Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5 ¶-termini and dephosphorylates DNA 3 ¶-termini. hPNKP is involved in both single-and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to ;-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC 50 of 0.06 Mmol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3 ¶-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 Mmol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

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A genetic screen using a library of 6,961 siRNAs led to the identification of SHP-1 (PTPN6), a tumor suppressor frequently mutated in malignant lymphomas, leukemias, and prostate cancer, as a potential synthetic lethal partner of the DNA repair protein polynucleotide kinase/phosphatase (PNKP). After confirming the partnership with SHP-1, we observed that codepletion of PNKP and SHP-1 induced apoptosis. A T-cell lymphoma cell line that is SHP-1 deficient (Karpas 299) was shown to be sensitive to a chemical inhibitor of PNKP, but resistance was restored by expression of wild-type SHP-1 in these cells. We determined that while SHP-1 depletion does not significantly impact DNA strand-break repair, it does amplify the level of reactive oxygen species (ROS) and elevate endogenous DNA damage. The ROS scavenger WR1065 afforded protection to SHP-1-depleted cells treated with the PNKP inhibitor. We propose that codisruption of SHP-1 and PNKP leads to an increase in DNA damage that escapes repair, resulting in the accumulation of cytotoxic double-strand breaks and induction of apoptosis. This supports an alternative paradigm for synthetic lethal partnerships that could be exploited therapeutically. Cancer Res; 72(22); 5934-44. Ó2012 AACR.

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