Wild-type Ovarian Cancer Cells Research Articles

Abstract 5941: Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer

Abstract PARP inhibitors (PARPi) targeting poly (ADP-ribose) polymerase are the first clinically approved drugs designed to apply synthetic lethality in BRCA1 mutant/deficient cancer. However, the treatment can cause reversion of BRCA mutation which results in drug resistance. To explore new strategies to improve antitumor efficacy of PARPi, we applied PARPi in combination with a small molecule NSC59984 for cancer therapy. NSC59984 is a small molecular targeting mutant p53 degradation and activating p73 via ROS-ERK2-MDM2 pathway. The treatments with radiation or hydrogen peroxide showed that NSC59984 enhanced DNA comet tails and gamma-H2AX, correlated to reduction of rad51 foci in cancer cells. These results suggest that NSC59984 impairs DNA damage repair via abrogation of homologous recombination (HR). We further applied the combinational treatment of NSC59984 and PARPi in BRCA1 wild-type ovarian cancer cells and found that NSC59984 synergized with PARPi to reduce cell viability, inhibit colony formation, and increase cell death. The combination treatment enhanced DNA damage and correlated with reduction of BRCA1 at the protein level. These results, taken together, suggest that the induction of BRCAness causes a synthetical lethality with PARPi in BRCA1 wild-type cancer cells. Cell cycle profiling showed that the cells were arrested at the G2/M phase in response to combinational treatment. At the G2 phase arrest, the high doses of the combinational drugs led to cell death via mitotic catastrophe, and the intermediate doses induced cellular senescence defined by senescence phenotypic hallmarks including senescence-associated beta-gal staining and a secretome consistent with senescence-associated secretory phenotype (SASP). Therapy-induced senescence (TIS) in cancer cells is considered as one of the mechanisms of tumor recurrence and drug resistance. To reduce the risk of TIS in treatment, we applied senolytic treatment to target senescent cells in the combinational treatment. The senolytic drug ABT263 treatment eliminated the senescent cancer cells from the combinational treatment. ABT263, NSC59984 + PARPi combinational treatment further reduced cell viability. Our study provides a rationale for small molecular compounds targeting HR deficiency in combination with PARPi to treat BRCA1wild-type ovarian cancer cells, and additional senolytic treatment may be required to limit resistance by removal of the TIS. Citation Format: Shengliang Zhang, Lanlan Zhou, Leiqing Zhang, Maximilian Pinho-Schwermann, Benedito Carneiro, John Sedivy, Wafik S. El-Deiry. Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5941.

Apatinib combined with olaparib induces ferroptosis via a p53-dependent manner in ovarian cancer.

PARP inhibitors combined with antiangiogenic drugs have been reported to improve outcomes in BRCA wild-type ovarian cancer patients, the mechanism of the combination is unclear. In this study, we explored the mechanism of apatinib combined with olaparib in the treatment of ovarian cancer. In this study, human ovarian cancer cell lines A2780 and OVCAR3 were used as experimental objects, and the expression of ferroptosis-related protein GPX4 after treatment with apatinib and olaparib was detected by Western blot. The SuperPred database was used to predict the target of the combined action of apatinib and olaparib, and the predicted results were verified by Western blot experiment to explore the mechanism of ferroptosis induced by apatinib and olaparib. Apatinib combined with olaparib-induced ferroptosis in p53 wild-type cells, and p53 mutant cells developed drug resistance. The p53 activator RITA sensitized drug-resistant cells to ferroptosis induced by apatinib combined with olaparib. Apatinib combined with olaparib-induced ferroptosis via a p53-dependent manner in ovarian cancer. Further studies showed that apatinib combined with olaparib-induced ferroptosis by inhibiting the expression of Nrf2 and autophagy, thereby inhibiting the expression of GPX4. The Nrf2 activator RTA408 and the autophagy activator rapamycin rescued the combination drug-induced ferroptosis. This discovery revealed the specific mechanism of ferroptosis induced by apatinib combined with olaparib in p53 wild-type ovarian cancer cells and provided a theoretical basis for the clinical combined use of apatinib and olaparib in p53 wild-type ovarian cancer patients.

Anticancer Effect of Active Component of Astragalus Membranaceus Combined with Olaparib on Ovarian Cancer Predicted by Network-Based Pharmacology.

In China, a traditional Chinese medicine formulation called astragalus membranaceus (AM) has been utilised for more than 20 years to treat tumors with extraordinary effectiveness. The fundamental mechanisms, nevertheless, are still not well understood. The aim of this study is identifying its possible therapeutic targets and to evaluate the effects of AM in combination with a PARP inhibitor (olaparib) in the treatment of BRCA wild-type ovarian cancer. Significant genes were collected from Therapeutic Target Database and Database of Gene-Disease Associations. The components of AM were analyzed using the Traditional Chinese Medicine System Pharmacology (TCMSP) database to screen the active ingredients of AM based on their oral bioavailability and drug similarity index. In order to find intersection targets, Venn diagrams and STRING website diagrams were employed. STRING was also used to create a protein-protein interaction network. In order to create the ingredient-target network, Cytoscape 3.8.0 was used. DAVID database was utilized to carry out enrichment and pathway analyses. The binding ability of the active compounds of AM to the core targets of AM-OC was verified with molecular docking using AutoDock software. Experimental validations, including cell scratch, cell transwell, cloning experiment, were conducted to verify the effects of AM on OC cells. A total of 14 active ingredients of AM and 28 AM-OC-related targets were screened by network pharmacology analysis. The ten most significant Gene Ontology (GO) biological function analyses, as well as the 20 foremost Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways were selected. Moreover, molecular docking results showed that bioactive compound (quercetin) demonstrated a good binding ability with tumor protein p53 (TP53), MYC, vascular endothelial growth factor A (VEGFA), phosphatase and tensin homolog (PTEN), AKT serine/threonine kinase 1 (AKT1) and cyclin D1 (CCND1) oncogenes. According to experimental methods, in vitro OC cell proliferation and migration appeared to be inhibited by quercetin, which also increased apoptosis. In addition, the combination with olaparib further enhanced the effect of quercetin on OC. Based on network pharmacology, molecular docking, and experimental validation, the combination of PARP inhibitor and quercetin enhanced the anti-proliferative activity in BRCA wild-type ovarian cancer cells, which supplies the theoretical groundwork for additional pharmacological investigation.

HDAC8-Selective Inhibition by PCI-34051 Enhances the Anticancer Effects of ACY-241 in Ovarian Cancer Cells.

HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.

Semi-Synthetic Analogues of Cryptolepine as a Potential Source of Sustainable Drugs for the Treatment of Malaria, Human African Trypanosomiasis, and Cancer.

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

Enhancing the sensitivity of ovarian cancer cells to olaparib via microRNA-20b-mediated cyclin D1 targeting.

We previously reported that cyclin D1 silencing interferes with RAD51 accumulation and increases the sensitivity of BRCA1 wild-type ovarian cancer cells to olaparib. However, the mechanisms associated with cyclin D1 overexpression in ovarian cancer are not fully understood. TargetScan predicted the potential binding sites for microRNA-20b (miR-20b) and the 3'-untranslated region of cyclin D1 mRNA; thus, we used luciferase reporter assay to verify those binding sites. The Kaplan-Meier method and log-rank test were used to examine the relationship between miR-20b and progression-free survival of ovarian cancer patients in The Cancer Genome Atlas (n = 367) dataset. In vitro experiments were performed to evaluate the effects of miR-20b on cyclin D1 expression, cell cycle and response to olaparib. A peritoneal cavity metastasis model of ovarian cancer was established to determine the effect of miR-20b on the sensitivity of olaparib. Immunohistochemistry was performed to evaluate molecular mechanisms. In this work, we demonstrated that miR-20b down-regulates cyclin D1, increases the sensitivity of ovarian cancer cells to olaparib, reduces the expression of RAD51, and induces cell cycle arrest in G0/G1 phase. Ovarian cancer patients with higher expression of miR-20b had significantly longer progression-free survival. These results indicate that miR-20b may be a potential clinical indicator for the sensitivity of ovarian cancer to olaparib and the survival of ovarian cancer patients. Our findings suggest that miR-20b may have therapeutic value in combination with olaparib treatment for ovarian cancer.

Enhanced Efficacy of Combined Therapy with Checkpoint Kinase 1 Inhibitor and Rucaparib via Regulation of Rad51 Expression in BRCA Wild-Type Epithelial Ovarian Cancer Cells.

PurposeThis study aimed to evaluate anticancer effects of combination treatment with poly(ADP-ribose) polymerase (PARP) and checkpoint kinase 1 (Chk1) inhibitors in BRCA wild-type ovarian cancer. PARP inhibitors can function as DNA-damaging agents in BRCA wild-type cancer, even if clinical activity is limited. Most epithelial ovarian cancers are characterized by a TP53 mutation causing dysfunction at the G1/S checkpoint, which makes tumor cells highly dependent on Chk1-mediated G/M phase cell-cycle arrest for DNA repair.Materials and MethodsWe investigated the anticancer effects of combination treatment with prexasertib (LY2606368), a selective ATP competitive small molecule inhibitor of Chk1 and Chk2, and rucaparib, a PARP inhibitor, in BRCA wild-type ovarian cancer cell lines (OVCAR3 and SKOV3).ResultsWe found that combined treatment significantly decreased cell viability in all cell lines and induced greater DNA damage and apoptosis than in the control and/or using monotherapies. Moreover, we found that prexasertib significantly inhibited homologous recombination–mediated DNA repair and thus showed a marked anticancer effect in combination treatment with rucaparib. The anticancer mechanism of prexasertib and rucaparib was considered to be caused by an impaired G2/M checkpoint due to prexasertib treatment, which forced mitotic catastrophe in the presence of rucaparib.ConclusionOur results suggest a novel effective therapeutic strategy for BRCA wild-type epithelial ovarian cancer using a combination of Chk1 and PARP inhibitors.

Abstract 1379: Entinostat induces PARPi sensitivity across multiple ovarian cancer models

Abstract Introduction: Ovarian cancer is a rare, but fatal disease and a leading cause of gynecologic cancer death, in the United States. Poly ADP ribose polymerase inhibitors (PARPi) are promising drugs, most effective in patients with tumors deficient in homologous recombination (HR) genes, mainly BRCA1/2. Our group has published that histone deacetylase inhibitors (HDACi) sensitized HR proficient BRCA wild-type ovarian cancer cells to PARPi, however the mechanism still remains elusive and the efficacy needed to be tested across multiple models. We recently tested Entinostat with and without Olaparib in three different ovarian cancer models, including human/mouse established cell lines, PDX derived primary cell lines and immune-competent/immune-compromised xenograft as well as PDX-derived preclinical mouse models. Objective: To delineate the efficacy of Entinostat to sensitize ovarian tumors to Olaparib across multiple models and determine the underlying mechanism. Methods: HR proficient BRCA wild-type ID8/SKOV3 and PDX derived primary cell lines were treated with Entinostat and Olaparib, alone or in combination and analyzed for cell proliferation. DNA damage was studied using Comet assay. Expression of cell proliferation, DNA damage and repair protein were studied using Western blot analysis and immunofluorescence. Further, ID8-treated immune-competent mouse models, SKOV3-treated immune-compromised mouse model and HGSOC patient derived PDX mouse model generated in our lab were treated with vehicle, Entinostat, Olaparib, or the combination. The mice were monitored for toxicity and body weight measured twice weekly, till tissue harvest or survival. Results: Cell proliferation was significantly decreased (p&lt0.05) in Entinostat-Olaparib combination treated groups in all cell lines tested. Comet assay showed significantly longer comet tail length in combination treated cells compared to control (p&lt0.05). Western blot and Immunofluorescence showed that combination treatment significantly decreased BRCA1, PCNA, RAD51 and increased cleaved PARP, ΓH2AX (p&lt0.05). Further, SKOV3- xenograft mouse model showed decreased tumor burden ((p&lt0.07), ID8-xenograft mouse tumors showed decreased Ki67 (p&lt0.05) and HGSOC derived PDX model showed longer survival when treated with Entinostat-Olaparib combination therapy. Conclusion: Entinostat in combination with Olaparib decreased cell proliferation, increased DNA damage, induced HR deficiency in vitro, reduced tumor burden, decreased Ki67 in xenograft mouse model, and improved survival of HGSOC derived PDX mice, thereby potentially sensitizing homologous recombination proficient ovarian cancer to PARPi across multiple models. Citation Format: Vijayalaxmi G. Gupta, Shariska Petersen, Jeff Hirst, Katherine Roby, Harsh Pathak, Meghan Kusch, Andrew Wilson, Andrew Godwin, Dineo Khabele. Entinostat induces PARPi sensitivity across multiple ovarian cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1379.

Abstract 3501: Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer

Abstract Introduction: Ovarian cancer is a rare but often fatal disease and a leading cause of gynecologic cancer death, in the United States. Poly ADP ribose polymerase inhibitors (PARPi) are promising new drugs that are most effective in patients with tumors with altered homologous recombination (HR) DNA repair genes, particularly BRCA1/2. To expand their clinical utility, PARPi are being tested in combination with other drugs. Our group has published that histone deacetylase inhibitors (HDACi) sensitize HR proficient BRCA wild-type ovarian cancer cells to PARPi. We have developed an investigator-initiated phase 1/2 clinical trial of the PARPi olaparib combined with the HDACi entinostat in patients with recurrent, HR proficient/BRCA wild-type ovarian cancer. Objective: To test the effects of a novel combination of olaparib and entinostat in preclinical models of BRCA wild-type ovarian cancer. Methods: HR proficient BRCA wild-type SKOV3 and ID8 ovarian cells were treated with Entinostat followed by Olaparib alone or the combination of Entinostat and Olaparib. Treated and control cells were analyzed for cell proliferation by sulforhodamine B assays, clonogenicity, and DNA damage by Comet assays. SKOV-3-luciferase cells were injected intraperitoneally into NOD-SCID mice. Mice were randomized into 4 treatment groups and treated with vehicle, entinostat, olaparib, or the combination. The mice were monitored for toxicity and by bioluminescence imaging (BLI). At sacrifice, tumor burden was quantified. Results: In cell culture assays, entinostat significantly enhanced the sensitivity of SKOV3 and ID8 cells to Olaparib. Entinostat combined with olaparib led to synergistic reductions in cell proliferation and clonogenicity in SKOV-3 and ID8 ovarian cancer cells, compared to each drug alone. Evidence of DNA damage was enhanced by the drug combination. In the SKOV3-luc xenograft mouse model, a significant reduction in tumor burden in the group of mice treated with the combination of entinostat and olaparib, compared to each drug alone. There was no significant toxicity with drug treatment. Conclusion: Entinostat sensitizes ovarian cancer cells to Olaparib in vitro and in vivo. Experiments in ID-luciferase and patient-derived xenograft models are ongoing. These preclinical models are powerful tools that will be used to discover markers and mechanisms of sensitivity and resistance to inform the parallel clinical trial. Citation Format: Vijayalaxmi G. Gupta, Yoskaly Lazo Fernandez, Katherine Roby, Harsh Pathak, Jeff Hirst, Andrew J. Wilson, Andrew Godwin, Dineo Khabele. Entinostat enhances the efficacy of olaparib in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3501.

Cyclin D1 silencing impairs DNA double strand break repair, sensitizes BRCA1 wildtype ovarian cancer cells to olaparib

ObjectivePoly(ADP-ribose) polymerase inhibitors (PARPi) are active in cancer cells that have impaired repair of DNA by the homologous recombination (HR) pathway. Strategies that disrupt HR may sensitize HR-proficient tumors to PARP inhibition. As a component of the core cell cycle machinery, cyclin D1 has unexpected function in DNA repair, suggesting that targeting cyclin D1 may represent a plausible strategy for expanding the utility of PARPi in ovarian cancer. MethodsBRCA1 wildtype ovarian cancer cells (A2780 and SKOV3) were treated with a combination of CCND1 siRNA and olaparib in vitro. Cell viability was assessed by MTT. The effects of the combined treatment on DNA damage repair and cell cycle progression were examined to dissect molecular mechanisms. In vivo studies were performed in an orthotopic ovarian cancer mouse model. Animals were treated with a combination of lentivirus-mediated CCND1 shRNA and olaparib or olaparib plus scrambled shRNA. Molecular downstream effects were examined by immunohistochemistry. ResultsSilencing of cyclin D1 sensitized ovarian cancer cells to olaparib through interfering with RAD51 accumulation and inducing cell cycle G0/G1 arrest. Treatment of lentivirus-mediated CCND1-shRNA in nude mice statistically significantly augmented the olaparib response (mean tumor weight ± SD, CCND1-shRNA plus olaparib vs scrambled shRNA plus olaparib: 0.172 ± 0.070 g vs 0.324 ± 0.044 g, P< 0.05). ConclusionsSilencing of cyclin D1 combined with olaparib may lead to substantial benefit for ovarian cancer management by mimicking a BRCAness phenotype, and induction of G0/G1 cell cycle arrest.

Abstract 4817: EP-100 sensitizes BRCA wild-type ovarian cancer cells to PARP inhibitor

Abstract 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. 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. We first examined the cytotoxic effects of EP-100 alone on 8 ovarian cancer cell lines with variable expression level of LHRH receptors and the IC50 values ranged from 0.5 to 2.0 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). The combination indices (CI) were &amp;lt; 0.9 at the ED50 level. Furthermore, the nuclear foci of γH2AX were significantly increased in combination group of EP100 and olaparib after 24 h incubation compared with control, EP-100 alone, or olaparib alone groups (p &amp;lt; 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 &amp;lt; 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 &amp;lt; 0.008), EP-100 alone (0.62 ± 0.28 g, p &amp;lt; 0.008), and olaparib alone (0.50 ± 0.22 g, p &amp;lt; 0.01). EP-100 has efficacy in preclinical models of ovarian cancer, especially in combination with olaparib. Our findings suggested that combining EP-100 with olaparib could be a promising therapeutic strategy for ovarian cancer. Citation Format: Shaolin Ma, Sunila Pradeep, Sherry Wu, Mark Seungwook Kim, Wei Hu, Selanere Mangala, Robert L. Coleman, Anil K. Sood. EP-100 sensitizes BRCA wild-type ovarian cancer cells to PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4817.

Abstract 5826: Enhanced anti-tumor effects of selinexor and niraparib in preclinical models of ovarian cancer

Abstract Introduction: Selinexor (KPT-330) is a first-in-class oral exportin-1 (XPO1/ CRM1) inhibitor that induces cell cycle arrest and apoptosis in cancer cells through reactivation of tumor suppressor proteins and inhibition of DNA damage repair genes. Here, we studied selinexor in combination with niraparib, an inhibitor of the DNA damage repair proteins PARP1 and 2, in preclinical models of ovarian cancer. Given that both compounds can inhibit DNA damage repair responses, we hypothesized the combination of selinexor and niraparib would enhance cancer cell death by accumulation of DNA damage that cannot be resolved in ovarian cancer. Methods: Selinexor and niraparib alone and in combination were tested in vitro on the BRCA wildtype ovarian cancer cell line A2780. Total RNA and protein were extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous A2780 xenograft mouse model was treated with selinexor [10 mg/kg, once per week (M) for three weeks] or niraparib [37.5 mg/kg, once-daily for five days per week (M-F) for three weeks] as single agents or in combination. Tumor growth and body weights were measured for 21 days. Tumors were harvested at the end of the study and analyzed by immunohistochemistry (IHC). Results: Selinexor and niraparib as single agents inhibited A2780 cell proliferation (selinexor IC50: 200nM; niraparib IC50: 800nM). The combination of selinexor and niraparib showed synergistic cytotoxicity in A2780 cells. Increased expression of phospho (S139) H2A.X with the combination confirmed induction of DNA damage. In vivo, the combination enhanced tumor inhibition (51.0% in combination versus 24.3% and 29.6% in selinexor and niraparib respectively) and improved median survival compared to vehicle or each agent alone (18 days in combination group versus 11, 14 and 16 days in vehicle, selinexor, and niraparib groups, respectively). IHC analysis showed enhanced nuclear p53 and p21 staining in selinexor treated tumors as well as in tumors treated with the combination. Increased apoptosis was observed in tumor samples treated with both selinexor and niraparib as compared to vehicle control or each agent alone. Conclusions: Selinexor plus niraparib demonstrated enhanced anti-tumor activity in preclinical models of human ovarian cancer. Since both drugs could inhibit DNA damage repair pathway, the drug combination efficiently accumulated DNA damage, which was associated with reduced cell proliferation, and induced apoptosis. This combination therapy warrants further investigation as a treatment option for patients with ovarian cancer. Citation Format: Hua Chang, Trinayan Kashyap, Sophie Debler, Thaddeus J. Unger, Sarah Wang, Keith Mikule, Jing Wang, Mansoor R. Mirza, Sharon Shacham, Yosef Landesman. Enhanced anti-tumor effects of selinexor and niraparib in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5826.

Ponatinib Shows Potent Antitumor Activity in Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) through Multikinase Inhibition.

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932-43. ©2018 AACR.

Mesoporous γ-Iron Oxide Nanoparticles for Magnetically Triggered Release of Doxorubicin and Hyperthermia Treatment.

Mesoporous iron-oxide nanoparticles (mNPs) were prepared by using a modified nanocasting approach with mesoporous carbon as a hard template. mNPs were first loaded with doxorubicin (Dox), an anticancer drug, and then coated with the thermosensitive polymer Pluronic F108 to prevent the leakage of Dox molecules from the pores that would otherwise occur under physiological conditions. The Dox-loaded, Pluronic F108-coated system (Dox@F108-mNPs) was stable at room temperature and physiological pH and released its Dox cargo slowly under acidic conditions or in a sudden burst with magnetic heating. No significant toxicity was observed in vitro when Dox@F108-mNPs were incubated with noncancerous cells, a result consistent with the minimal internalization of the particles that occurs with normal cells. On the other hand, the drug-loaded particles significantly reduced the viability of cervical cancer cells (HeLa, IC50 =0.70 μm), wild-type ovarian cancer cells (A2780, IC50 =0.50 μm) and Dox-resistant ovarian cancer cells (A2780/AD, IC50 =0.53 μm). In addition, the treatment of HeLa cells with both Dox@F108-mNPs and subsequent alternating magnetic-field-induced hyperthermia was significantly more effective at reducing cell viability than either Dox or Dox@F108-mNP treatment alone. Thus, Dox@F108-mNPs constitute a novel soft/hard hybrid nanocarrier system that is highly stable under physiological conditions, temperature-responsive, and has chemo- and thermotherapeutic modes of action.

Increased γ-H2AX and Rad51 DNA Repair Biomarker Expression in Human Cell Lines Resistant to the Chemotherapeutic Agents Nitrogen Mustard and Cisplatin

Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms. However, alkylating agents which induce DNA interstrand crosslinks (ICL) are amongst the most effective anticancer agents and often form the mainstay of many anticancer therapies. The effectiveness of these drugs can be limited by the development of drug resistance in cancer cells and many studies have demonstrated that alterations in DNA repair kinetics are responsible for drug resistance. In this study we developed two cell lines resistant to the alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow cytometry to quantitate the number of γ-H2AX and Rad51 foci in the nuclei of cells after drug exposure. γ-H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases and Rad51 was used to measure the activity of homologous recombination in the repair of ICL. In the drug-resistant derivative cell lines there was overall a significant increase in the number and persistence of both γ-H2AX and Rad51 foci in the nuclei of cells over a 72-hour period, when compared to the non-resistant parental cell lines (ANOVA p < 0.0001). In a Pt-resistant ovarian cancer cell line (A2780cis^R) a similar enhancement of DNA repair was observed when compared to the non-drug-resistant wild-type ovarian cancer cells (A2780) following exposure to HN2. Our data suggest that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and human tumours may be of experimental and clinical benefit. We concede, however, that examination of a larger population of cell lines and tumours is required to fully evaluate the validity of this approach.

Abstract 5433: Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells.

Abstract Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensitivity to paclitaxel. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is the cardiac isoform of the most important glycolytic regulator, phosphofructokinase-2, From the TCGA database of &amp;gt;300 ovarian cancers, the PFKFB2 gene ranked in the top 33% of overexpressed genes and in the top 19% of genes with copy number gain. We found that PFKFB2 mRNA was upregulated in 10 of 12 ovarian cancer cell lines when expression of the gene was compared to that in normal ovarian epithelial cells in culture. Knockdown of PFKFB2 with siRNA markedly inhibited cell proliferation and increased paclitaxel sensitivity in p53 wild type ovarian cancer cell lines as demonstrated in short-term cytotoxicity and long-term clonogenic assays. Liposome encapsulated PFKFB2 siRNA significantly inhibited growth (P&amp;lt;0.05) of human ovarian cancer xenografts in two ovarian cancer models (Hey A8 and A2780); even greater growth inhibition was observed when PFKFB2 siRNA treatment was combined with paclitaxel. Cell growth inhibition in cell culture was linked to G1 arrest and to a modest increase in apoptosis. Induction of p21Cip1 and p27Kip1 protein by PFKFB2 knockdown contributed to the G1 arrest, since silencing p21Cip1 and p27Kip1 dramatically reduced PFKFB2 knockdown-induced G1 arrest. PFKFB2 knockdown also modulated the expression of CDK2 and the phosphorylation of p27Kip1. Induction of p53 protein contributed to both PFKFB2 knockdown-induced G1 arrest and to apoptosis, since silencing p53 markedly alleviated PFKFB2 knockdown-induced G1 arrest and apoptosis. PFKFB2 knockdown inhibited expression of the ABC transporter family members ABCC4 and ABCG2 at the level of transcription, possibly contributing to enhanced paclitaxel retention and increased cytotoxicity after PFKFB2 knockdown. Taken together, these results suggest that PFKFB2 protein may regulate cell growth and sensitivity to paclitaxel by multiple mechanisms, particularly in p53 wild type ovarian cancers. As methods to deliver siRNA evolve in the clinic, PFKFB2 may provide a novel target for enhancing sensitivity to paclitaxel-based chemotherapy in patients with low grade serous ovarian cancer where p53 function remains intact. Citation Format: Shu Zhang, Weiqun Mao, Anil K. Sood, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Zong-Fang Li, Robert C. Bast, Xiao-Feng Le. Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells. [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 5433. doi:10.1158/1538-7445.AM2013-5433

Abstract 3158: Transcription factor SOX11 decreases viability in ovarian carcinoma cells.

Abstract Objective: The Cancer Genome Atlas (TCGA) evaluated nearly 500 advanced serous ovarian carcinomas and identified four gene expression subclasses. The transcription factor SOX11 was markedly overexpressed in the proliferative subtype and associated with phosphorylation of other cell cycle related proteins including CHEK2, WWTR1, and EIF4EBP1. We tested the hypothesis that over-expression of SOX11 will decrease proliferation of ovarian cancer cells through transcriptional repression. Methods: SOX11 was overexpressed via lipofectamine transfection in the TP53 wild-type ovarian cancer cell line SKOV3 and the TP53 mutated ovarian cancer cell line OVCAR3. Cell proliferation was assessed 48 hours after transfection using the VisionBlue fluorescence-based viability assay. Cell cycle analysis was performed utilizing a propidium iodide (PI) assay followed by fluorescence-activated cell sorting (FACS). Finally migration of SOX11-transfected cells was analyzed in comparison to mock-transfected controls via monolayer scratch assay. Results: Successful overexpression of the SOX11 protein was confirmed using GFP fluoroscopy and western blot 24 and 48 hours following transfection. Cell viability was reduced by 14-63% at 48 hours in SOX11-transfected SKOV3 cells compared to mock controls (P = 0.01) in a dose dependent fashion. Cell cycle was unaffected by SOX11 overexpression. Finally, the monolayer scratch assay demonstrated that cells overexpressing SOX11 migrated more slowly than mock controls (median velocity 3.92 vs. 6.19 um/hr, P = 0.001). Data from OVCAR3 cells are currently being analyzed and will be presented. Conclusions: Overexpression of SOX11 resulted in decreased cell viability without affecting cell cycle. Further studies will assess the mechanism of this decreased viability which may involve diversion toward an apoptotic pathway when SOX11 is overexpressed or regulation by transcription repressors such as WWTR1. SOX11 suppression has been shown in lymphoma to be associated with more aggressive tumors through mechanisms associated with lysophosphatidic acid growth regulation, offering mechanistic associations with ovarian cancer. Ongoing work will determine the relationship between SOX11 and TP53 mutation frequently found in high-grade serous carcinoma. These studies will help to explain the differential outcomes seen across high-grade serous ovarian carcinoma. Citation Format: Caryn M. St. Clair, Stephanie L. Wethington, Maria Bisogna, Fanny Dao, Petar Jelinic, Douglas A. Levine. Transcription factor SOX11 decreases viability in ovarian carcinoma cells. [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 3158. doi:10.1158/1538-7445.AM2013-3158

Abstract 3277: Pifithrin-α and veliparib synergize in the sensitization of p53 wild-type ovarian cells to cisplatin therapy.

Abstract Objectives. Veliparib (ABT), a PARP1/2 inhibitor, has been studied in ovarian adenocarcinoma treatment, particularly in conjunction with platinum therapy, and sensitizes cells to cisplatin (Cis). Pifithrin-α (PFT), a transcriptional inhibitor of p53, sensitizes p53 wild-type ovarian cancer cells to antimicrotubule agent-induced apoptosis. No studies have evaluated the interaction of PFT with platinum drugs or PARP inhibitors. We evaluated the potential synergistic interactions between PFT and ABT/Cis in p53 intact ovarian cancer cells. Methods. We conducted clonogenic assays using BG-1, a human ovarian adenocarcinoma cell line with WT p53 activity. Cells were seeded in 6-well plates and, after 24 hours, treated with combinations of 5μM PFT and multiple doses of ABT and Cis. After 8 days, colony counts were obtained with automated GelDoc software. To identify synergistic interactions, Combination Indices (CI) were calculated with Calcusyn software. Results. At the lowest dose of Cis (0.625μM), relative cell survival is 60.4% with Cis-PFT, 64.9% with Cis-ABT, and 24.5% with Cis-ABT-PFT. At the highest dose (10μM), relative cell survival is 34.3% with Cis-PFT, 5.25% with Cis-ABT, and 0.85% with Cis-ABT-PFT. At low concentrations of Cis, Cis-PFT showed mild to moderate antagonism. At Cis concentrations &amp;gt;2.5μM, moderate synergism developed. At all concentrations, PFT-ABT showed moderate to strong antagonism. Cis-ABT-PFT showed moderate to strong synergism at all concentrations. Conclusions. PFT sensitizes BG-1 cells to Cis in a dose dependent pattern, improving synergism at increasing doses of both Cis and PFT. In contrast, PFT shows marked antagonism to the cytotoxic effects of ABT, independent of ABT dose. The combination of ABT+PFT shows strong, dose-independent synergism in the sensitization of BG-1 cells to Cis and significantly reduces relative survival, suggesting the potential for improved tumor control at reduced doses of cisplatin in p53 WT ovarian cancer. Pifithrin-α and veliparib synergize in the sensitization of BG-1 cells to cisplatin therapy Cis (μM) ABT (μM) PFT (μM) CI Synergy Cisplatin + Pifithrin-α 0.625 − 5 1.729 −−− 1.25 − 5 1.342 −− 2.5 − 5 0.865 + 5 − 5 0.469 +++ 10 − 5 0.390 +++ Veliparib + Pifithrin-α − 0.625 5 1.803 −−− − 1.25 5 3.325 −−−− − 2.5 5 1.647 −−− − 5 5 2.657 −−− − 10 5 1.503 −−− Cisplatin + Veliparib 0.625 1.25 − 0.566 +++ 1.25 2.5 − 0.794 ++ 2.5 5 − 0.863 + 5 10 − 0.656 +++ 10 20 − 0.823 ++ Cisplatin + Veliparib + Pifithrin-α 0.625 1.25 5 0.293 ++++ 1.25 2.5 5 0.418 +++ 2.5 5 5 0.392 +++ 5 10 5 0.291 ++++ 10 20 5 0.262 ++++ Synergism: CI&amp;lt;1, “+” Antagonism: CI&amp;gt;1, “−” Citation Format: Margaret Whicker, Brian Liang, Z. Ping Lin, Alan Sartorelli, Elena Ratner. Pifithrin-α and veliparib synergize in the sensitization of p53 wild-type ovarian cells to cisplatin therapy. [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 3277. doi:10.1158/1538-7445.AM2013-3277

Abstract 3057: CDK5 modulates the cell cycle, apoptosis and paclitaxel sensitivity in human ovarian cancer cells with wild-type p53 function

Abstract Cyclin-dependent kinase 5 (CDK5) is a ubiquitously expressed serine/threonine kinase that was initially identified in bovine brain extracts and plays an important role in normal neuron development and neurodegenerative diseases. This molecule has recently been shown to possess many cellular functions in non-neuronal tissues as well. Using a kinome siRNA screen, we identified CDK5 as a kinase that modulates paclitaxel sensitivity in human ovarian cancer cells. The aim of this report is to identify mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancer cells. Despite nucleotide sequence homology with human CDK1, a role of CDK5 in regulating the cell cycle remains unclear. Here we demonstrate dual roles of CDK5 in regulating the cell cycle and apoptosis. We have found that knockdown of CDK5 with multiple siRNAs markedly inhibits cell proliferation and increases paclitaxel sensitivity in several p53 wild type ovarian cancer cell lines using assays for cell viability and clonogenic growth. CDK5 knockdown with or without paclitaxel treatment induces G1 arrest of the cell cycle and apoptotic cell death linked to a significant induction of p53, p21Cip1 and p27Kip1 proteins. Induction of G1 arrest following CDK5 knockdown depends on induction of p21Cip1 and p27Kip1, since silencing p21Cip1 and/or p27Kip1 dramatically reduces CDK5 knockdown-induced G1 arrest. A CDK5 knockdown-induced increase of p53 expression contributes both to CDK5 knockdown-induced G1 arrest and to apoptosis, as silencing p53 markedly diminishes CDK5 knockdown-induced G1 arrest and apoptosis. As expected, a CDK5 knockdown-induced increase in p21Cip1 expression depends on p53-mediated transcriptional activation demonstrated by QRT-PCR and reporter luciferase assays. CDK5 knockdown activates caspase-3 and a pan-caspase inhibitor completely blocks CDK5 knockdown-induced apoptosis. Significant Inhibition of Bcl-2 expression is found in CDK5 knockdown-induced apoptosis and additional inhibition of Bcl-2 expression is found with combined CDK5 and paclitaxel treatment. Taken together, these data suggest that CDK5 can modulate paclitaxel sensitivity through regulating both the cell cycle and apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells with wild-type p53 function, providing a novel approach for enhancing the efficacies of paclitaxel-based regimens for low grade ovarian cancer in the clinic. 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 3057. doi:1538-7445.AM2012-3057

Abstract 4901: Impact of the TGF-β1 isoform on p53 signalling in ovarian cancer cell lines in response to cisplatin

Abstract Transforming growth factor β is a pleiotropic cytokine which inhibits cell growth, induces cellular differentiation, and can lead to cellular apoptosis under certain conditions. TGF-β is also involved in epithelial-mesenchymal transition (EMT), a phenomenon by which cells acquire a dedifferentiated phenotype and gain motility and invasive properties. In the present study, we investigated the impact of TGF-β1 on p53 mediated cellular signalling in ovarian cancer cell lines in response to cisplatin. Four cell lines with different p53 status: A2780 (wild-type p53), A2780CP (derived from A2780, resistant to cisplatin, one p53 allele mutated V172F and one p53 allele wild-type), OVCAR-3 (p53 mutated R248Q) and SKOV-3 (p53 negative) were used in the present study. Cells were co-treated with TGF-β1 (10ng/mL) and cisplatin (10μM) for 24 hours and protein levels were analysed by western blotting. Our results show that TGF-β1 inhibits cisplatin-induced apoptosis in SKOV-3 cells as demonstrated by reduced PARP cleavage and caspase-3 activation. However, the inhibition of cisplatin-induced apoptosis was not observed in cells with wild type or mutated p53. Co-treatment of TGF-β1 and cisplatin decreased the levels of phosphorylated/active Akt compared to cisplatin alone, suggesting that TGF-β1 promotes cisplatin-induced apoptosis in A2780 cells in part through p53. In addition, Mdm2 was cleaved in response to cisplatin treatment only in p53 wild-type expressing A2780 cells. The levels of the caspase inhibitor XIAP were also decreased in A2780 cells when treated with cisplatin. Together, these results suggest that TGF-β1 promotes apoptosis when used in combination with cisplatin in p53 wild-type ovarian cancer cells. 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 4901. doi:1538-7445.AM2012-4901