Radiation-induced RAD51 Foci Formation Research Articles

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

PALB2 or BARD1 loss confers homologous recombination deficiency and PARP inhibitor sensitivity in prostate cancer

PARP inhibitors were recently approved for treatment of molecularly-defined subsets of metastatic castrate-resistant prostate cancer (mCRPC) patients. Although the PARP inhibitor olaparib was approved for use in patients with a mutation in one of fourteen genes, the mutation frequency of the genes varies widely in mCRPC and the impact of the less commonly altered genes on PARP inhibitor sensitivity is uncertain. We used functional approaches to directly test the impact of PALB2 and BARD1 loss on homologous recombination (HR) function and PARP inhibitor sensitivity in prostate cancer cell lines. PALB2 or BARD1 loss led to decreased HR function as measured by loss of radiation-induced Rad51 foci formation as well as decreased HR capacity in a cell-based reporter assay. PALB2 or BARD1 loss also significantly increased sensitivity to the PARP inhibitors olaparib and rucaparib across a panel of prostate cancer cell lines. These data support PALB2 and BARD1 loss as markers of clinically relevant PARP inhibitor sensitivity and highlight the potential to use functional approaches to complement and extend findings from clinical trials of targeted agents.

BRCA2-DSS1 interaction is dispensable for RAD51 recruitment at replication-induced and meiotic DNA double strand breaks

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

Abstract 6164: Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer

Abstract Nucleophosmin1 (NPM1) is rapidly recruited to sites of DNA double-strand breaks and is required for radiation-induced Rad51 foci formation. Inhibition of NPM1 or Rad51 is a radiosensitizing event. YTR107 is a small molecule, identified in a forward chemical genetics functional structure/activity screening, that binds to the amino terminus of NPM1 and prevents NPM1 recruitment to damaged chromatin. In silico docking studies of YTR107 and the crystal structure of the NPM1 N-terminal oligomerization domain revealed that YTR107 docked to monomeric NPM1 in a pocket crucial for forming an interface between neighboring subunits of the NPM1 pentamer. YTR107 inhibited radiation-induced Rad51 foci formation and repair of DNA DSBs, as measured by a neutral comet assay. Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines, HT29 colorectal cancer cells, D54 glioblastoma cells, PANC1 pancreatic cancer cells, and 2 breast cancer cell lines (dose modifying factors of > 1.5; cells irradiated with 300 kVp/10mA X-rays @ 2 Gy/min). Radiosensitization was found to be NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line. YTR107 produced a 4.7 fold increase in HT29 xenograft growth delay (time required for the tumor to increase 4-fold in volume (p = 0.001) following 10 mg/kg YTR107 administered 30 min prior to administering 3 Gy to the tumor for 7 consecutive days). YTR107 increased the survival of A549 tumor-bearing mice determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107 and 3 Gy vs 20% for mice treated with 3 Gy alone, p = 0.0012 (0 or 20 mg/kg YTR107 administered 1 hr prior to 0 or 2.2 Gy being administered to the tumor for 7 consecutive days). Female C57BL/6J mice were administered 0 or 10 mg/kg i.p. YTR107 for 5 consecutive days. Thirty five days after injection mice were euthanized and subjected to necropsy by a veterinarian trained in veterinary pathology. Gross and histological examination of liver, lung, thymus, heart, spleen, cerebellum, pancreas, small intestine, kidney, and adrenal gland did not reveal evidence of toxicity. Both a complete blood count and a white blood cell differential count were performed. No significant differences were noted. Mice were subjected to five daily injections of 30 mg/kg YTR107. This dose level was also tolerated without overt toxicity after 35 days. Mice were injected with 0 or 30 mg/kg YTR107 (i.p.) and 30 min later administered 0 or 16-Gy to the thorax. Pulmonary fibrotic lesions were quantified using respiratory-gated micro CT 136 days later. YTR107 did not increase the severity of radiation-induced lung fibrosis and did not affect body weights or life span of unirradiated or irradiated mice (p > 0.05) over the 136-day study. Thus, this study provides support for development of YTR107 as a therapeutic radiation sensitizer and was supported in part by R44CA228756-02 Citation Format: Konjeti R. Sekhar, Geri Traver, Peter A. Crooks, Michael L. Freeman. Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6164.

Homozygous hypomorphic BRCA2 variant in primary ovarian insufficiency without cancer or Fanconi anaemia trait

BackgroundPrimary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms....

Abstract 2812: Status of TGFbeta signaling determines PARP inhibitor sensitivity in head and neck cancer

Abstract Although human papilloma virus (HPV) status is the most informative prognostic marker in head and neck squamous cell carcinoma (HNSCC), the mechanisms that provide the survival advantage are poorly understood. Here we demonstrate that defects in transforming growth factor β (TGFβ) signaling confer a profound vulnerability in DNA repair for HPV+ HNSCC. We interrogated HNSCC profiled by The Cancer Genome Atlas (TCGA) with a 77 gene chronic TGFβ signature, defined as those genes that were significantly and reciprocally altered in MCF10A cultured for 7 days with TGFβ or a small molecule inhibitor of TGFβ type 1 receptor kinase (TBRIi). The 50 TGFβ-upregulated genes and 27 TGFβ-downregulated genes were negatively correlated (P<0.0001; R2=-0.68) in single-sample gene set enrichment analysis confirmed that the signature is informative in HNSCC. Unsupervised clustering of 294 HNSCC TCGA patient specimens using the TGFβ signature showed that HPV+ cancers were clustered by loss of TGFβ regulation. Patients whose cancers exhibit this profile had significantly better overall survival (P=0.002), independent of HPV status. We previously found that TGFβ signaling suppresses miR-182, which is a reported inhibitor of homologous recombination (HR). HPV+ cell lines increased expression of miR-182 compared to HPV- cell lines. Thus we evaluated radiation induced RAD51 foci formation, a biomarker of HR, in viable explants of patient-derived xenografts (PDX) treated with or without TBRIi. HPV+ PDX showed significantly fewer RAD51 foci than HPV- samples (P<0.005). HPV- PDX specimens treated with TBRIi also reduced RAD51 foci formation. We then established an HPV- cell line containing reporters to measure the frequency of DNA double strand break (DSB) repair with HR or alternative end-joining (Alt-EJ) following DSB induction with I-SceI endonuclease. TBRIi decreased HR events. In contrast, Alt-EJ was significantly increased (P<0.05). Poly (ADP-ribose) polymerase 1 (PARP1) is required for DSB repair with Alt-EJ. TCGA data shows dramatically increased expression of PARP1 in HPV+ HNSCC (P<0.0001). Hence, we speculated that loss of TGFβ signaling increase reliance on PARP1 mediated Alt-EJ to cope with DSB. Consistent with this, HPV+ cell lines were more sensitive to olaparib; while olaparib in combination with TGFβ inhibitor significantly increased cell death in HPV- cell lines by flow cytometry analysis of annexin V positive cells, but not in HPV+ cell lines. Notably, antagonizing miR-182 in HPV+ cells significantly reduced olaparib sensitivity (P<0.05). Together these data indicate HPV suppression of TGFβ signaling in turn shifts DNA repair from error-free HR to hazardous Alt-EJ through upregulated miR-182. Importantly, TGFβ inhibitors can recapitulate this DNA repair deficiency in hard to treat HPV- HNSCC. Citation Format: Qi Liu, Lin Ma, Luis Palomero, Miquel Àngel Pujana, Trevor Jones, Patrick Ha, John Murnane, Mary Helen Barcellos-Hoff. Status of TGFbeta signaling determines PARP inhibitor sensitivity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2812.

Dual targeting of hypoxia and homologous recombination repair dysfunction in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcome and few validated drug targets. Two prevalent features of TNBC, tumor hypoxia and derangement of homologous recombination (HR) repair, are potentially exploitable for therapy. This study investigated whether hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may inhibit growth of TNBC by simultaneously addressing these two targets. We measured in vitro activity of HAP of DNA breakers (tirapazamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in TNBC cell lines and isogenic models, and related this to measures of HR repair and expression of prodrug-activating enzymes. Antitumor activity of HAP was examined in isogenic BRCA2-knockout xenograft models and compared with platinum chemotherapy. All five HAP selectively inhibited growth of TNBC cell lines under hypoxia. Sensitivity to HAP was not strongly associated with BRCA1 genotype. However, HAP sensitivity was enhanced by suppression of HR (assessed by radiation-induced RAD51 focus formation) when BRCA1 and PALB2 were knocked down in a common (MDA-MB-231) background. Furthermore, knockout of BRCA2 markedly sensitized DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and PR-104 and significantly augmented sterilization of clonogens by these agents in xenografts, both as monotherapy and in combination with radiotherapy, but had less effect on activity of the benzotriazine di-N-oxide SN30000. PR-104 monotherapy was more effective than cisplatin at inhibiting growth of BRCA2-knockout tumors at equitoxic doses. This study demonstrates the potential for HAP of nitrogen mustards to simultaneously exploit hypoxia and HR defects in tumors, with translational implications for TNBC and other HR-deficient malignancies.

Combined inhibition of Wee1 and PARP1/2 for radiosensitization in pancreatic cancer.

While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX), and homologous recombination repair (HRR) were determined. Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization, whereas treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, whereas AZD1775 produced moderate, yet significant, radiosensitization (P < 0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P < 0.0001) evidenced by a 13-day delay in tumor volume doubling (vs. radiation alone) and complete eradication of 20% of tumors. Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization. Clin Cancer Res; 20(19); 5085-96. ©2014 AACR.

Inhibition of Protein Phosphatase 2A Radiosensitizes Pancreatic Cancers by Modulating CDC25C/CDK1 and Homologous Recombination Repair

To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we conducted an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target. We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacologic (LB100, a small molecule entering phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo. PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P < 0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γ-H2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss. Collectively, our data show that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer.

A novel small molecule RAD51 inactivator overcomes imatinib‐resistance in chronic myeloid leukaemia

RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and often contributes to drug-resistance. Abnormally elevated RAD51 function and hyperactive homologous recombination (HR) rates have been found in a panel of cancers, including breast cancer and chronic myeloid leukaemia (CML). Directly targeting RAD51 and attenuating the deregulated RAD51 activity has therefore been proposed as an alternative and supplementary strategy for cancer treatment. Here we show that a newly identified small molecule, IBR2, disrupts RAD51 multimerization, accelerates proteasome-mediated RAD51 protein degradation, reduces ionizing radiation-induced RAD51 foci formation, impairs HR, inhibits cancer cell growth and induces apoptosis. In a murine imatinib-resistant CML model bearing the T315I Bcr-abl mutation, IBR2, but not imatinib, significantly prolonged animal survival. Moreover, IBR2 effectively inhibits the proliferation of CD34+ progenitor cells from CML patients resistant to known BCR-ABL inhibitors. Therefore, small molecule inhibitors of RAD51 may suggest a novel class of broad-spectrum therapeutics for difficult-to-treat cancers.

RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination

Synthetic lethality is an approach to study selective cell killing based on genotype. Previous work in our laboratory has shown that loss of RAD52 is synthetically lethal with BRCA2 deficiency, while exhibiting no impact on cell growth and viability in BRCA2-proficient cells. We now show that this same synthetically lethal relationship is evident in cells with deficiencies in BRCA1 or PALB2, which implicates BRCA1, PALB2 and BRCA2 in an epistatic relationship with one another. When RAD52 was depleted in BRCA1- or PALB2-deficient cells, a severe reduction in plating efficiency was observed, with many abortive attempts at cell division apparent in the double-depleted background. In contrast, when RAD52 was depleted in a BRCA1- or PALB2-wildtype background, a negligible decrease in colony survival was observed. The frequency of ionizing radiation-induced RAD51 foci formation and double-strand break-induced homologous recombination (HR) was decreased by 3- and 10-fold, respectively, when RAD52 was knocked down in BRCA1- or PALB2-depleted cells, with minimal effect in BRCA1- or PALB2-proficient cells. RAD52 function was independent of BRCA1 status, as evidenced by the lack of any defect in RAD52 foci formation in BRCA1-depleted cells. Collectively, these findings suggest that RAD52 is an alternative repair pathway of RAD51-mediated HR, and a target for therapy in cells deficient in the BRCA1-PALB2-BRCA2 repair pathway.

Non-specific chemical inhibition of the Fanconi anemia pathway sensitizes cancer cells to cisplatin

BackgroundPlatinum compounds such as cisplatin and carboplatin are DNA crosslinking agents widely used for cancer chemotherapy. However, the effectiveness of platinum compounds is often tempered by the acquisition of cellular drug resistance. Until now, no pharmacological approach has successfully overcome cisplatin resistance in cancer treatment. Since the Fanconi anemia (FA) pathway is a DNA damage response pathway required for cellular resistance to DNA interstrand crosslinking agents, identification of small molecules that inhibit the FA pathway may reveal classes of chemicals that sensitize cancer cells to cisplatin.ResultsThrough a cell-based screening assay of over 16,000 chemicals, we identified 26 small molecules that inhibit ionizing radiation and cisplatin-induced FANCD2 foci formation, a marker of FA pathway activity, in multiple human cell lines. Most of these small molecules also compromised ionizing radiation-induced RAD51 foci formation and homologous recombination repair, indicating that they are not selective toward the regulation of FANCD2. These compounds include known inhibitors of the proteasome, cathepsin B, lysosome, CHK1, HSP90, CDK and PKC, and several uncharacterized chemicals including a novel proteasome inhibitor (Chembridge compound 5929407).Isobologram analyses demonstrated that half of the identified molecules sensitized ovarian cancer cells to cisplatin. Among them, 9 demonstrated increased efficiency toward FA pathway-proficient, cisplatin-resistant ovarian cancer cells. Six small molecules, including bortezomib (proteasome inhibitor), CA-074-Me (cathepsin B inhibitor) and 17-AAG (HSP90 inhibitor), synergized with cisplatin specifically in FA-proficient ovarian cancer cells (2008 + FANCF), but not in FA-deficient isogenic cells (2008). In addition, geldanamycin (HSP90 inhibitor) and two CHK1 inhibitors (UCN-01 and SB218078) exhibited a significantly stronger synergism with cisplatin in FA-proficient cells when compared to FA-deficient cells, suggesting a contribution of their FA pathway inhibitory activity to cisplatin sensitization.ConclusionOur findings suggest that, despite their lack of specificity, pharmaceutical inhibition of the FA pathway by bortezomib, CA-074-Me, CHK1 inhibitors or HSP90 inhibitors may be a promising strategy to sensitize cisplatin-resistant, FA pathway-proficient tumor cells to cisplatin. In addition, we identified four new small molecules which synergize with cisplatin. Further development of their analogs and evaluation of their combination with cisplatin may lead to the development of efficient cancer treatments.

BRCA1 and BRCA2 heterozygosity in embryonic stem cells reduces radiation-induced Rad51 focus formation but is not associated with radiosensitivity

Purpose: The breast cancer susceptibility genes BRCA1 (breast cancer 1) and BRCA2 (breast cancer 2) encode proteins involved in double-strand break (DSB) repair, whose functions include facilitating homologous recombination through interactions with Rad51, the human homologue of bacterial RecA. Homozygous deficiency inhibits Rad51 focus formation and enhances radiosensitivity, but the effects of heterozygosity have not been investigated in detail. The purpose of this work was to examine the effect of heterozygosity on Rad51 activation and clonogenicity following X-irradiation (XR).Materials and methods: We used quantitative assessment of immunofluorescent foci to assess Rad51 activation in wild type mouse embryonic fibroblasts (MEF) and in paired mutant and wild type BRCA1 and BRCA2 embryonic stem cells (ES cells). We measured radiosensitivity in the same cell lines using clonogenic survival assays.Results: ES cells exhibit higher numbers of cells with Rad51 foci post radiation than MEF, likely due to differences in cell cycle distribution. Compared to wild type cells, BRCA1 and BRCA2 heterozygous ES cells demonstrate lower numbers of Rad51 foci per nucleus 4 and 24 hours post radiation. This was not associated with significantly enhanced radiosensitivity.Conclusions: BRCA1/2 heterozygosity in ES cells is associated with a subtle reduction in Rad51 foci formation that is not associated with increased XR induced cytotoxicity.

Gene Expression Profile of BRCAness That Correlates With Responsiveness to Chemotherapy and With Outcome in Patients With Epithelial Ovarian Cancer

To define a gene expression profile of BRCAness that correlates with chemotherapy response and outcome in epithelial ovarian cancer (EOC). A publicly available microarray data set including 61 patients with EOC with either sporadic disease or BRCA(1/2) germline mutations was used for development of the BRCAness profile. Correlation with platinum responsiveness was assessed in platinum-sensitive and platinum-resistant tumor biopsy specimens from six patients with BRCA germline mutations. Association with poly-ADP ribose polymerase (PARP) inhibitor responsiveness and with radiation-induced RAD51 foci formation (a surrogate of homologous recombination) was assessed in Capan-1 cell line clones. The BRCAness profile was validated in 70 patients enriched for sporadic disease to assess its association with outcome. The BRCAness profile accurately predicted platinum responsiveness in eight out of 10 patient-derived tumor specimens, and between PARP-inhibitor sensitivity and resistance in four out of four Capan-1 clones. [corrected] When applied to the 70 patients with sporadic disease, patients with the BRCA-like (BL) profile had improved disease-free survival (34 months v 15 months; log-rank P = .013) and overall survival (72 months v 41 months; log-rank P = .006) compared with patients with a non-BRCA-like (NBL) profile, respectively. The BRCAness profile maintained independent prognostic value in multivariate analysis, which controlled for other known clinical prognostic factors. The BRCAness profile correlates with responsiveness to platinum and PARP inhibitors and identifies a subset of sporadic patients with improved outcome. Additional evaluation of this profile as a predictive tool in patients with sporadic EOC is warranted.

A gene expression profile of BRCAness that correlates with responsiveness to platinum and PARP inhibitors.

5004 Background: PARP inhibitors have been evaluated in patients with germline BRCA 1/2 mutations with impressive results. In addition to patients with germline BRCA 1/2 mutations, it has been suggested that PARP inhibition might also be a useful therapeutic strategy for the treatment of some patients with sporadic cancers. In order to better identify such patients, we defined a gene expression profile of “BRCAness” that correlates with responsiveness to platinum and PARP inhibitors. Methods: A publicly available microarray dataset including 61 patients with epithelial ovarian cancer with either sporadic disease or BRCA 1/2 mutations was used for development of the profile. Correlation with platinum responsiveness was assessed in platinum sensitive and resistant tumor biopsy specimens from 6 patients with BRCA germline mutations. Association with PARP inhibitor responsiveness and with radiation-induced RAD51 foci formation (a surrogate of homologous recombination) was assessed in Capan-1 cell line clones. The BRCAness profile was validated in 70 patients (35 patients who had been sequenced and known to be wildtype for BRCA 1/2, and 35 patients enriched for sporadic disease based on negative family history) in order to assess its association with outcome. Results: The BRCAness profile accurately predicted platinum responsiveness and mutation status in 8 out of 10 patient-derived tumor specimens, and between PARP-inhibitor sensitivity and resistance in 4 out of 4 Capan-1 clones. When applied to the 70 patients with sporadic disease, patients with BRCA-like (BL) profile had improved disease-free survival (34 months versus 15 months, log-rank p = 0.013) and overall survival (72 months versus 41 months, log-rank p = 0.006) compared to non-BRCA-like (NBL) patients, respectively. BRCAness profile maintained independent prognostic value in multivariate analysis, controlling for known clinical prognostic factors. Conclusions: The BRCAness profile correlates with responsiveness to platinum and PARP inhibitors and identifies a subset of sporadic patients with improved outcome. Use of this profile may ultimately permit identification of sporadic EOC patients who may benefit from PARP inhibitor therapy. No significant financial relationships to disclose.

Long-Term In vivo Effects of Cisplatin on γ-H2AX Foci Signaling in Peripheral Lymphocytes of Tumor Patients After Irradiation

This study determined the effects of cis-diamminedichloroplatinum(II) on radiation-induced foci formation of gamma-H2AX and Rad51 in lymphocytes. Twenty-eight cancer patients were irradiated for intrathoracic, pelvic, or head and neck tumors and received simultaneous cisplatin containing chemotherapy. The effect of cisplatin on radiation-induced gamma-H2AX and Rad51 foci as a response to ionizing radiation-induced DNA double-strand breaks was measured in lymphocytes after in vivo and in vitro radiochemotherapy. The role of DNA-dependent protein kinase and ataxia-telangiectasia mutated kinase in gamma-H2AX signaling, the consequences of altered gamma-H2AX foci formation on double-strand break end joining, was studied. Cisplatin decreased the number of induced gamma-H2AX foci in lymphocytes after in vivo or in vitro irradiation by 34% +/- 6% at days 0 to 3 after cisplatin (P < 0.0001) and remained significant until day 6. The variation in this cisplatin effect from patient to patient was larger than the retest error within the same patient (P = 0.01). The cisplatin effect was not accompanied by an inhibition of end joining of double-strand break as analyzed using gel electrophoresis of DNA under neutral conditions. Cisplatin also decreased radiation induced Rad51 foci formation in lymphocytes after stimulation of proliferation with phytohemagglutinin by 47% +/- 6% (P < 0.0001). Cisplatin has long-term effects on the early double-strand break response of gamma-H2AX and Rad51 foci formation after ionizing radiation. Inhibition of sensing and processing of double-strand break by gamma-H2AX and Rad51 foci formation are important mechanisms by which cisplatin can alter the radiation response.

Extensive Chromosomal Instability in Rad51d-Deficient Mouse Cells

Homologous recombination is a double-strand break repair pathway required for resistance to DNA damage and maintaining genomic integrity. In mitotically dividing vertebrate cells, the primary proteins involved in homologous recombination repair are RAD51 and the five RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. In the absence of Rad51d, human and mouse cells fail to proliferate, and mice defective for Rad51d die before birth, likely as a result of genomic instability and p53 activation. Here, we report that a p53 deletion is sufficient to extend the life span of Rad51d-deficient embryos by up to 6 days and rescue the cell lethal phenotype. The Rad51d-/- Trp53-/- mouse embryo-derived fibroblasts were sensitive to DNA-damaging agents, particularly interstrand cross-links, and exhibited extensive chromosome instability including aneuploidy, chromosome fragments, deletions, and complex rearrangements. Additionally, loss of Rad51d resulted in increased centrosome fragmentation and reduced levels of radiation-induced RAD51-focus formation. Spontaneous frequencies of sister chromatid exchange were not affected by the absence of Rad51d, but sister chromatid exchange frequencies did fail to be induced upon challenge with the DNA cross-linking agent mitomycin C. These findings support a crucial role for mammalian RAD51D in normal development, recombination, and maintaining mammalian genome stability.

C-Abl Tyrosine Kinase Is Not Essential for Ataxia Telangiectasia Mutated Functions in Chromosomal Maintenance

c-Abl is activated by DNA damage in an ataxia telangiectasia mutated (ATM)-dependent manner and plays important roles in growth arrest and apoptosis induced by DNA damage. c-Abl also interacts physically and functionally with Rad51, a key molecule in homologous recombinational (HR) DNA repair. To study further the roles of c-Abl in HR DNA repair, we generated c-Abl(-/-) and ATM(-/-)/c-Abl(-/-) mutant cell lines from a chicken B lymphocyte DT40 cell line, comparing the phenotypes of these mutants to those of ATM(-/-) DT40 cells that we had created previously. We found that the time course of radiation-induced Rad51 focus formation is abnormal in ATM(-/-) DT40 cells, consistent with the observation that ATM(-/-) DT40 cells display hypersensitivity to ionizing radiation and highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In contrast, c-Abl(-/-) cells did not show these ATM-related defects in their cellular response to radiation, nor did the disruption of c-Abl in ATM(-/-) DT40 cells exacerbate these ATM-related defects. However, c-Abl(-/-) DT40 cells, but not ATM(-/-) DT40 cells, were resistant to radiation-induced apoptosis, indicating an important role for c-Abl in this cellular response to ionizing radiation. These results therefore indicate that, although ATM plays an important role in genome maintenance, c-Abl is not essential for this ATM function. These findings suggest that c-Abl and ATM play important roles in the maintenance of the cell homeostasis in response to DNA damage that are, at least in part, independent.