PKR (TgPKR) and dominant-negative PKR (DNPKR) mice were previously developed in our lab. PKR knockout mice (PKRKO) were provided by Dr. Robert Silverman (Lerner Research Institute, Cleveland Clinic, Cleveland, OH). The study was approved by UF IACUC protocol #201102224. Sample preparation and RPPA processing was done as described (Kornblau et al., 2011). Proteomic profiling for PKR was performed on AML patient samples by RPPA using the PKR monoclonal M02 antibody (Abnova). Neutral Comet assay and Olive tail moment calculations were performed by Wimasis Image analysis (Munich, Germany). γ-H2AX, p-ATM, p-NBS-1 levels were evaluated by both western blot and flow cytometry.

Results:

High level of PKR expression in CD34+ blast cells from 414 newly diagnosed AML patients correlates with reduced remission duration and poor survival. Cells with decreased PKR expression or treated with the PKR inhibitor (PKRI) have a significantly more rapid induction of γ-H2AX formation, increased p-ATM and p-NBS1 following ionizing irradiation (IR). On the other hand, bone marrow hematopoietic stem progenitor cells (HSPCs) isolated from transgenic mice overexpressing human PKR (TgPKR) display a decreased level of p-ATm and p-NBS1 indicating a depressed DDR following IR. Significantly in human CD34+ leukemia blasts an inverse relationship exists between PKR expression and γ-H2AX or p-ATM. Results from the Neutral comet assay shows that REH cells with decreased PKR expression or HSPCs from PKRKO mice are able to repair DSBs at a significantly faster rate than HSPCs from WT mice, which repair DSB at faster rate than those from TgPKR mice. In addition treatment of TgPKR cells or CD34+ AML blasts with PKRI can restore the efficiency of DSB repair.

Conclusions:

Our findings demonstrate that PKR inhibits/retards the DDR and DNA DSB repair following genotoxic stress, the consequence of which results in genomic instability. This finding may explain, at least in part, our observation that high PKR expression in primary AML blast cells is associated with poor survival of patients with AML. Therefore pharmacological inhibition of PKR may represent a novel therapeutic strategy that targets and reduces genomic instability in these cells and may improve the clinical outcomes in such AML patients.