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EP - 100 sensitizes BRCA wild - type ovarian cancer cells to PARP inhibitor Shaolin Ma 1,2 , Sunila Pradeep 1 , Sherry Wu 1 , Mark Seungwook Kim 1 , Wei Hu 1 , Selanere, Mangala 1 , Robert L. Coleman 1 , Anil K Sood 1,3 1 1Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center; 2 Reproductive Medicine Research Center, the Sixth Affiliated Hospital of Sun Yat- Sen University; 3 Center for RNA Interference and Non-Coding RNA, The University of Texas MD Anderson Cancer Center Abstract Objective: EP-100 is a synthetic lytic peptide specific for targeting LHRH receptors on cancer cells that is being clinically tested for treatment of ovarian cancer. In this study, we aimed to identify combination approaches with EP-100 in ovarian cancer models. Method: We carried out a series of in vitro (MTT assay, immunoblot analysis, Q- PCR, reverse phase protein array (RPPA), comet assay and immunofluorescence staining) and in vivo (orthotopic mouse model) experiments in ovarian cancer models to determine the biological effects of EP-100 alone and in combination with standard of care drugs. Results: We first examined the cytotoxic effects of EP-100 alone on 8 ovarian cancer cell lines with variable expression levels of LHRH receptors and the IC 50 values ranged from 0.5 to 3.0 μM. We then tested the effect of combining EP- 100 with standard drugs (e.g., cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib) on ovarian cancer cells. Of those drugs, we found that combination of EP-100 and olaparib was highly synergistic in drug sensitive and multi-drug resistant ovarian cancer cell lines (HeyA8, HeyA8-MDR, A2780, and A2780CP20). Furthermore, the nuclear foci of γH2AX were significantly increased in combination group of EP-100 and olaparib after 24 h incubation compared with control, EP-100 alone, or olaparib alone groups (p < 0.0001, ANOVA). In addition, there was increased DNA accumulation in tails using comet assay after treating cells with EP-100 and olaparib for 24 h (p < 0.001, ANOVA). RPPA results identified that PI3K/AKT pathway can be blocked significantly by the combination treatment. The study in HeyA8 xenograft mouse models showed that mice treated with EP-100 and olaparib had the lowest tumor weight (0.06 ± 0.05 g) compared with those treated with vehicle (1.19 ± 0.39 g, p < 0.008), EP- 100 alone (0.62 ± 0.28 g, p < 0.008), and olaparib alone (0.50 ± 0.22 g, p < 0.01). Conclusion: EP-100 has efficacy in preclinical models of ovarian cancer, especially in combination with olaparib. Our findings suggest that combining EP- 100 with a PARP inhibitor such as olaparib may be a promising therapeutic strategy for ovarian cancer. Background Objective Results Results Results Methods Conclusions Acknowledgements EP-100 is a fusion peptide consisting of the LHRH ligand and a lytic peptide to specifically target cancer cells that express LHRH-R 1 LHRH-R is a potential molecular target for cancer therapy which is overexpressed in many human tumors whereas it is not expressed or expressed at very low levels in adjacent normal tissues 2,3 • To evaluate the therapeutic effects of EP-100 on ovarian cancer cells using in vitro studies and orthotopic mouse xenograft models of ovarian cancer • To extend the utility of EP-100, identify effective combination therapies with EP- 100 for ovarian cancer and explore the potential mechanisms of this combination in ovarian cancer models Detect cell viability in the presence of EP-100 using MTT assay Western blot analysis to determine protein expression RPPA assay to identify the downstream mechanisms of the combination treatment Comet assay to detect the DNA damage status Immunofluorescence staining to characterize the localization of target protein LHRH-R was depleted by shRNA plasmids transfection EP-100 has an antitumor effect in ovarian cancer models while depletion of LHRH-R reduces the therapeutic effect of EP-100. EP-100 sensitizes ovarian cancer cells to the treatment of PARPi olaparib which can be abrogated upon downregulation of LHRH-R. The combination is highly synergistic with CI values of < 0.9. Combined treatment with EP-100 and olaparib leads to increased DNA damage in ovarian cancer cells. Error bars represent SD. References EP-100 can sensitize ovarian cancer cells to treatment with PARPi olaparib in vitro and in vivo through enhancing DNA damage accumulation and suppressing PI3K/AKT pathway and inhibition of BRCA1. 1. Curtis, K. K. et al. Novel LHRH-receptor-targeted cytolytic peptide, EP-100: first-in-human phase I study in patients with advanced LHRH- receptor-expressing solid tumors. Cancer Chemother Pharmacol 73, 931-941, doi:10.1007/s00280-014-2424-x (2014). 2. Fekete, M., Wittliff, J. L. & Schally, A. V. Characteristics and distribution of receptors for [D-TRP6]-luteinizing hormone-releasing hormone, somatostatin, epidermal growth factor, and sex steroids in 500 biopsy samples of human breast cancer. J Clin Lab Anal 3, 137-147 (1989). 3. Engel, J. B., Schally, A. V., Dietl, J., Rieger, L. & Honig, A. Targeted therapy of breast and gynecological cancers with cytotoxic analogues of peptide hormones. Mol Pharm 4, 652-658, doi:10.1021/mp0700514 (2007). Research supported by the MD Anderson Ovarian Cancer Spore and the American Cancer Society. RPPA assay revealed that PI3K/AKT pathway was the top inhibitory pathway in the presence of EP-100. Western blot analysis showed that EP-100 inhibits the expression of BRCA1. EP-100 and olaparib synergize to suppress tumor growth in a BRCA1/2 wild-type ovarian cancer xenograft model. Error bars represent SD.
