PKR inhibits DDR signaling in primary CD34⁺ AML cells, primary murine Lin⁻ BM cells, and human leukemic cell lines. (A) p(Ser1981)-ATM (p-ATM) and γ-H2AX were evaluated in CD34⁺ AML blast cells isolated from the PB and BM of 6 AML patients by IF microscopy (×63). Treatment with 0.5 μM PKRI for 8 hours increased p-ATM and γ-H2AX staining, both 30 minutes after 5 Gy IR. Images are of CD34⁺ cells from a single AML patient sample that are representative of results from 6 AML patients. (B-C) The percent of primary CD34⁺ AML blasts cells positive for p-ATM (B) and p(Ser343)-NBS1 (p-NBS1) (C) after 5 Gy IR was increased by 8 hours of treatment with 0.5 μM PKRI as measured by flow cytometry. Results are an average of CD34⁺ AML cells from 6 patients. (D) At the indicated times following 5 Gy IR treatment, primary CD34⁺ AML cells treated with PKRI displayed an increased rate and extent of γ-H2AX foci formation following IR. The number of γ-H2AX foci per cell was counted by IF microscopy of 30 randomly selected cells for each AML patient sample and the averages graphed. (E-F) Quantitative real-time polymerase chain reaction was used to measure PKR expression in CD34⁺ AML blasts from 6 patients relative to healthy donor CD34⁺ BM cells. The percent of γ-H2AX (E) or p-ATM positive (F) cells 30 minutes after 5 Gy IR was measured by flow cytometry and plotted against the relative quantity (RQ) of PKR expression for each of the 6 AML patient samples. (G-H) Treatment of primary human CD34⁺ cells isolated from BM of healthy donors with 0.5 μM PKRI for 8 hours increased the percentage of cells positive for γ-H2AX (G) and p-ATM (H) after 5 Gy IR as measured using flow cytometry. Results are an average of 3 independent experiments. (I) Lin⁻ BM cells were isolated from WT, PKRKO, or TgPKR mice and irradiated with 5 Gy. At the indicated times after IR, the percent of cells positive for γ-H2AX was measured by flow cytometry. (J) p-ATM and (K) p-NBS1 were decreased in Lin⁻ cells from BM of TgPKR mice compared with cells from WT mice. (L) Western blotting of REH cells demonstrates that p-ATM and p-NBS1 are increased in cells with reduced PKR expression by shRNA knockdown (sh-PKR) compared with cells expressing a control shRNA (sh-ctrl), both under normal growth conditions and 30 minutes after treatment with 5 Gy IR. (M) REH cells pretreated with PKRI (0.5 μM for 8 hours), IFN-γ (10 ng/mL for 24 hours), or poly(I:C) (5 μg/mL for 6 hours) demonstrate that increased PKR expression and activity decreased p-ATM and p-NBS1 30 minutes after IR. *P < .05; **P < .01. DAPI, 4,6 diamidino-2-phenylindole; M.W., molecular weight.