Supplementary Figure 3 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Supplementary Tables 1-2 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Supplementary Table 1 from Imaging DNA Damage In Vivo Using γH2AX-Targeted Immunoconjugates

Supplementary Figure Legends 1-6 from Imaging DNA Damage In Vivo Using γH2AX-Targeted Immunoconjugates

Abstract DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow 111In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, 111In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings. Cancer Res; 71(13); 4539–49. ©2011 AACR.

DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophoreand radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (gH2AX), which forms foci at sites of DNA double-strand breaks. Anti-gH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-gH2AX-Tat with gH2AX foci. In comparison with nonspecific antibody conjugates, In-anti-gH2AX-Tat was retained longer in cells. Anti-gH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-gH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of gH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target gH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings. Cancer Res; 71(13); 4539–49. 2011 AACR.

Authors' A Gray Institu Oxford, Un Institute o 3Departme University, University o

Abstract Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor–resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor–resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair–independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair–independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. Cancer Res; 70(15); 6268–76. ©2010 AACR.

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.

Authors' Toxicology, for Cancer upon Tyne Netherland Research U Oncology a Medicum, N Medical On 7Science fo Note: Sup Research O Current ad University o Correspon Roosevelt D 324; Fax: 4

1015 Objectives DNA double strand breaks (DSB) are the most deleterious lesion caused by ionizing radiation (IR). Following DSB formation, the histone, H2AX, is phosphorylated by DNA-PKcs, ATM, or ATR and designated γH2AX. γH2AX forms foci at sites of DSB and is detectable by immunofluorescence microscopy. We have synthesised Cy3-labelled TAT-immunoconjugates targeted against γH2AX for imaging DNA damage in vivo. Methods The formation of γH2AX in response to IR in 4 breast cancer cell lines was investigated using Western blot analysis. TAT-peptide (GRKKRRQRRRPPQGYG; with nuclear localisation sequence) was conjugated to anti-γH2AX antibody using EDC/NHSchemistry. Cy3-conjugation was achieved using Cy3-TPP ester. MDA-MB-468 cells were exposed to IR (4Gy) and then to Cy3-anti-γH2AX-TAT (1μg/mL) or Cy3-mouseIgG-TAT (as a non-specific immunoconjugate control) for 1h. Samples were prepared for fluorescence microscopy. Cy3-labelled anti-γH2AX-TAT (20µg) or mouse-IgG-TAT was administered to mice bearing MDA-MB-468 tumour xenografts. In vivo fluorescence images were obtained 30min, 24 and 48h after tumours were exposed to IR (4Gy). Results TAT:IgG conjugation ratio was 5:1. Cy3:IgG ratio was 6-7:1. HPLC demonstrated that the final compound was >97% pure and free of unconjugated Cy3. Foci were visualised using Cy3-anti-γH2AX-TAT but not using Cy3-mouseIgG-TAT in irradiated MDA-MB-468 cells. Cy3-anti-γH2AX-TAT, but not Cy3-mouseIgG-TAT, accumulated in irradiated tumours at 48h. Conclusions The evaluation of γH2AX in vivo may be feasible using immunoconugates such as Cy3-anti-γH2AX-TAT and could be used to predict response to anticancer therapy.

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.

X-ray repair cross-complementing protein-1 (XRCC1)-deficient cells are sensitive to DNA damaging agents and have delayed processing of DNA base lesions. In support of its role in base excision repair, it was found that XRCC1 forms a tight complex with DNA ligase IIIalpha and also interacts with DNA polymerase beta (Pol beta) and other base excision repair (BER) proteins. We have isolated wild-type XRCC1-DNA ligase IIIalpha heterodimer and mutated XRCC1-DNA ligase IIIalpha complex that does not interact with Pol beta and tested their activities in BER reconstituted with human purified proteins. We find that a point mutation in the XRCC1 protein which disrupts functional interaction with Pol beta, affected the ligation efficiency of the mutant XRCC1-DNA ligase IIIalpha heterodimer in reconstituted BER reactions. We also compared sensitivity to hydrogen peroxide between wild-type CHO-9 cells, XRCC1-deficient EM-C11 cells and EM-C11 cells transfected with empty plasmid vector or with plasmid vector carrying wild-type or mutant XRCC1 gene and find that the plasmid encoding XRCC1 protein, that does not interact with Pol beta has reduced ability to rescue the hydrogen peroxide sensitivity of XRCC1- deficient cells. These data suggest an important role for the XRCC1-Pol beta interaction for coordinating the efficiency of the BER process.

Base excision repair (BER) is one of the major pathways for repair of simple DNA base lesions and is carried out through a series of coordinated reactions relying on several different enzymatic activities and accessory proteins. Imbalance of BER activities has been reported to be linked to genetic instability and cancer. To experimentally address the mechanisms orchestrating BER, we monitored both the overall rate and the rate-limiting steps in the repair in cell-free extracts of five different endogenously occurring DNA lesions (abasic site, uracil, 8-oxoguanine, hypoxanthine and 5,6-dihydrouracil) and the effect of addition of rate-limiting BER components on the rate and co-ordination of BER reactions. We find that several mechanisms including regulation of DNA glycosylase turnover and involvement of poly(ADP-ribose) polymerase participate in synchronization of the repair events. We also find that repair of different DNA lesions involves different mechanisms for optimizing repair rates without accumulation of intermediates. Repair of some lesions such as 8-oxoguanine is regulated by glycosylase turnover and progress without substantial accumulation of repair intermediates. However, during repair of the apurinic/apyrimidinic (AP) sites or 5,6-dihydrouracil, poly(ADP-ribose) polymerase plays an important role in the coordination of the rates of repair reactions.

In mammalian cells, DNA ligase IIIalpha and DNA ligase I participate in the short- and long-patch base excision repair pathways, respectively. Using an in vitro repair assay employing DNA ligase-depleted cell extracts and DNA substrates containing a single lesion repaired either through short-patch (regular abasic site) or long-patch (reduced abasic site) base excision repair pathways, we addressed the question whether DNA ligases are specific to each pathway or if they are exchangeable. We find that immunodepletion of DNA ligase I did not affect the short-patch repair pathway but blocked long-patch repair, suggesting that DNA ligase IIIalpha is not able to substitute DNA ligase I during long-patch repair. In contrast, immunodepletion of DNA ligase IIIalpha did not significantly affect either pathway. Moreover, repair of normal abasic sites in wild-type and X-ray cross-complementing gene 1 (XRCC1)-DNA ligase IIIalpha-immunodepleted cell extracts involved similar proportions of short- and long-patch repair events. This suggests that DNA ligase I was able to efficiently substitute the XRCC1-DNA ligase IIIalpha complex during short-patch repair.

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.