PARP Inhibitor AZD2281 Research Articles

HMGA2 as a functional antagonist of PARP1 inhibitors in tumor cells.

Poly(ADP‐ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage‐induced PARP1 activity. We used human triple‐negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage‐induced PARP1 activity, which was dependent on functional DNA‐binding AT‐hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

PARP-1–Targeted Radiotherapy in Mouse Models of Glioblastoma

The DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) is overexpressed in glioblastoma, with overall low expression in healthy brain tissue. Paired with the availability of specific small molecule inhibitors, PARP-1 is a near-ideal target to develop novel radiotherapeutics to induce DNA damage and apoptosis in cancer cells, while sparing healthy brain tissue. Methods: We synthesized an 131I-labeled PARP-1 therapeutic and investigated its pharmacology in vitro and in vivo. A subcutaneous tumor model was used to quantify retention times and therapeutic efficacy. A potential clinical scenario, intratumoral convection-enhanced delivery, was mimicked using an orthotopic glioblastoma model combined with an implanted osmotic pump system to study local administration of 131I-PARPi (PARPi is PARP inhibitor). Results:131I-PARPi is a 1(2H)-phthalazinone, similar in structure to the Food and Drug Administration-approved PARP inhibitor AZD-2281. In vitro studies have shown that 131I-PARPi and AZD-2281 share similar pharmacologic profiles. 131I-PARPi delivered 134.1 cGy/MBq intratumoral injected activity. Doses to nontarget tissues, including liver and kidney, were significantly lower. Radiation damage and cell death in treated tumors were shown by p53 activation in U87-MG cells transfected with a p53-bioluminescent reporter. Treated mice showed significantly longer survival than mice receiving vehicle (29 vs. 22 d, P < 0.005) in a subcutaneous model. Convection-enhanced delivery demonstrated efficient retention of 131I-PARPi in orthotopic brain tumors, while quickly clearing from healthy brain tissue. Conclusion: Our results demonstrate 131I-PARPi's high potential as a therapeutic and highlight PARP's relevance as a target for radionuclide therapy. Radiation plays an integral role in brain tumor therapy, and radiolabeled PARP therapeutics could ultimately lead to improvements in the standard of care.

APR-246 (PRIMA-1MET) strongly synergizes with AZD2281 (olaparib) induced PARP inhibition to induce apoptosis in non-small cell lung cancer cell lines

APR-246 (PRIMA-1Met) is able to bind mutant p53 and restore its normal conformation and function. The compound has also been shown to increase intracellular ROS levels. Importantly, the poly-[ADP-ribose] polymerase-1 (PARP-1) enzyme plays an important role in the repair of ROS-induced DNA damage. We hypothesize that by blocking this repair with the PARP-inhibitor AZD2281 (olaparib), DNA damage would accumulate in the cell leading to massive apoptosis.We observed that APR-246 synergistically enhanced the cytotoxic response of olaparib in TP53 mutant non-small cell lung cancer cell lines, resulting in a strong apoptotic response. In the presence of wild type p53 a G2/M cell cycle block was predominantly observed. NOXA expression levels were significantly increased in a TP53 mutant background, and remained unchanged in the wild type cell line. The combined treatment of APR-246 and olaparib induced cell death that was associated with increased ROS production, accumulation of DNA damage and translocation of p53 to the mitochondria. Out data suggest a promising targeted combination strategy in which the response to olaparib is synergistically enhanced by the addition of APR-246, especially in a TP53 mutant background.

Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor

ABSTRACTPoly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

In vivo studies of the PARP inhibitor, AZD-2281, in combination with fractionated radiotherapy: An exploration of the therapeutic ratio

Background and purposePre-clinical data have shown that PARP inhibitors (PARPi) may increase the efficacy of radiotherapy in prostate cancer. However, it is uncertain as to whether PARPi lead to clonogenic kill when combined with radiotherapy (RT). Material and methodsWe tested the PARP inhibitor AZD-2281 as a radiosensitizing agent under oxic and hypoxic conditions for clonogenic survival in vitro and in vivo using the human prostate cancer cell line, 22Rv1. In addition, the effects of PARPi+RT on normal tissue were investigated using a crypt clonogenic assay. ResultsAZD-2281 inhibited cellular PARP activity under both oxic and hypoxic conditions. The addition of AZD-2281 radiosensitized 22Rv1 cells under oxia, acute hypoxia and chronic hypoxia in vitro. The combination of AZD-2281 with fractionated radiotherapy resulted in a significant growth delay and clonogenic kill in vivo. No increased gut toxicity was observed using this combined PARPi+radiotherapy regimen. ConclusionsThis is the first preclinical study to demonstrate direct clonogenic kill in vivo by the addition of AZD-2281 to radiotherapy. As we did not observe gut toxicity, the use of PARPi in the context of prostate cancer radiotherapy warrants further investigation in clinical trials.

Abstract 7: PARP and Bcl-2 co-inhibition in small cell lung cancer (SCLC)

Abstract Background: Small cell lung cancer (SCLC) accounts for 10-15% of all lung cancers. SCLC is initially responsive to chemotherapy, but quickly relapses. With few other treatment options available, the 5-year survival rate remains less than 5%. Bcl-2 is an anti-apoptotic protein that is overexpressed in SCLC and is associated with chemotherapy resistance. Previous studies have shown Bcl-2 inhibition to be effective in SCLC cell lines; however, Bcl-2 antagonists, such as GX15-070 (obatoclax mesylate), have been disappointing in clinical trials. We have recently shown that PARP-1, a DNA binding protein involved in single and double strand break repairs, is overexpressed in SCLC and that SCLC models are sensitive to PARP inhibition. As recent work illustrated a synergistic interaction between Bcl-2 antagonists and PARP inhibitors in pancreatic cancer cell lines, we hypothesize that a similar effect may occur in SCLC. Methods: Thirteen SCLC cell lines were treated with GX15-070 ± the PARP inhibitor AZD2281 (olaparib), and proliferation was assayed by CellTiter-Glo. In an effort to identify potential biomarkers of response to combination therapy, drug sensitivities (IC50 values) were correlated with proteomic profiling data generated by reverse phase protein array (RPPA), which measured the expression of 193 total and/or phospho-proteins. Results: As expected, SCLC cell lines exhibited a range of sensitivities to single agent GX15-070 in vitro (0.02-0.40 μM). When AZD2281 (PARP inhibitor) was added to a low dose of GX15-070 (0.03 μM), an additive effect was observed in 6 of 10 cell lines tested. Baseline proteomic profiling by RPPA revealed a number of proteins that correlated with sensitivity to Bcl-2 inhibition, including a pathway connecting high insulin receptor substrate-1 (IRS) to high Bcl-xL, (IRS-1, SGK, FOXO3, BIM, and Bcl-xL). Bcl-xL is also a target of GX15-070. Conclusion: Some SCLC cell lines were sensitive to Bcl-2 inhibition. In contrast to pancreatic cancer, a synergistic interaction was not observed when GX15-070 was combined with AZD2281. This combination showed at best an additive interaction in 6 of 10 SCLC cell lines tested, indicating limited value to the SCLC patient population as a whole. Interestingly, high IRS-1 and Bcl-xL expression may serve as biomarkers of response to GX15-070, suggesting a subset of SCLC patients that may be more sensitive to Bcl-2 inhibition. Our baseline proteomic analysis also suggests novel Bcl-2 inhibitor combinations. Metformin, for example, elevates expression of IRS-1, and its use could further sensitize SCLC to Bcl-2 inhibition. We are currently testing this novel combination in pre-clinical models of SCLC Citation Format: Janaya Shelly, Robert J. Cardnell, Fatemeh Masrorpour, Lixia Diao, Jing Wang, Lauren Byers. PARP and Bcl-2 co-inhibition in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2015-7

The PARP inhibitor AZD2281 (Olaparib) induces autophagy/mitophagy in BRCA1 and BRCA2 mutant breast cancer cells.

PARP inhibitors are considered promising anti-cancer agents and currently being tested in clinical trials in hereditary breast cancer patients harboring mutations in BRCA1 and BRCA2 genes. In this study, we investigated the antiproliferative effects and mechanism of PARP inhibitors ABT-888 (Veliparib), BSI-201 (Iniparib) and AZD228 (Olaparib) in breast cancer cell lines with BRCA1 or BRCA2 mutations and 9 different BRCA wild-type cell lines with BRCA1 allelic loss. We found that AZD2281 was the most potent in the PARP inhibitors and induces significant growth inhibition (~95%) in BRCA1 mutant (HCC-1937, MDA-MB-436, and SUM-149PT) and BRCA2 mutant (HCC-1428) cell lines. AZD2281 treatment also resulted in growth inhibition ranging from 20 to 50% in cells with BRCA1 allelic loss, including ER(+), HER2/Neu(+) and triple-negative breast cancer (TNBC) cells, but showed no effect in cells without with type BRCA without allelic loss. Knocking down of BRCA1 or BRCA2 in TNBC cells with BRCA1 allelic loss by RNA interference significantly enhanced AZD2281-induced growth inhibition and induced significant autophagy that was associated with mitophagy in cells with BRCA mutations. Inhibition of autophagy by gene knockdown significantly diminished AZD2281-induced mitophagy and apoptosis, indicating that autophagic process mediates some of the downstream effects of PARP inhibitors. In conclusion, our data provide the first evidence of PARP inhibitor AZD2281 autophagy and mitophagy in breast cancer cell lines with BRCA mutations or BRCA-allelic loss. In addition, our results indicate that the patients with BRCA1 allelic loss may also benefit from PARP inhibitor therapy if BRCA is further inhibited.

Combination of AZD2281 (Olaparib) and GX15-070 (Obatoclax) results in synergistic antitumor activities in preclinical models of pancreatic cancer

In this study, we explored the antitumor activities of the PARP inhibitor AZD2281 (Olaparib) and the pan-Bcl-2 inhibitor GX15-070 (Obatoclax) in six pancreatic cancer cell lines. While both agents were able to cause growth arrest and limited apoptosis, the combination of the two was able to synergistically cause growth arrest and non-apoptotic cell death. Furthermore, in an in vivo xenograft model, the combination caused substantially increased tumor necrosis compared to either treatment alone. Our results support further investigation of the combination of Bcl-2 and PARP inhibitors for the treatment of pancreatic cancer.

PARP1 Is Overexpressed in Nasopharyngeal Carcinoma and Its Inhibition Enhances Radiotherapy

Nasopharyngeal carcinoma is a rare but highly invasive cancer. As options of agents for effective combination chemoradiotherapy for advanced nasopharyngeal carcinoma are limited, novel therapeutic approaches are desperately needed. The ubiquitin ligase CHFR is known to target PARP1 for degradation and is epigenetically inactivated in nasopharyngeal carcinoma. We present evidence that PARP1 protein is indeed overexpressed in nasopharyngeal carcinoma cells in comparison with immortalized normal nasopharyngeal epithelial cells. Tissue microarray analysis also indicated that PARP1 protein is significantly elevated in primary nasopharyngeal carcinoma tissues, with strong correlation with all stages of nasopharyngeal carcinoma development. We found that the PARP inhibitor AZD2281 (olaparib) increased DNA damage, cell-cycle arrest, and apoptosis in nasopharyngeal carcinoma cells challenged with ionizing radiation or temozolomide. Isobologram analysis confirmed that the cytotoxicity triggered by AZD2281 and DNA-damaging agents was synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing radiation in animal xenograft models. These observations implicate that PARP1 overexpression is an early event in nasopharyngeal carcinoma development and provide a molecular basis of using PARP inhibitors to potentiate treatment of nasopharyngeal carcinoma with radio- and chemotherapy.

Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

Abstract 5584: Finding predictive markers for tumor response to classical cytotoxic drugs.

Abstract As attempts to find predictive markers for cytotoxic drugs have been relatively unsuccessful using patient tumor samples (1), we are using a mouse model for BRCA1-deficient mammary tumors to search for such markers. Although these tumors originate in inbred mice from an inactivating deletion of the Brca1 and p53 genes, their response to doxorubicin (2), docetaxel (2), topotecan (3) and PARP inhibitors (4) varies. We have shown that in this tumor model 5 of the 22 tumors respond poorly to docetaxel, because of upregulated drug-transporting P-glycoproteins (5). These 5 were not found by standard gene expression analytical tools, such as significance analysis of microarrays (SAM), and we have shown that SAM only detects resistance markers if present in more than half the tumor samples (5). Better algorithms for the detection of resistance determinants in a subfraction of tumors are being developed by De Ronde and Wessels (5). We are still searching for the cause of the poor docetaxel response in 17 of the 22 tumors. 1. Borst,P. and Wessels,L. (2010). Do predictive signatures really predict response to cancer chemotherapy? Cell Cycle 9, 4836-4840. 2. Rottenberg,S., et al (2007). Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer. Proc. Natl. Acad. Sci. U. S. A. 104, 12117-12122. 3. Zander,S., et al (2010). Sensitivity and acquired resistance of BRCA1; -53-deficient mouse mammary tumors to the topoisomerase I inhibitor topotecan. Cancer Res. 70, 1700-1710. 4. Rottenberg,S., et al (2008). High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc. Natl Acad. Sci. U. S. A 105, 17079-17084. 5. Rottenberg,S., et al (2012). Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Res 72, 2350-2361. Citation Format: Piet Borst, Sven Rottenberg. Finding predictive markers for tumor response to classical cytotoxic drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5584. doi:10.1158/1538-7445.AM2013-5584

Abstract P6-07-31: Assessing germline Homologous Recombination pathway deficiency in BRCA1 mutation carriers using Single Cell Network Profiling.

Abstract Background: Inherited alterations in BRCA1/2 genes increase genomic instability and cancer susceptibility. DNA sequencing detects BRCA1/2 mutations, but has the following limitations; 1) mutations may have unknown functional significance, 2) epigenetic alterations and mutations in other Homologous Recombination (HR) pathway components are not detected, and 3) the combined effects of pathway mutations are not understood. Thus a functional assessment of HR competence at the single cell level remains an unmet need as BRCA1/2 sequencing does not holistically inform on functionality of the HR pathway. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, at the single cell level, extracellular surface markers and functional changes in intracellular signaling in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). In this study, we tested the ability of SCNP to detect and quantify functional changes in HR signaling using peripheral blood mononuclear cell (PBMC) samples from BRCA1 mutation carriers (MUT) and wild type (WT) subjects. Methods: HR pathway activity was examined in PBMCs from BRCA1 MUT (n = 21) or WT (n = 20) subjects. Cell lines carrying BRCA1 MUTor WT genes were used as controls. PBMCs were stimulated with anti-CD3 and anti-CD28 for 24 hours to induce T cell proliferation then treated with PARP inhibitor (PARPi) AZD2281 +/− Temozolomide (TMZ) for 48h or 72h to induce DNA damage. DNA damage response (DDR) readouts were measured in both CyclinA2- and CyclinA2+ T cell subsets. Measurements included induced levels of p21, p53 and phosphorylation (p−) of p-H2AX, p-DNA-PKcs, p-RPA2/32, and p-BRCA1. Results: As expected based on the mechanism of action of PARPi, higher levels of induced p-H2AX and p53 were observed in CyclinA2+ cells of BRCA1 MUT versus WT cell line controls. In PBMCs, T cell proliferation (%CyclinA2+) was positively associated with PARPi induced DDR readouts. After controlling for proliferation, statistically significant differences in PARPi induced DDR signaling were observed between BRCA1 MUT and WT samples in many simultaneously assessed readouts including p-H2AX, p53 and p21 (increased in MUT), particularly in CyclinA2+ cells. Additionally, BRCA1 MUT samples displayed lower basal p-BRCA1 but higher induced p-BRCA1 levels compared to BRCA1 WT samples. Conclusions: SCNP was able to detect and quantify functional differences between PBMC samples from BRCA1 MUT (haploinsufficient) and WT donors by quantitatively assessing DDR signaling in CyclinA2+ T cells. Once verified on a larger data set, the assay could form the basis for the development of screening tests to identify subjects at higher risk of developing cancer or stratification tests to inform on cancer patient selection for treatment with PARP inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-31.

MiR-96 Downregulates REV1 and RAD51 to Promote Cellular Sensitivity to Cisplatin and PARP Inhibition

Cell survival after DNA damage relies on DNA repair, the abrogation of which causes genomic instability. The DNA repair protein RAD51 and the trans-lesion synthesis DNA polymerase REV1 are required for resistance to DNA interstrand cross-linking agents such as cisplatin. In this study, we show that overexpression of miR-96 in human cancer cells reduces the levels of RAD51 and REV1 and impacts the cellular response to agents that cause DNA damage. MiR-96 directly targeted the coding region of RAD51 and the 3'-untranslated region of REV1. Overexpression of miR-96 decreased the efficiency of homologous recombination and enhanced sensitivity to the PARP inhibitor AZD2281 in vitro and to cisplatin both in vitro and in vivo. Taken together, our findings indicate that miR-96 regulates DNA repair and chemosensitivity by repressing RAD51 and REV1. As a therapeutic candidate, miR-96 may improve chemotherapeutic efficacy by increasing the sensitivity of cancer cells to DNA damage.

Abstract 2539: The PARP inhibitor AZD2281 induces autophagy and complete cell death in BRCA mutant breast cancer cell lines

Abstract BRCA1 and BRCA2 are well-known DNA repair proteins that are usually mutated or deleted in breast tumors. According to the literature, 5 (12%) of 41 breast cancer cell lines have BRCA mutations and 28 (68%) of the 41 cell lines have BRCA1 allelic loss. PARP inhibitors are known to inhibit tumor growth in BRCA mutant tumors. The purpose of this study was to investigate the effects of PARP inhibitors in BRCA mutant breast cancer cell lines. We found that AZD2281 as a single agent induced complete cell death in BRCA1 mutant (HCC-1937, MDA-MB-436, and SUM-149PT) and BRCA2 mutant (HCC-1428) cell lines. AZD2281 induced an average growth inhibition of 33% in BRCA wild-type cell lines with BRCA1 allelic loss. Downregulation of BRCA1 or BRCA2 by shRNA sensitized the triple-negative breast cancer cell lines BT-20 and MDA-MB-231 to AZD2281 treatment. We also investigated whether autophagy is induced in response to PARP inhibition. AZD2281 treatment induced LC3-II expression, an indication of autophagy. Transmission electron micrographs demonstrated autophagosome formation after AZD2281 treatment. On transmission electron micrographs, we observed a decrease in mitochondria related to autophagosome formation after AZD2281 treatment. To show AZD2281 induced autophagy is targeting mitochondria, we labeled mitochondria with yellow fluorescein protein using lentiviral stable transfection. Flow cytometry measurement demonstrated a decrease in fluorescein-labeled mitochondria after AZD2281 treatment. Inhibition of autophagy in a AZD2281-treated BRCA mutant breast cancer cell line rescued mitochondrial degradation and partially inhibited apoptosis. In conclusion, we showed that autophagy plays an important role in AZD2281-induced cell death in BRCA mutant breast cancer cell lines by degradation of mitochondria besides DNA degradation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2539. doi:1538-7445.AM2012-2539

510 5-azacitidine restores and amplifies the response to bicalutamide and radiotherapy on preclinical models of androgen receptor expressing or deficient prostate tumors

Pre-clinical data have shown that PARP inhibitors (PARPi) may increase the efficacy of radiotherapy in prostate cancer. However, it is uncertain as to whether PARPi lead to clonogenic kill when combined with radiotherapy (RT).We tested the PARP inhibitor AZD-2281 as a radiosensitizing agent under oxic and hypoxic conditions for clonogenic survival in vitro and in vivo using the human prostate cancer cell line, 22Rv1. In addition, the effects of PARPi + RT on normal tissue were investigated using a crypt clonogenic assay.AZD-2281 inhibited cellular PARP activity under both oxic and hypoxic conditions. The addition of AZD-2281 radiosensitized 22Rv1 cells under oxia, acute hypoxia and chronic hypoxia in vitro. The combination of AZD-2281 with fractionated radiotherapy resulted in a significant growth delay and clonogenic kill in vivo. No increased gut toxicity was observed using this combined PARPi + radiotherapy regimen.This is the first preclinical study to demonstrate direct clonogenic kill in vivo by the addition of AZD-2281 to radiotherapy. As we did not observe gut toxicity, the use of PARPi in the context of prostate cancer radiotherapy warrants further investigation in clinical trials.

Mismatch repair proficiency as a predictor of response to the PARP inhibitor AZD2281.

e13644 Background: Microsatellite instability (MSI), the result of defective DNA mismatch repair (MMR), is observed in approximately 15% of colorectal cancers. It can either occur sporadically thro...

Pre-clinical activity of the PARP inhibitor AZD2281 in human breast cancer cell lines and in combination with DNA damaging agents.

Abstract Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast cancer. AZD2281, a potent, oral, PARP inhibitor has been shown to have clinical activity in patients with BRCA mutant breast cancers. Laboratory studies have suggested that non-BRCA mutant breast cancers may also be sensitive to PARP inhibition in tumors as a consequence of deficiencies in other homologous recombination (HR) repair components. Using a large panel of human breast cancer cell lines we tested the hypotheses that (1) there may be a subset of non-BRCA mutant breast cancers that are sensitive to single-agent AZD2281 and (2) AZD2281 would potentiate the cytotoxic effects of the DNA damaging agent cisplatin. Methods: 43 human cell lines representing known molecular subgroups of breast cancer (i.e. ER+, HER2 amplified, “triple-negative”), and 3 immortalized breast lines were treated in duplicate in adherent plates with AZD2281 using two-fold dilutions over 6 concentrations for 6 days. Dose response curves were generated using a cell count assay to calculate the IC50 of AZD2281. In addition, a subset of cell lines that grow under anchorage independent conditions were grown in triplicate in the presence and absence of 1 µM AZD2281 in soft agar for at least 3 weeks and growth inhibition was calculated as per cent of untreated control. Cell lines (both sensitive and resistant to single agent AZD2281) were also evaluated in combination with cisplatin in a cell count assay to assess the interaction between the two agents.&amp;#x2028; Results: The majority of breast cancer cell lines evaluated in the short term 2-D growth assay did not show significant growth inhibition (IC50 &amp;lt; 1 µM) following AZD2281 treatment, including a known BRCA mutant cell line, suggesting this assay may not be ideal for determining sensitivity to AZD2281. However, in the longer term anchorage independent clonogenic assay, approximately half of the cell lines evaluated demonstrated an IC50 &amp;lt; 1 µM. Of note, the majority of the cell lines representing a “triple-negative” phenotype appeared sensitive to AZD2281 in this assay. In addition, pre-treatment with AZD2281 prior to cisplatin, potentiated the growth inhibition seen with cisplatin in both AZD2281 sensitive and resistant cell lines. Additional studies evaluating predictive markers other than BRCA status are ongoing. Conclusion: The PARP inhibitor AZD2281 has significant pre-clinical activity in human breast cancer cell lines. In a clonogenic assay, cell lines representing the “triple negative” subtype were especially sensitive to AZD2281 supporting clinical development in this population, regardless of BRCA status. In addition, these pre-clinical data support the hypothesis that PARP inhibition may potentiate the effects of chemotherapy induced DNA damage and provide further rationale for clinical development. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1038.

High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs

Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative ("triple-negative") mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.

557 POSTER Pre-clinical activity of the PARP inhibitor AZD2281 in homologous recombination repair deficient triple negative breast cancer

557 POSTER Pre-clinical activity of the PARP inhibitor AZD2281 in homologous recombination repair deficient triple negative breast cancer

Selective Inhibition of BRCA2-Deficient Mammary Tumor Cell Growth by AZD2281 and Cisplatin

To assess efficacy of the novel, selective poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor AZD2281 against newly established BRCA2-deficient mouse mammary tumor cell lines and to determine potential synergy between AZD2281 and cisplatin. We established and thoroughly characterized a panel of clonal cell lines from independent BRCA2-deficient mouse mammary tumors and BRCA2-proficient control tumors. Subsequently, we assessed sensitivity of these lines to conventional cytotoxic drugs and the novel PARP inhibitor AZD2281. Finally, in vitro combination studies were done to investigate interaction between AZD2281 and cisplatin. Genetic, transcriptional, and functional analyses confirmed the successful isolation of BRCA2-deficient and BRCA2-proficient mouse mammary tumor cell lines. Treatment of these cell lines with 11 different anticancer drugs or with gamma-irradiation showed that AZD2281, a novel and specific PARP inhibitor, caused the strongest differential growth inhibition of BRCA2-deficient versus BRCA2-proficient mammary tumor cells. Finally, drug combination studies showed synergistic cytotoxicity of AZD2281 and cisplatin against BRCA2-deficient cells but not against BRCA2-proficient control cells. We have successfully established the first set of BRCA2-deficient mammary tumor cell lines, which form an important addition to the existing preclinical models for BRCA-mutated breast cancer. The exquisite sensitivity of these cells to the PARP inhibitor AZD2281, alone or in combination with cisplatin, provides strong support for AZD2281 as a novel targeted therapeutic against BRCA-deficient cancers.