The American Cancer Society and National Cancer Institute estimates that there will be over 22,240 new cases of ovarian cancer with over 14,000 deaths from ovarian cancer this year.1 The median age for diagnosis for sporadic disease is 60 years with those with a genetic tendency towards ovarian cancer often being diagnosed in their 50’s. Ovarian cancer represents tumors of epithelial, germ cell and sex cord-stromal origin. Epithelial ovarian cancer which arises from arise from the surface epithelium of the ovary represents about 90% of ovarian cancers. The most important risk factors for the development of ovarian cancer are age, family history and infertility while lactation, pregnancy, and tubal ligation are mildly protective.2 The ovarian epithelium cancers can be divided into four types: papillary serous, endometroid, mucinous and clear cell. Clear cell represents represent 6% of the epithelial cells types and is the most aggressive and resistant to therapy.3 Following cytoreductive surgery for ovarian cancer the use of platinum – taxane based chemotherapy has traditionally been the gold standard for chemotherapy.1 Although most ovarian cancers are chemosensitive at diagnosis drug acquired resistance frequently appears leading to progression and ultimately death.

The hallmark of cancer development is the development of clonal expansions genetic mutations which continue to accrue additional genetic mutations and eventually genomic instability. This led to the idea that perhaps one could take advantage of this genetic instability in cancer cells an instability not present in non-cancer cells.4 Thus, providing a pathway to targeted therapy. In a paper by Farmer that appeared in Nature, the feasibility of using PARP inhibitors as targeted therapy was shown.5 It has reported that cells with BRCA1 or BRCA2 mutations which are important for homologous recombination were 1000 times more sensitive to PARP inhibition than those cell without a defect in homologous recombination.6 Papers by De Soto and another by Calabrese illustrated the clinical feasibility of using PARP inhibitors as chemosensitizers in the treatment of human cancer.7, 8 Poly (ADP-Ribose) Polymerase (PARP) is a key enzyme utilized in the repair of single strand DNA damage via the Base Excision Repair pathway. PARP inhibition produces persistent single-strand DNA breaks which during mitosis become double stranded breaks at the replication fork.9 These double stranded breaks may be repaired by homologous recombination. However, many cancers are deficient in their ability to repair by homologous recombination due to deficiencies in BRCA1, BRCA2 and other enzymes needed in this repair pathway. This leads to the accumulation of chromosomal damage and the death of cancer cells. Because most non-cancer cells are efficient in homologous recombination, they are relatively unaffected by the inhibition of PARP. Myelosuppression however dies occur with PARP inhibitors in addition to somnolence.10, 11 Today, PARP inhibitors are approved for maintenance therapy in ovarian cancer following platinum based therapy.12 PARP inhibitors are also treatment friendly as Olaparib and Veliparib both have the advantage of being available orally.13, 14 Here we show the effective use of PARP inhibiters as chemosensitizers against clear cell carcinoma with taxane, platinum and other agents used to treat ovarian cancer.

Cell Lines & Medications

ES-2, TOV-112D, TOV-21G, and SKOV-3 and human epithelial ovarian cancer cell lines where obtained from American Type Tissue Culture in Manassas VA. Cisplatin, Carboplatinum, Doxorubicin, 5-Fluorouracil, Gemcitabine and Paclitaxel where purchased from Sigma-Aldrich, St. Louis MO. The PARP-1 inhibitor AG14361 was obtained from Organix inc. in Woburn MA while ABT888 (Veliparib) and AZD2281 (Olaparib) were obtained from Selleck Chemical in Houston TX. Cells were grown in DMEM media with 10% fetal bovine serum in a 5% CO₂ incubator at 37 ⁰C. Cells at 80-85% confluence were trypsinized , washed with PBS and plated for each experiment

Determining IC50 Values

Standard curves where made by plating 25k to 300k cells for each cell line for 4-12 hours and after ascertaining that the cells where attached exposed to a solution of 1 mg/ml thiazolyl blue tetrazolium (MTT) for 30 minutes. This was followed by decanting the MTT and adding propranolol to each well for 30 minutes. The absorption for each well was read with the Perkin-Elmer 1420 multi-label counter. Each data point was replicated at least 10 times. IC50 values were then determined by plating cells in 12 well plates and after incubation for 4-12 hours the cells were exposed for 72 hours to various doses of drug. There were at least two controls containing vehicle for each experiment. The number of cells in each well at each specific drug concentration was then determined by the previously described MTT assay. Each experiment was replicated at least 9 times with the tenth time being a direct cell count by hemacytometer. The IC 50 values were then calculated by graphing the % inhibition vs log(dose) using Sigma Plot and standard 3rd order (sigmoidal) equations with the required r2 value having to be > 0.975.

The “PARP in vivo Pharmacodynamic Assay II kit” from Trevigen inc, Gaithersburg MD was used to determine the amount of PARP activity in each cell line prior to and after treatment with PARP inhibitors.

The IC50 values of AG14361, Veliparib and Olaparib against clear cell, serous and mucinous epithelial ovarian cancer cell lines was evaluated. The ES-2 clear cell ovarian carcinoma was derived from a 47 year old African American female with low to moderate level resistance to cisplatin and doxorubicin.15 This clear ovarian cancer was very sensitive to PARP inhibition with the IC50 values for AG14361, Veliparib and Olaparib being 6.2 µM, 38 µM and 97 µM. A second clear cell ovarian carcinoma TOV21G was derived from a 62 year old female was also found to be very sensitive to PARP inhibition.16 The following are the IC50 values of the PARP inhibitors against the TOV21G ovarian cancer cell line: AG14361 8.9 µM, Veliparib 78 µM and Olaparib 150 µM. A serous ovarian cancer cell line SKOV-3 derived from a 64 year old female with tumor resistance to cisplatin and doxorubicin was tested against AG14361, Veliparib and Olaparib with the IC50 values being 15.2 µM, 82 µM and 17.6 µM. The Ov-90 cell line was another serous cell ovarian cancer cell line evaluated which was derived from a 64 year old French female.17 The OV-90 cell line is Her2/neu + and like the clear cell line TOV-21G has a deletion at 3p24. Interestingly, this same locus has been found to be associated with breast cancer. The IC50 values for the Ov-90 cell line were 5.2 µM , 35 µM and 15 µM for AG14361, Veliparib and Olaparib respectively. Next the ability of the PARP inhibitors to inhibit endometrioid type ovarian cancer was evaluated through the TOV112D ovarian cancer cell line. This cell line was derived from a 42 year old French female and was her2/neu +.16 The IC50 values after exposure to the PARP inhibitors were similar to those obtained for the serous cell lines the results are summarized in Table 1.

Evidence suggests that the PARP inhibitors may be able to chemo- sensitize breast and brain cancers to DNA damaging agents. The IC50 values for five commonly used chemotherapeutic agents used to treat ovarian cancer cisplatin, carboplatin, doxorubicin, 5-fluorouracil, gemcitabine and paclitaxel where determined for the ES-2, TOV112D and TOV21G cell lines Table 2 . Next, we determined the IC50 value of these same agents when used in combination with a constant 10 µM of AG14361. The value of 10 µM was chosen as it is a blood level which represents a blood level which might clinically be reached not only for AG14361 but Veliparib and Olaparib, thus potentially allowing for the translation of these results into the clinic.

It has been shown that up to 50% of ovarian cancers may have trouble repairing double strand breaks via homologous recombination.18 The addition of adjuvant chemotherapy in clear cell carcinoma does not show any survival difference following surgical excision in stage I and stage II cancer and little improvement in stage III or IV.19 Thus, the need for improved cytotoxicity of adjuvant chemotherapy towards clear cell ovarian cancer is needed. Clear cell ovarian cancer has been compared to triple negative breast cancer due to its absence of both estrogen and progesterone receptors and rapid growth.20 Dysfunctional homologous recombination is a determinant in platinum and PARP sensitivity and possibly taxane sensitivity providing a rational for the use of PARP inhibitors in conjunction with these other agents in treating ovarian cancer.21 Loss of the ability to perform homologous recombination may be due to either genetic mutation or epigenetic influences.22 The increased amount of double stranded breaks leads to rapid chromosomal instability and cellular death. Among the proteins that may have mutations or be epigenetically silenced are BRCA1, BRCA2, PALB2, ATM, CHEK1, CHEK2, and RAD51.23 The loss of homologous recombination is also associated with TP53 mutation and hence, a loss of cell cycle regulation.24 Which may be important in clear cell ovarian cancer which is an exceptionally fast growing cancer. Additionally, PARP may act as a chemosensitizer by also partially disrupting spindle assembly.25 PARP inhibitors may be especially useful for those cancers that are resistant to prior platinum and other treatments but, are limited in their ability to chemosensitize these agents in cancers that are refractory to specific chemoagents26 . Refractory behavior is often seen in ovarian cancer that has a back mutation in previously dysfunctional enzymes needed for homologous recombination.27 The use of PARP inhibitors may also be useful in treating those clear cell carcinomas that are cross resistant to platinum, topoisomerase and ionizing radiation therapy providing a grim outcome for these patients. This type of cross resistance is often due elevated glutathione-S-transferase which detoxifies these agents.15 The use of PARP inhibitors would allow for a cytotoxic effect for the reduced levels of chemotherapeutic agents.

The use of PARP inhibitors maybe useful as single agent maintenance therapy in clear cell carcinoma as the sensitivity to PARP is at least as great as serous cell ovarian cancer which has been shown to be amiable to maintenance therapy.28

The use of PARP inhibitors to treat clear cell ovarian cancer in conjunction with platinum agents, taxane or topoisomerase inhibitors may allow for using a lower dose of these toxic agents and thus reduce the side effects. Additionally, the use of PARP inhibitors as chemosensitizers may allow for the more targeted and efficient killing of clear cell ovarian cancer cells. PARP inhibitors are exceptional chemosensitizers of clear cell ovarian cancer to treatment with most standard chemotherapy agents. Gemcitabine however is however antagonized by PARP inhibitors. This is the first paper to specifically show the effectiveness of PARP inhibitors against clear cell ovarian cancer.

Table 2

	ES-2 Clear cell	TOV112D Endometrioid	TOV-21G Serous
Cisplatin	1.18 µM	5.4 µM	17.5 µM
Doxorubicin	0.046 µM	0.038 µM	0.082 µM
5-Fluorouracil	20.7 µM	19.9 µM	40. 5 µM
Gemcitabine	0.00012 µM	0.0046 µM	8.6 µM
Paclitaxel	0.018 µM	0.008 µM	0.075 µM

Table 3 Underhill C. Toulmonde M. Bonnefoi H. A review of PARP inhibitors: from bench to bedside Annals of Oncology 2011 22 2 268 279 0923-7534 10.1093/annonc/mdq322 Elsevier BV https://dx.doi.org/10.1093/annonc/mdq322 Nagasawa Saya Ikeda Kazuhiro Horie-Inoue Kuniko Sato Sho Itakura Atsuo Takeda Satoru Hasegawa Kosei Inoue Satoshi Systematic Identification of Characteristic Genes of Ovarian Clear Cell Carcinoma Compared with High-Grade Serous Carcinoma Based on RNA-Sequencing International Journal of Molecular Sciences 2019 20 18 4330 4330 1422-0067 10.3390/ijms20184330 MDPI AG https://dx.doi.org/10.3390/ijms20184330 Brett M. Reid Brett M. Reid Jennifer B. Permuth Thomas A. Sellers Jennifer B. Permuth Thomas A. Sellers Epidemiology of ovarian cancer: a review Cancer Biology & Medicine 2017 14 1 9 32 2095-3941 10.20892/j.issn.2095-3941.2016.0084 Cancer Biology and Medicine https://dx.doi.org/10.20892/j.issn.2095-3941.2016.0084 Spriggs D R Longo D L PARP Inhibitors in Ovarian Cancer Treatment Provencher D M Lounis H Champoux L Characterization of four novel epithelial cancer cell lines In Vitro Cell. Dev. Biol. Anim 2000 36 357 361 Soto De Wang J A Tominaga X Y The inhibition of treatment of breast cancer with poly(ADP-ribose) polymerase (PARP-1) inhibitors Int. J. Biol. Sci 2006 2 4 179 185 Ledermann Jonathan Harter Philipp Gourley Charlie Friedlander Michael Vergote Ignace Rustin Gordon Scott Clare Meier Werner Shapira-Frommer Ronnie Safra Tamar Matei Daniela Macpherson Euan Watkins Claire Carmichael James Matulonis Ursula Olaparib Maintenance Therapy in Platinum-Sensitive Relapsed Ovarian Cancer New England Journal of Medicine 2012 366 1382 1392 0028-4793, 1533-4406 Massachusetts Medical Society 10.1056/nejmoa1105535 https://dx.doi.org/10.1056/nejmoa1105535 Fong P C Yap T A Boss D S Tutt A Wu P ADP-ribose) polymerase inhibition: Frequent durable responses in BRCA carrier correlating with platinum -free survival JCO 2010 28 2512 2519 Lounis Hafida Mes-Masson Anne-Marie Dion France Bradley W Edward C Seymour Robert J Provencher Diane Tonin Patricia N Mapping of chromosome 3p deletions in human epithelial ovarian tumors Oncogene 1998 17 18 2359 2365 0950-9232, 1476-5594 10.1038/sj.onc.1202152 Springer Science and Business Media LLC https://dx.doi.org/10.1038/sj.onc.1202152 Chang Paul Jacobson Myron K. Mitchison Timothy J. Poly(ADP-ribose) is required for spindle assembly and structure Nature 2004 432 7017 645 649 0028-0836, 1476-4687 10.1038/nature03061 Springer Science and Business Media LLC https://dx.doi.org/10.1038/nature03061 Mateo J. Lord C.J. Serra V. Tutt A. Balmaña J. Castroviejo-Bermejo M. Cruz C. Oaknin A. Kaye S.B. de Bono J.S. A decade of clinical development of PARP inhibitors in perspective Annals of Oncology 2019 30 9 1437 1447 0923-7534 10.1093/annonc/mdz192 Elsevier BV https://dx.doi.org/10.1093/annonc/mdz192 Calabrese C R Almassy R Barton S Anticancer chemosensitization and radiosensitization by the novel poly(ADP-ribose) polymerase-1 inhibitor AG14361 J Natl Cancer Inst 2004 96 56 67 Moore K N Pothuri B Monk B Coleman R L PARP inhibition in recurrent ovarian cancer Clinical advances in hematology & Oncology 2020 18 10 10 Swisher E. M. Sakai W. Karlan B. Y. Wurz K. Urban N. Taniguchi T. Secondary BRCA1 Mutations in BRCA1-Mutated Ovarian Carcinomas with Platinum Resistance Cancer Research 2008 68 8 2581 2586 0008-5472, 1538-7445 10.1158/0008-5472.can-08-0088 American Association for Cancer Research (AACR) https://dx.doi.org/10.1158/0008-5472.can-08-0088 Gien Lilian T. Mackay Helen J. The Emerging Role of PARP Inhibitors in the Treatment of Epithelial Ovarian Cancer Journal of Oncology 2010 2010 1 6 1687-8450, 1687-8469 10.1155/2010/151750 Hindawi Limited https://dx.doi.org/10.1155/2010/151750 McCabe Nuala Lord Christopher J. Tutt Andrew NJ Martin Niall Smith Graeme C.M. Ashworth Alan BRCA2-deficient CAPAN-1 cells are extremely sensitive to the inhibition of poly (ADP-ribose) polymerase: An issue of potency Cancer Biology & Therapy 2005 4 9 934 936 1538-4047, 1555-8576 10.4161/cbt.4.9.2141 Informa UK Limited https://dx.doi.org/10.4161/cbt.4.9.2141 Lee H Y Hong J H Byun J H Kim H J Baek S K Clinical characteristics of clear cell ovarian cancer: A retrospective multicenter experience of 308 patients in South Korea Cancer Res Treat 2020 52 1 277 283 Press J Z Luca De Boyd A N Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities BMC Cancer 2008 8 17 28 Vita De Lawrence V Rosenberg T S Cancer Principles & Practice of Oncology 2019 Wolters Kluwer Publishers Campos Susana M. Ghosh Sue A Current Review of Targeted Therapeutics for Ovarian Cancer Journal of Oncology 2010 2010 1 11 1687-8450, 1687-8469 10.1155/2010/149362 Hindawi Limited https://dx.doi.org/10.1155/2010/149362 Mukhopadhyay A. Elattar A. Cerbinskaite A. Wilkinson S. J. Drew Y. Kyle S. Los G. Hostomsky Z. Edmondson R. J. Curtin N. J. Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors Clinical Cancer Research 2010 16 8 2344 2351 1078-0432, 1557-3265 10.1158/1078-0432.ccr-09-2758 American Association for Cancer Research (AACR) https://dx.doi.org/10.1158/1078-0432.ccr-09-2758 Papa Anselmo Caruso Davide Strudel Martina Tomao Silverio Tomao Federica Update on Poly-ADP-ribose polymerase inhibition for ovarian cancer treatment Journal of Translational Medicine 2016 14 1 267 267 1479-5876 10.1186/s12967-016-1027-1 Springer Science and Business Media LLC https://dx.doi.org/10.1186/s12967-016-1027-1 Gentles Lucy Goranov Bojidar Matheson Elizabeth Herriott Ashleigh Kaufmann Angelika Hall Sally Mukhopadhyay Asima Drew Yvette Curtin Nicola O’Donnell Rachel Exploring the Frequency of Homologous Recombination DNA Repair Dysfunction in Multiple Cancer Types Cancers 2019 11 3 354 354 2072-6694 10.3390/cancers11030354 MDPI AG https://dx.doi.org/10.3390/cancers11030354 Lau D H Lewis A D Ehsan M N Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin Cancer Res 1991 51 5181 5188 Yamashita Y. Ovarian cancer: new developments in clear cell carcinoma and hopes for targeted therapy Japanese Journal of Clinical Oncology 2015 45 5 405 407 0368-2811, 1465-3621 10.1093/jjco/hyu221 Oxford University Press (OUP) https://dx.doi.org/10.1093/jjco/hyu221 Farmer Hannah McCabe Nuala Lord Christopher J. Tutt Andrew N. J. Johnson Damian A. Richardson Tobias B. Santarosa Manuela Dillon Krystyna J. Hickson Ian Knights Charlotte Martin Niall M. B. Jackson Stephen P. Smith Graeme C. M. Ashworth Alan Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy Nature 2005 434 7035 917 921 0028-0836, 1476-4687 10.1038/nature03445 Springer Science and Business Media LLC https://dx.doi.org/10.1038/nature03445 Kummar S Kinders R Gutierrez M E Phase 0 clinical trial of the poly(ADP-Ribose) polymerase inhibitor ABT-888 in patients with advanced malignancies JCO 2009 27 2705 2711 Hennessy B T Timms K M Carey M S Somatic mutations in BRCA1 and BRCA2 could expand the number of patients that benefit from poly(ADP-ribose) polymerase inhibitors in ovarian cancer JCO 2010 28 3570 3576