BRCA Status does not Predict Synergism of a ... - ACS Publications

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BRCA Status does not Predict Synergism of a Carboplatin and Olaparib Combination in High-grade Serous Ovarian Cancer Cell Lines Yen Ting Shen, James C. Evans, Gaetano Zafarana, Christine Allen, and Micheline Piquette-Miller Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00246 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Molecular Pharmaceutics

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BRCA Status does not Predict Synergism of a Carboplatin and Olaparib Combination in High-grade Serous Ovarian Cancer Cell Lines

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Yen Ting Shen†, James C. Evans†, Gaetano Zafarana†‡, Christine Allen†, Micheline PiquetteMiller*†

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†Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada

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‡Genetics and Genome Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G

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1X8, Canada

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Corresponding Author

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Prof. Micheline Piquette-Miller Leslie Dan Faculty of Pharmacy University of Toronto 144 College Street Toronto, Ontario M5S 3M2 Canada

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E-mail: [email protected]

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Synergy; PARP inhibitor; BRCA; Carboplatin; Olaparib; Drug Combination; Ovarian Cancer

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Over 50% of epithelial ovarian cancers express the BRCAness profile that leads to a

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dysfunctional homologous recombination repair system. The combination of a dysfunctional

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homologous recombination repair system and a poly (ADP-ribose) polymerase (PARP) inhibitor

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results in a synthetic lethal phenotype. The PARP inhibitor olaparib, approved as a monotherapy

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for patients with a germline BRCA mutation, has shown promising results in preclinical studies

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when combined with DNA damaging agents such as carboplatin. However, dose-limiting

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toxicities have hindered the use of combination therapy with olaparib in the clinical setting. By

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concurrent administration of carboplatin and olaparib at various molar ratios of drugs, the aim of

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this study was to explore the optimal dosing ratio of carboplatin-olaparib combinations in a

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comprehensive panel of eight BRCA proficient and deficient high-grade serous ovarian cancer

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(HGSOC) cell lines. Overall, synergy was observed in BRCA1/2 mutated or defective cell lines

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when olaparib was combined at lower molar ratios of olaparib to carboplatin. Immunostaining of

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γH2AX foci revealed increased DNA damage as a result of this synergistic drug combination in

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the UWB1.289 paired cell lines. In vitro activity of the individual agents, carboplatin and

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olaparib did not correlate with PARP1 expression in each cell line. Importantly, synergism was

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also observed in a subset of BRCA wild type cell lines (OV90 and PEO4) suggesting therapeutic

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benefits of this combination beyond BRCA dependent synthetic lethality. Administration of

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drugs at synergistic ratios has the potential to increase efficacy and reduce toxicity.

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Introduction

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Each year, more than 200,000 women are diagnosed with ovarian cancer worldwide. Ovarian

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cancer is the leading cause of death amongst gynaecological cancers and the fourth highest cause

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of cancer associated deaths in women1. While early stage ovarian cancer is associated with a

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favorable prognosis, most patients are diagnosed at an advanced stage (e.g. stage III or IV) due

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to lack of effective screening methods2. Over 90% of ovarian cancers are epithelial in origin3, of

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which, 75% are high-grade serous ovarian cancer (HGSOC) which is an aggressive and highly

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metastatic subtype4,5. The current first line therapy for HGSOC consists of cytoreductive surgery

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followed by a carboplatin/paclitaxel combination, with each drug administered at its maximum

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tolerated dose (MTD) every 3 weeks for 6 cycles6. While most patients respond well initially to

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this regimen, 80% of patients relapse within 18 months with a resistant disease7.

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Given the high rates of recurrence, there is a significant unmet need for new and effective

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treatment approaches for patients with HGSOC. Inhibitors of the poly (ADP-Ribose) polymerase

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(PARP) enzyme have emerged as an effective treatment option for ovarian cancer. PARP

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inhibitors are a new class of small molecule drugs that have been developed to capitalize on

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deficiencies in the breast cancer susceptibility gene (BRCA)8, which are observed in over 50% of

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women with ovarian cancer. BRCA mutations are recognized as a hallmark of ovarian cancer

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given that germline or somatic mutations in BRCA1/2 genes occur in 10-20% of ovarian cancer

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patients9, and approximately 35% of ovarian cancers exhibit the “BRCAness” phenotype due to

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epigenetic silencing of BRCA proteins10,11. BRCA1/2 proteins are involved in the homologous

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recombination (HR) pathway of DNA double strand break repair12. Given their role, a reduction

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in BRCA1/2 function leads to deficiencies in HR and a reduced capacity to repair DNA double

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strand breaks. In the absence of functional HR repair, the DNA repair mechanism is driven

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towards single strand break repair and non-homologous end joining (NHEJ). As well, in BRCA2

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defective hamster cells, it has been shown that PARP1 is hyperactivated13. PARP1 is a critical

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enzyme in the base excision repair mechanism that corrects DNA single strand breaks14.

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Enzymatic inhibition of PARP1 traps the PARP1 protein at the DNA damage site, and the

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resulting protein-DNA complex leads to a stalled replication fork and the subsequent formation

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of DNA double strand breaks. While PARP inhibition alone generally inflicts limited

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cytotoxicity, PARP inhibition in cells with a defective HR repair mechanism results in an

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accumulation of DNA damage and increased cytotoxicity. Indeed, olaparib, a widely studied

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PARP inhibitor has shown promising anti-tumor activity in clinical trials with BRCA mutated

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patients15,16. In a Phase II efficacy study with olaparib monotherapy, notable anti-tumor activity

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was observed in ovarian cancer patients with confirmed germline BRCA1/2 mutations17. This

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lead to the FDA approval of olaparib (Lynparza®) for advanced ovarian cancer patients with

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deleterious or suspected germline BRCA mutations. In 2016, the PARP inhibitor rucaparib also

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received approval as monotherapy for advanced ovarian cancer patients with either germline or

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somatic BRCA mutations18. More recently, the clinical use of PARP inhibitors has been further

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expanded to ovarian cancer patients without BRCA mutations. In 2017, niraparib (Zejula™)

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received FDA approval as maintenance therapy for recurrent, platinum sensitive, epithelial

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ovarian, fallopian tube or primary peritoneal cancer regardless of BRCA status19. Furthermore,

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approval of olaparib was expanded in 2017 to include indication as maintenance therapy for

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complete or partial platinum sensitive ovarian cancer irrespective of the BRCA status20.

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One strategy to further increase the efficacy of PARP inhibitors, particularly in patients with

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resistant disease, is to target multiple pathways through combination with other cytotoxic

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agents21. The combination of a PARP inhibitor and a platinum-based drug, such as cisplatin or


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carboplatin in patients with BRCA defects has the potential to enhance therapeutic efficacy via

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platinum mediated DNA damage and the inhibition of DNA repair pathways resulting in

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synergistic cytotoxic effects. Indeed, several preclinical22,23 and clinical24–27 studies of platinum-

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PARP inhibitor combinations have resulted in favourable outcomes in BRCA deficient animal

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models and patients. Despite promising results from clinical trials, dose-limiting toxicities

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remain an issue in patients administered with a PARP inhibitor in combination with cytotoxic

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agents, resulting in drug discontinuation and/or sub-optimal dosing24,26,27. A possible approach to

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overcome this limitation is to utilize ratiometric dosing strategies. It has been shown in vitro that

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different ratios of pharmacological agents can impart either synergistic, additive, or antagonistic

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effects 28–30. Therefore, delivering drug combinations at their optimal synergistic ratio has the

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potential to reduce toxicity by the administration of lower drug doses while maintaining efficacy.

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One such example is Vyxeos™, a liposomal formulation of a cytarabine:daunorubicin

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combination that is administered at a synergistic 5:1 molar ratio for the treatment of acute

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myeloid leukemia31. FDA approval of this formulation was based on a Phase III trial

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demonstrating that Vyxeos treatment, which includes lower total drug doses, resulted in

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significantly improved outcomes when compared to the conventional drug regimen32.

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Building on Vyxeos’ success which supports the use of ratiometric dosing strategies, the present

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work utilized multiple in vitro models of BRCA proficient and deficient ovarian cancer to

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optimize the combination of the platinum compound carboplatin with the PARP inhibitor

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olaparib. A panel of ovarian cancer cell lines that varied in terms of histology, genetic profile and

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origin of derivation was selected to represent the high degree of heterogeneity associated with

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HGSOC (Table 1). The synergistic potential of the combination was assessed using the well-

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established combination index (CI) method21, and the extent of DNA damage was evaluated by



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immunostaining for γH2AX formation. We also examined whether in vitro activity of the

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individual agents, carboplatin and olaparib, was dependent on the level of PARP1 expression in

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each cell line.

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Table 1. Characteristics of the panel of ovarian cancer cell lines. Abbreviation: T2, cell doubling

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time. + categorized as BRCA proficient; - categorized as BRCA deficient. * Cell lines derived

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from the same patient. PEO1 was derived from treatment naïve patient and contains nonsense

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mutation Y1655X resulting in loss of BRCA2 expression. PEO4 was derived in the same patient

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after platinum therapy and acquired a reversion mutation which restored BRCA2 expression33.

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Histology

T2

BRCA status

Serous

16 h

BRCA1/2 wild type

UWB1.289+BRCA1 UWB1.289 OV90

Xenograft tumor Tumor Tumor Ascites

BRCA expression +

51 h 51 h 40 h

BRCA1 restored BRCA1 mutated BRCA1/2 wild type

+ +

OVCAR8

Tumor

Serous Serous High-grade serous Serous

26 h

-

COV362

Tumor

56 h

PEO1*

Ascites

84 h

BRCA2 mutated

-

PEO4*

Ascites

High-grade Serous High-grade serous High-grade serous

BRCA1 hypermethylated BRCA1 mutated

104 h

BRCA2 restored

+

HeyA8

-

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Materials and Methods

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Materials

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Olaparib was purchased from Tongchuang Pharma, Suzhou Co. Ltd (China). Thiazolyl blue

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tetrazolium bromide, sodium pyruvate, G-418 disulfate salt, and carboplatin were obtained from

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Sigma Aldrich (Oakville, ON). RPMI-1640, DMEM media, fetal bovine serum (FBS), penicillin-


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streptomycin solution, and phosphate buffered saline (PBS) (pH 7.4) were obtained from Life

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Technologies (Burlington, ON). Media199 solution and MCDB105 media powder was

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purchased from Sigma Aldrich (Oakville, ON, CA). Mammary Epithelial Cell Growth Medium

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(MEGM) Kit was obtained from Lonza (Mississauga, ON). Primary rabbit polyclonal anti-

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PARP1 antibody (9542) and RIPA buffer (9806) were purchased from Cell Signaling

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Technology (Danvers, MA). Primary rabbit polyclonal anti-histone H3 antibody (07-690) and

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primary rabbit anti-phospho histone H2A.X antibody (05-636) were obtained from EMD

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Millipore (Etobicoke, ON, CA). Primary mouse monoclonal anti-actin antibody was purchased

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from Sigma Aldrich (Oakville, ON).

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Cell Culture

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UWB1.289, UWB1.289+BRCA1, and OV90 cell lines were obtained from the American Type

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Culture Collection (ATCC; Manassas, VA). PEO1, PEO4, and COV362 from the European

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Collection of Cell Cultures (ECACC, Public Health England; Salisbury) were purchased through

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Sigma Aldrich. HeyA8 was obtained from M. D. Anderson Cancer Center (Houston, TX).

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OVCAR8 was obtained from the Biological Testing Branch of the National Cancer Institute

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(NCI; Frederick, MD). All cell lines were cultured in media as recommended by the suppliers

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under 5% CO2 at 37oC with 90% relative humidity. The UWB cell lines were cultured in a 1:1

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mixture of RPMI-1640 and MEGM™ medium supplemented with 5 components of MEGM

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mammary epithelial cell growth factors (BPE, hEGF, Insulin, Hydrocortisone, and GA-1000)

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and 3% FBS. The media for the UWB1.289+BRCA1 cell line was further supplemented with

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200 µg/mL of G-418 disulfate salt to maintain the expression of BRCA1 protein. PEO1 and

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PEO4 cells were grown in RPMI-1640 media with 10% FBS and supplemented with 2mM of

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sodium pyruvate. HeyA8 and OVCAR8 cells were cultured in RPMI-1640 media with 10% FBS.



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OV90 cells were maintained in a 1:1 mixture of MCDB 105 and Media 199, supplemented with

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15% FBS. COV362 cells were grown in DMEM media with 10% FBS. All media were

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supplemented with penicillin-streptomycin solution to a final concentration of 100 units/mL for

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penicillin and 100 µg/mL for streptomycin. All cell lines were authenticated by STR analysis (S1

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Table).

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Evaluation of Cytotoxicity

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Cytotoxicity of carboplatin and olaparib alone and in combination were evaluated using the MTT

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assay. Cells were seeded onto 96-well plates and allowed to form a monolayer overnight. The

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seeding density was determined separately for each cell lines to ensure the growth threshold was

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not exceeded in the control group (1000 cells/well for HeyA8 and OVCAR8, 3000 cells/well for

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UWB1.289 and UWB1.289+BRCA1, 4000 cells/well for PEO1 and PEO4, 5000 cells/well for

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OV90 and COV362). Cells were then exposed to nine serial dilutions of carboplatin or olaparib

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as single drug monotherapy or as combination therapy for 72 hours. Following treatment, 10 µL

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of 5 mg/mL MTT solution was added in each well and incubated for 3 hours at 37oC. The

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absorbance (A) at 570 nm was determined using a spectrophotometer (SPECTRAmax M2,

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Sunnyvale, CA). Cell viability was determined by the following formula:

=

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The concentration that resulted in 50% cell death (IC50 or fraction affected, Fa= 0.5) was

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determined by curve fitting using the Hill equation.

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Calculation of the Combination Index Values


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Synergism was evaluated using the CI equation, a standard method established by

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Chou&Talaley21. The prerequisite for the use of this method is the identification of the IC50

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values for the single drug treatments, in this case carboplatin and olaparib, and their combination

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as described previously21. The CI values were then calculated using the following formula:

CI =

+ +

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where D1 and D2, respectively, are the IC50 of carboplatin and olaparib in the combination, Dm1

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and Dm2 are the IC50 values for each of carboplatin and olaparib alone. The nature of the

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combination effect of the drug pair are indicated as CI1, antagonism; CI=1,

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additive. The extent of the drug-drug interactions is further described as CI< 0.7: strong

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synergism; 0.7-0.85: moderate synergism; 0.85-0.9: slight synergism; 1.0: additive, >1.0:

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antagonism.

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Drug Treatment

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For the evaluation of DNA damage and determination of chromatin-bound PARP1 level, cells

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were treated with drug combination at 10:1 and 1:2 carboplatin to olaparib ratios. The

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carboplatin concentrations for the combinations were determined by the respective IC50 of

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UWB1.289 (20µM) and UWB1.289+BRCA (21µM) cell lines. The olaparib concentration was

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then determined according to the treatment ratios (ie. carboplatin:olaparib 20 µM:2 µM or 20

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µM:40µM for UWB1.289 cells). The concentrations for olaparib monotherapy were 64 µM and

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32 µM for UWB1.289 and UWB1.289+BRCA, respectively.

199

200



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Western blotting

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The protein levels of PARP1 were analyzed in whole cell lysates and the degree of DNA-protein

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complex formation upon PARP1 inhibition was measured in the chromatin bound fraction using

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the immunoblotting method. Untreated cells were collected at sub-confluency and lysed in RIPA

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buffer (Cell Signaling, 9806). Following incubation at 4oC for an hour, the cell lysates were

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sonicated and centrifuged at 16,000 g for 10 minutes at 4oC and the supernatant collected. For

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preparation of the chromatin bound fractions, 20,000 cells were seeded in T75 flasks and allowed

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to form a monolayer overnight. Cells were then treated with the carboplatin-olaparib

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combination at 10:1 and 1:2 molar ratios for 72 hours. For fractionation, a Subcellular

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Fractionation Kit was used (Thermo Fisher, 78840) following the manufacturer’s instructions.

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Standard procedures were used for immunoblotting as previously described34. Actin and Histone

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H3 were used as loading controls for whole cell lysate and chromatin bound fraction,

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respectively. The expression of BRCA1 and BRCA2 was confirmed in the panel of ovarian

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cancer cell lines (Supplemental Figure S1) using mouse monoclonal anti-BRCA1 (ab16780)

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and rabbit polyclonal anti-BRCA2 (ab123491) antibodies using previously described methods35.

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Ku80 was used as the loading control for BRCA1/2 Western blotting analysis. Density of the

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protein band was quantified using Alpha Ease FC software.

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Evaluation of DNA damage of the UWB cells

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The extent of double strand DNA damage induced by the drug combination was determined by

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measuring the formation of γH2AX foci. Cells were seeded at 300,000 cells/well in 6-well plates

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with 20×20mm cover slips and allowed to attach overnight. Following 72-hour treatments with

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carboplatin and olaparib monotherapy at their respective IC50 concentrations or at carboplatin-

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olaparib combinations of 10:1 and 1:2, cells were fixed on the cover slip using 4%


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paraformaldehyde for 20 minutes. The cover slips were inverted and incubated with the anti-

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γH2AX antibody overnight. DAPI was used to label to nucleus. Cover slips were mounted on to

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slides and analyzed using Zen 2011 software and the foci quantified using Image J (NIH, 1.51h).

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Statistical analysis

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Data were expressed as mean ± standard deviation (SD) of three independent experiments (n=3).

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The difference between two means was analysed using a student t-test. For comparison between

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three or more groups, one-way analysis of variance (ANOVA) was performed followed by

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Dunnett’s post-test. For all experiments, p

BRCA Status does not Predict Synergism of a ... - ACS Publications

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