Abstract LB-1: PKR promotes genetic instability and evolution of MDS to acute leukemia in mice by negatively regulating DDR and DNA repair in a novel mechanism

Abstract

Abstract The interferon-inducible, dsRNA-dependent protein kinase, PKR, is reported to be involved in diverse cytoplasmic functions. However, PKR has recently been reported to be expressed in the nucleus of high-risk MDS and leukemia patients but its nuclear function is not known. We confirm that increased nuclear activated PKR does occur in AML cell lines compared to normal MEF and bone marrow stem/progenitor cells. Increased nuclear PKR is associated with impaired DNA damage response (DDR) and double strand break (DSB) repair following ionizing irradiation (IR). Knock-down and pharmacological inhibition of PKR reveals that activated nuclear PKR may function to suppress the γ-H2AX response characteristic of DDR following IR. Mechanistically, we find that nuclear PKR binds and co-immunoprecipitates with ATM in a mechanism that inhibits ATM phosphorylation. This leads to inefficient initiation of DDR (as measured by retarded γ-H2AX response) as well as inhibition of interaction between ATM, γ-H2AX, and MRN as part of the DDR complex. As a result, activated nuclear PKR retards DDR and leads to insufficient DSB repair as detected by prolonged Olive Tail Moment in a neutral Comet assay. However, since DNA repair can be restored by inhibiting PKR (by either knock-down or treatment with a PKR inhibitor), this supports a negative regulatory role for PKR in DDR and DSB repair Additional studies further demonstrate that PKR may block DDR by inhibiting ATM activity indirectly by binding with and activating PP2A, a known ATM protein phosphatase. Furthermore, in support of PKR inhibiting DSB repair, in vivo studies reveal that age-related or IR-induced genetic instability is more prevalent in PKR Tg vs WT mice and markedly lower in PKR knock-out mice as measured by the loss of GPI-linked CD24 on reticulocytes (a measure of increased somatic mutation rates due to loss of X-linked GPI activity). Interestingly, an increased somatic mutation rate and a more rapid evolution from MDS to acute leukemia is observed for double transgenic TgPKR/NHD13 vs Wt/NHD13 mice, which is significantly higher than evolution observed in PKRnull/NHD13 mice. Collectively, these data identify a novel function for PKR in suppressing DDR and DSB repair, which may enhance genetic instability and play a fundamental role in the evolution of MDS to acute leukemia. Citation Format: Xiaodong Cheng, Michael Byrne, Richard Lynn Bennett, Stratford William May. PKR promotes genetic instability and evolution of MDS to acute leukemia in mice by negatively regulating DDR and DNA repair in a novel mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-1. doi:10.1158/1538-7445.AM2014-LB-1