Discovery of increased number or interval growth of brain metastases on same-day GammaKnife™ planning MRI: Predicting factors and patient outcomes.

Purpose: To determine factors associated with increased risk of finding new and/or enlarged brain metastases (BM) on GammaKnife™ (GK) MRI and their impact on patient outcomes. Results: 43.9% of patients showed BM growth, 32.9% had additional brain metastases (aBM), and 18.1 % had both. Initial brain metastasis velocity (iBMV) was associated with finding aBM. Time between diagnostic MRI (dMRI) and GK MRI was associated with interval growth and each day increased this risk by 2%. Prior brain metastasectomy and greater time between either dMRI or latest extracranial RT and GK MRI predicted both aBM and BM growth. aBM and/or BM growth led to management change in 1.8% of cases and were not associated with OS or incidence of distant intracranial failure. Conclusions: Number of metastases seen on dMRI and iBMV predicted both aBM and/or BM growth, however, these factors did not significantly affect survival or incidence of distant intracranial failure.

Radiation-Induced Cerebral Microbleeds in Pediatric Patients With Brain Tumors Treated With Proton Radiation Therapy.

PURPOSE: Proton beam radiation therapy (PBT) has been increasingly used to treat pediatric brain tumors; however, limited information exists regarding radiation-induced cerebral microbleeds (CMBs) among these patients. The purpose of this study was to evaluate the incidence, risk factors, and imaging appearance of CMBs in pediatric patients with brain tumors treated with PBT. MATERIALS AND METHODS: A retrospective study was performed of 100 pediatric patients with primary brain tumors treated with PBT. CMBs were diagnosed by examination of serial magnetic resonance imaging scans, including susceptibility-weighted imaging. Radiation therapy plans were analyzed to determine doses to individual CMBs. Clinical records were used to determine risk factors associated with the development of CMBs in these patients. RESULTS: The mean age at time of PBT was 8.1 years. The median follow-up duration was 57 months. The median time to development of CMBs was 8 months (mean, 11 months; range, 3-28 months). The percentage of patients with CMBs was 43%, 66%, 80%, 81%, 83%, and 81% at 1 year, 2 years, 3 years, 4 years, 5 years, and >5 years from completion of proton radiation therapy. Most of the CMBs (87%) were found in areas of brain exposed to ≥30 Gy. Risk factors included maximum radiation therapy dose (P = .001), percentage and volume of brain exposed to ≥30 Gy (P = .0004, P = .0005), and patient age at time of PBT (P = .0004). Chemotherapy was not a significant risk factor (P = .35). No CMBs required surgical intervention. CONCLUSIONS: CMBs develop in a high percentage of pediatric patients with brain tumors treated with proton radiation therapy within the first few years after treatment. Significant risk factors for development of CMBs include younger age at time of PBT, higher maximum radiation therapy dose, and higher percentage and volume of brain exposed to ≥30 Gy. These findings demonstrate similarities with CMBs that develop in pediatric patients with brain tumor treated with photon radiation therapy.

Synthetic lethal targeting of PTEN-deficient cancer cells using selective disruption of polynucleotide kinase/phosphatase.

A recent screen of 6,961 siRNAs to discover possible synthetic lethal partners of the DNA repair protein polynucleotide kinase/phosphatase (PNKP) led to the identification of the potent tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Here, we have confirmed the PNKP/PTEN synthetic lethal partnership in a variety of different cell lines including the PC3 prostate cancer cell line, which is naturally deficient in PTEN. We provide evidence that codepletion of PTEN and PNKP induces apoptosis. In HCT116 colon cancer cells, the loss of PTEN is accompanied by an increased background level of DNA double-strand breaks, which accumulate in the presence of an inhibitor of PNKP DNA 3'-phosphatase activity. Complementation of PC3 cells with several well-characterized mutated PTEN cDNAs indicated that the critical function of PTEN required to prevent toxicity induced by an inhibitor of PNKP is most likely associated with its cytoplasmic lipid phosphatase activity. Finally, we show that modest inhibition of PNKP in a PTEN knockout background enhances cellular radiosensitivity, suggesting that such a "synthetic sickness" approach involving the combination of PNKP inhibition with radiotherapy may be applicable to PTEN-deficient tumors.

Genetic screening for synthetic lethal partners of polynucleotide kinase/phosphatase: potential for targeting SHP-1-depleted cancers.

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.

Mechanism of action of an imidopiperidine inhibitor of human polynucleotide kinase/phosphatase.

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphatase activity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNA from binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNA against PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.

Identification of a small molecule inhibitor of the human DNA repair enzyme polynucleotide kinase/phosphatase.

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 gamma-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 IC50 of 0.06 micromol/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 micromol/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.