Ovarian Cancer Cell Line Models Research Articles

Sialyl-Tn serves as a potential therapeutic target for ovarian cancer

BackgroundOvarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer.MethodsWe aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples.ResultsOur in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis.ConclusionsOur preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective.

Abstract A038: Epigenetic compound library screen of ovarian clear cell carcinoma cell line models identifies decreased cell viability following treatment with the Bruton tyrosine kinase inhibitor ibrutinib

Abstract Ovarian clear cell carcinoma (OCCC) is a difficult to treat histological subtype of ovarian cancer, demonstrating resistance to standard chemotherapy. New approaches are required in order to identify compounds with the potential to target this tumor. OCCC are characterized by mutations in ARID1A and/or ARID1B (AT-rich interactive domain-containing proteins) that encode for members of the mammalian SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex. We have used an epigenetic library consisting of 160 compounds that targets epigenetic readers, writers, erasers, and transcriptional modulators, to screen seven OCCC cell line models (RMG1, JHOC5, OVTOKO, OVMANA, OVISE, OV207 and TOV21G) and three non-OCCC ovarian cancer cell line models (A2780, COV434 and OVCAR8). Cell lines were seeded in triplicate into 96-well plates and treated with all 160 compounds at both 5 µM and 0.5 µM, as well as with a DMSO vehicle control, for 72 hours. Following treatment, cell viability was determined using a MTS assay and each data point normalized to the DMSO control. Based on the results of viability assays, drugs were sought that preferentially targeted OCCC relative to non-OCCC cell lines. Drugs that increased cell proliferation in two or more of the OCCC lines were removed from the analysis, as were drugs that did not impact upon viability. Drug 152, ibrutinib, inhibited cell viability between 53 to 78% in 6 of the 7 OCCC cell lines at 5 µM, with an average inhibition of 64%. In non-OCCC cell lines, the average cell viability after treatment with 5 µM ibrutinib was 91% (range, 84 – 103%). Ibrutinib is a small molecule inhibitor of Bruton tyrosine kinase (BTK) that has been approved by the FDA for treatment of a number of hematological malignancies including mantle cell lymphoma, small lymphocytic lymphoma and chronic lymphocytic leukemia. BTK functions in B-cell receptor signaling, regulating B cell proliferation and survival. Ibrutinib works by irreversibly blocking the kinase activity of BTK. Interestingly, ibrutinib binding sites are also present in other tyrosine kinases, including EGFR, raising the possibility of off-target effects that can function to decrease tumor cell viability. In addition to hematological malignancies, ibrutinib is being investigated in clinical trials for the potential to impact on solid tumors including non-small cell lung cancer, certain oesophagogastric carcinoma, advanced carcinoid and pancreatic neuroendocrine tumors, squamous cell carcinoma of the head and neck, renal cell carcinoma and glioblastoma, including in some cases in combination with other therapeutic drugs including cisplatin, temozolomide and nivolumab. Following organoid drug testing, off-label use of ibrutinib in a patient with low-grade serous ovarian cancer has recently shown promising results. Research is on-going to determine the effectiveness of ibrutinib in pre-clinical models of OCCC, including analyses of the effectiveness of ibrutinib in ARID1A and ARID1B wild-type and mutant CRISPR-engineered isogenic cell line panels of OCCC. Citation Format: Yue Ma, Kristie Ann Dickson, Natisha Field, Tao Xie, Nham Tran, Deborah Joy Marsh. Epigenetic compound library screen of ovarian clear cell carcinoma cell line models identifies decreased cell viability following treatment with the Bruton tyrosine kinase inhibitor ibrutinib [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A038.

Abstract B095: Sacituzumab govitecan-based drug combinations overcome platinum/PARP inhibitor resistance in ovarian cancer models

Abstract Background: Ovarian cancer causes more deaths than any other gynaecological cancer per year in the USA. Sacituzumab govitecan (SG) is a TROP2-targeted antibody-drug conjugate, approved for the treatment of triple-negative and hormone receptor-positive breast cancers. TROP2 is commonly overexpressed in ovarian cancer and represents an attractive, potential therapeutic target in ovarian cancer. This study investigated the in vitro activity of SG alone and in combination with targeted therapies/chemotherapies in ovarian cancer cell line models. Methods: This study utilized the PEO1 and PEO4 cell lines, which were derived from the same patient, with PEO4 acquiring a secondary restorative BRCA2 mutation to confer platinum resistance. Olaparib-resistant PEO1 (PEO1-Ola) cells were generated through continuous exposure to olaparib, to a maximum 13 μM, for four months. The anti-proliferative effects of SG, talazoparib, olaparib, carboplatin, and paclitaxel were tested in PEO1, PEO1-Ola, and PEO4 cell lines. Synergism was assessed using Combenefit software with the Loewe additivity model. Induction of caspase 3/7 activation, a marker of apoptosis, was tested with single agents and combinations using kinetic, fluorescent microscopy with the Incucyte S3 imaging system. Results: Olaparib and carboplatin resistance did not result in their cross-resistance. PEO1-Ola showed similar sensitivity to carboplatin as PEO1 parental cells, while carboplatin-resistant PEO4 cells displayed clinically achievable sensitivity to olaparib and talazoparib. SG was highly effective in all models tested. SG in combination with carboplatin or talazoparib was synergistic across all cell lines and resulted in induction of apoptosis. Conclusions: This pre-clinical study showed that TROP2-targeting ADC-based drug combinations warrant further investigation for the treatment of ovarian cancer. Citation Format: Neil T Conlon, Elena Möhrle, Luna Stockmann, John Crown. Sacituzumab govitecan-based drug combinations overcome platinum/PARP inhibitor resistance in ovarian cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B095.

A Regulatory Loop Involving miR-200c and NF-κB Modulates Mortalin Expression and Increases Cisplatin Sensitivity in an Ovarian Cancer Cell Line Model

Ovarian cancer is currently the most lethal gynecological cancer. At present, primary debulking surgery combined with platinum-based chemotherapy is the standard treatment strategy for ovarian cancer. Although cisplatin-based chemotherapy has greatly improved the prognosis of patients, the subsequent primary or acquired drug resistance of cancer cells has become an obstacle to a favorable prognosis. Mortalin is a chaperone that plays an important role in multiple cellular and biological processes. Our previous studies have found that mortalin is associated with the proliferation and migration of ovarian cancer cells and their resistance to cisplatin-based chemotherapy. In this study, microRNA (miR)-200b/c downregulated mortalin expression and inhibited the proliferation and migration of the paired cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) epithelial ovarian cancer cell lines. Moreover, miR-200c increased the sensitivity of ovarian cancer cells to cisplatin treatment by regulating mortalin levels. Nuclear factor (NF)-κB directly regulated mortalin and miR-200b/c expression levels, while NF-κB and miR-200b/c jointly regulated the expression of mortalin. The combination of cisplatin and miR-200c significantly enhanced the therapeutic effects on ovarian cancer in vivo, suggesting that miR-200c may serve as a potential therapeutic agent for ovarian cancer.

Abstract 3790: A study of evolutionary dynamics induced by carboplatin, olaparib and paclitaxel in high-grade serous ovarian cancer cell line models

Abstract Background High-grade serous ovarian cancer (HGSOC) is characterized by extensive intratumoral heterogeneity, which is associated with drug resistance. Our aim was to study the effects of drugs used in the treatment of HGSOC - carboplatin (C), paclitaxel (P) and olaparib (O) - on clonal dynamics in experimental models. Methods We tracked clonal evolution in PEO1, a BRCA2-mutant HGSOC cell line model, by transfecting an initial population of 1.714x106 cells with a barcode (BC) library (CloneTrackerXP). Barcoded populations of cells (18.73x106 cells/flask) were grown in HYPERflasks till confluence, and then replicates were exposed to C (3163 nM) for 5 weeks, P (6.32 nM) for 4 weeks, and O (28467 nM) for 5 weeks (as single agents), reducing the cell population to < 10% (controls kept till cell confluence in the absence of drugs). After that, drug pressure was removed, allowing cells to regrow fully. NGS was used to quantify BCs in the final cell populations and, to get information on the temporal dynamics, also in dead floating cells collected from the weekly culture media change. Heterogeneity of the cell populations was estimated using the Shannon diversity index (SDI). Results The SDI in the controls was 6.306 (95% Bootstrap CI 6.304-6.310) for C and 6.192 (95% Bootstrap CI 6.190-6.197) for P and O. After exposure to C, P and O, the SDI was reduced to 3.208, 2.520 and 4.753 (95% Bootstrap CI 3.209-3.211, 2.517-2.523, 4.750-4.759) respectively, using aggregated replicates (pairwise 2-sided Hutcheson t-test: p < 2.2e-16 pre-drug exposure vs C, P and O). The number of unique BCs in controls vs C, P and O were 1799 vs 545, 1463 vs 288 and 1463 vs 1270, respectively, demonstrating reduction of heterogeneity and change in frequency of clones following drug selection pressure. Cell populations exposed to C and O share some similar clone selection (approximately 60%). This is important as, although the degree of heterogeneity caused by individual drugs are different (C and P lead to less heterogeneous populations than O), there is some concordance between the remaining clones determined by DNA damaging/DNA damage response inhibitors compared to clone selection following exposure to antimicrotubule agents. The selection pattern of BCs among replicates of the different drugs was similar with conservation of BCs with the highest fitness, demonstrating that the initial conditions are determinants of selection under drug exposure. Conclusion Our work, for the first time, quantifies and shows that heterogeneity followed by O therapy is greater than by C and P. Furthermore, the emergent population of cells post-selection pressure was more similar in the C and O groups when compared to P-exposed cells, suggesting correlation in the mechanism of action of drugs and drug resistance/tolerance. Our results also indicate that evolutionary dynamics are largely deterministic and, therefore, can be predictable. Citation Format: Alvaro H. Ingles Russo Garces, Salvatore Milite, Javier Fernandez-Mateos, Bingjie Chen, Lisa Pickard, Adam Stewart, Sara Diaz Sanchez, Rachel Lau, Erica Oliveira, Susana Banerjee, Andrea Sottoriva, Udai Banerji. A study of evolutionary dynamics induced by carboplatin, olaparib and paclitaxel in high-grade serous ovarian cancer cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3790.

Reactivity of NK Cells Against Ovarian Cancer Cells Is Maintained in the Presence of Calcium Phosphate Nanoparticles.

Calcium phosphate nanoparticles (CaP-NPs) are biodegradable carriers that can be functionalized with biologically active molecules. As such, they are potential candidates for delivery of therapeutic molecules in cancer therapies. In this context, it is important to explore whether CaP-NPs impair the natural or therapy-induced immune cell activity against cancer cells. Therefore, in this study, we have investigated the effects of different CaP-NPs on the anti-tumor activity of natural killer (NK) cells using different ovarian cancer (OC) cell line models. We explored these interactions in coculture systems consisting of NK cells, OC cells, CaP-NPs, and therapeutic Cetuximab antibodies (anti-EGFR, ADCC-inducing antibody). Our experiments revealed that aggregated CaP-NPs can serve as artificial targets, which activate NK cell degranulation and impair ADCC directed against tumor targets. However, when CaP-NPs were properly dissolved by sonication, they did not cause substantial activation. CaP-NPs with SiO2-SH-shell induced some activation of NK cells that was not observed with polyethyleneimine-coated CaP-NPs. Addition of CaP-NPs to NK killing assays did not impair conjugation of NK with OC and subsequent tumor cytolytic NK degranulation. Therapeutic antibody coupled to functionalized CaP-NPs maintained substantial levels of antibody-dependent cellular cytotoxic activity. Our study provides a cell biological basis for the application of functionalized CaP-NPs in immunologic anti-cancer therapies.

PARP Inhibitors Display Differential Efficacy in Models of BRCA Mutant High-Grade Serous Ovarian Cancer.

Several poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors are now in clinical use for tumours with defects in BReast CAncer genes BRCA1 or BRCA2 that result in deficient homologous recombination repair (HRR). Use of olaparib, niraparib or rucaparib for the treatment of high-grade serous ovarian cancer, including in the maintenance setting, has extended both progression free and overall survival for women with this malignancy. While different PARP inhibitors (PARPis) are mechanistically similar, differences are apparent in their chemical structures, toxicity profiles, PARP trapping abilities and polypharmacological landscapes. We have treated ovarian cancer cell line models of known BRCA status, including the paired cell lines PEO1 and PEO4, and UWB1.289 and UWB1.289+BRCA1, with five PARPis (olaparib, niraparib, rucaparib, talazoparib and veliparib) and observed differences between PARPis in both cell viability and cell survival. A cell line model of acquired resistance to veliparib showed increased resistance to the other four PARPis tested, suggesting that acquired resistance to one PARPi may not be able to be rescued by another. Lastly, as a proof of principle, HRR proficient ovarian cancer cells were sensitised to PARPis by depletion of BRCA1. In the future, guidelines will need to emerge to assist clinicians in matching specific PARPis to specific patients and tumours.

Lapatinib and poziotinib overcome ABCB1-mediated paclitaxel resistance in ovarian cancer.

Conventional frontline treatment for ovarian cancer consists of successive chemotherapy cycles of paclitaxel and platinum. Despite the initial favorable responses for most patients, chemotherapy resistance frequently leads to recurrent or refractory disease. New treatment strategies that circumvent or prevent mechanisms of resistance are needed to improve ovarian cancer therapy. We established in vitro paclitaxel-resistant ovarian cancer cell line and organoid models. Gene expression differences in resistant and sensitive lines were analyzed by RNA sequencing. We manipulated candidate genes associated with paclitaxel resistance using siRNA or small molecule inhibitors, and then screened the cells for paclitaxel sensitivity using cell viability assays. We used the Bliss independence model to evaluate the anti-proliferative synergy for drug combinations. ABCB1 expression was upregulated in paclitaxel-resistant TOV-21G (q < 1x10-300), OVCAR3 (q = 7.4x10-156) and novel ovarian tumor organoid (p = 2.4x10-4) models. Previous reports have shown some tyrosine kinase inhibitors can inhibit ABCB1 function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant ABCB1-overexpressing ovarian cancer cell lines to paclitaxel. We observed synergy when we combined poziotinib or lapatinib with paclitaxel in resistant TOV-21G and OVCAR3 cells. Silencing ABCB1 expression in paclitaxel-resistant TOV-21G and OVCAR3 cells reduced paclitaxel IC50 by 20.7 and 6.2-fold, respectively. Furthermore, we demonstrated direct inhibition of paclitaxel-induced ABCB1 transporter activity by both lapatinib and poziotinib. In conclusion, lapatinib and poziotinib combined with paclitaxel synergizes to inhibit the proliferation of ABCB1-overexpressing ovarian cancer cells in vitro. The addition of FDA-approved lapatinib to second-line paclitaxel therapy is a promising strategy for patients with recurrent ovarian cancer.

General transcription inhibitors as novel therapeutic agents in clear cell ovarian carcinoma

<h3>Objectives:</h3> General transcription inhibitors are being evaluated as novel agents to treat various malignancies in isolation and combination with other agents. Cancer cells have been demonstrated to have ‘transcriptional oncogene addiction' due to their reliance on master transcription factors (MTFs) for survival. General transcription inhibitors have been shown to inhibit these master transcription factors leading to cell death, making them ideal drug targets. Our goal was to see if general transcription inhibitors had activity within clear cell ovarian cancer (CCOC) cell line models, to identify novel therapeutic options that warrant further evaluation in a disease that is frequently chemoresistant. <h3>Methods:</h3> We performed integrated multi-omic analyses of genome-wide transcriptomic, epigenomic and knock-out screen data to prioritize candidate MTFs for CCOC. Three prototypic human CCOC cell lines: RMGII, JHOC5 and OVMANA were used to study responses to general transcription inhibitors. 3000-5000 cells were seeded in 96-well plates and then were treated with serial dilutions of transcription inhibitors: THZ1, THZ531 and JQ1; inhibitors of CDK7, CDK12/13 and BRD1/2/4 respectively with three technical replicates for 72 hours. Cell viability was quantified using the Cell-titer Glo reagent and results were analyzed using GraphPad Prism Software preforming non-linear regression to obtain dose response curves and IC50 values. <h3>Results:</h3> Using integrated analyses of gene expression, landscapes of active chromatin, and functional dependencies prioritized HNF1B and PAX8 as candidate master regulators for CCOC. All three transcription inhibitors showed activity in the three CCOC lines studied. CCOC models were the most sensitive to THZ1 with IC50 values for THZ1 at 11nM, 14nM and 22nM in RMGII, OVMANA and JHOC5 respectively (SD: 8,12,12nM). IC50 for THZ531 was 51nM, 157nM and 361nM for RMGII, OVMANA and JHOC5 (SD: 35,32,12nM) and JQ1 demonstrated the least activity overall with IC50 of 39nM, 105nM and 294nM for the respective cell lines (RMGII, OVMANA, JHOC5, (SD:23, 47, 107nM)). <h3>Conclusions:</h3> General transcription inhibitors have activity in clear cell ovarian cancer cell lines. Mechanistic studies are exploring the role of PAX8 and HNF1B in determining CCOC sensitivity to these drugs. Further exploration of synergy with agents such as PARP inhibitors and standard chemotherapeutics is underway in <i>in vitro</i> models to identify potential actionable drug combinations for clinical trial development.

Pharmacological Inhibition of BAD Ser99 Phosphorylation Enhances the Efficacy of Cisplatin in Ovarian Cancer by Inhibition of Cancer Stem Cell-like Behavior.

Platinum-based chemotherapy has been the standard treatment for ovarian cancer patients for approximately four decades. However, the prognosis of patients with advanced ovarian carcinoma remains dismal, mainly attributed to both dose-limiting toxicities of cisplatin and the high rate of chemo-resistant disease recurrence. Herein, both patient-derived and experimentally generated cisplatin-sensitive and -resistant ovarian cancer cell line models were used to delineate BADSer99 phosphorylation as an actionable target in ovarian cancer. BADSer99 phosphorylation was negatively associated with cisplatin sensitivity in ovarian cancer, and the inhibition of BADSer99 phosphorylation by point mutation induced apoptosis and reduced cisplatin IC50. In addition, BAD phosphorylation was also shown to be associated with cancer stem cell-like properties. Henceforth, a novel small molecule which inhibits BAD phosphorylation specifically at Ser99 (NPB) was utilized. NPB promoted apoptosis and reduced 3D growth of bulk cancer cells and inhibited cancer stem cell-like properties in both cisplatin-sensitive and -resistant ovarian cancer cells. The combination of cisplatin with NPB exhibited synergistic effects in vitro. NPB in combination with cisplatin also achieved an improved outcome compared to either monotreatment in vivo, including suppression of the cancer stem cell population, an effect not observed with cisplatin treatment. Furthermore, NPB exhibited strong synergistic effects with the AKT inhibitor AZD5363, and significantly reduced its IC50 in cells resistant to cisplatin treatment. These findings identify BADSer99 phosphorylation as an actionable and pharmacologically relevant target to improve outcomes of cisplatin treated ovarian cancer.

Characterization of isogenic ovarian cancer cell line models of acquired resistance to the clinical ATR inhibitor AZD6738

Objective: Clinical ATR kinase inhibitors (ATRi) are an emerging class of therapeutics being used to treat diverse solid tumor malignancies, including ovarian cancer. This study is focused on improving our understanding of ATRi resistance through characterizing isogenic ovarian cancer cell line models of acquired resistance to the clinical ATR inhibitor AZD6738.

Abstract 1061: Increasing potency of anticancer drugs through Sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer

Abstract Rational : The development of personalized therapies against ovarian cancer remains highly challenging even in current modern oncology. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to peptide ligands that selectively target receptors abundantly and/or exclusively expressed in these tumor cells. Increased expression of Sortilin (SORT1), a scavenging receptor also known as neurotensin receptor-3, has been observed in invasive ovarian cancer biopsies. In addition, increased SORT1 expression correlated with breast tumor grades. Procedures : In light of these observations, we developed a peptide conjugation platform to target SORT1 and to increase cell selectivity and internalization of anticancer agents. As a proof-of-concept, doxorubicin and docetaxel were conjugated to a Sortilin-binding proprietary peptide (TH19P01). Results : In vitro, significant intracellular delivery of the doxorubicin-TH19P01 conjugate (TH1904) and docetaxel-TH19P01 conjugate (TH1902) were observed in a ES-2 ovarian cancer cell line model while preserving efficient doxorubicin or docetaxel cytotoxic mechanism. TH19P01 and drug conjugate uptake in ovarian cancer cells assays were reduced when SORT1 expression was specifically silenced using siRNA or upon competition with the SORT1 ligands neurotensin and progranulin. Importantly, drug-TH19P01 conjugates were found to bypass the P-glycoprotein (P-gp) efflux pump in MDCK-MDR1 cells overexpressing P-gp as the uptake of conjugates was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, TH1904 and TH1902 caused a more potent inhibition of human ovarian tumor xenografts grown in mice and were better tolerated than their unconjugated parent drugs. Conclusion : These results strongly support future clinical development of this targeted technology platform to generate novel personalized therapeutics with specific indications in Sortilin-positive cancers. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Larocque, Alain Zgheib, Sophie Kozelko, Richard Béliveau, Christian Marsolais, Borhane Annabi. Increasing potency of anticancer drugs through Sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer [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 1061.

Abstract 3000: Evaluating the efficacy of a novel class of covalent microtubule stabilizers in taxane resistant ovarian cancer

Abstract Ovarian cancer has few targeted therapies and high rates of metastasis. Taxane microtubule stabilizers, including paclitaxel, are a mainstay in the treatment of adult solid tumors as single agents and in combination with other cytotoxic chemotherapies, targeted therapies, and immunotherapies. The standard of care for ovarian cancer patients comprises of combination chemotherapy consisting of a taxane with a platinum agent administered intravenously or intraperitoneally. Although this approach is highly effective initially, up to 70% of ovarian cancer patients will acquire taxane resistance. Therefore, it is essential to identify drugs that retain efficacy in clinically relevant models of taxane resistance, including those with elevated P-glycoprotein (Pgp) expression. The development of a microtubule stabilizer that retains efficacy in taxane resistant ovarian cancer would be optimal since microtubule disruption is proven to be an effective target in this disease. The taccalonolides are a novel class of covalent and irreversible microtubule stabilizers that circumvent clinically relevant forms of taxane resistance in vitro and in vivo breast cancer models. However, the taccalonolides have a narrow therapeutic window and short serum half-life that would benefit from a more targeted delivery. We hypothesized that localized intraperitoneal delivery of the taccalonolides in ovarian cancer models may be an optimal strategy for the treatment of intraperitoneally disseminated disease. We have found that the taccalonolides are less susceptible than paclitaxel to Pgp-mediated drug resistance in a human ovarian cancer cell line model. Additional experiments have demonstrated that the taccalonolides retain long-term antiproliferative and cytotoxic efficacy following acute treatment of human and murine ovarian cancer cells, demonstrating superior cellular persistence as compared to other classes of microtubule stabilizing drugs. We are currently evaluating the efficacy of the taccalonolides in ovarian cancer models using localized, intraperitoneal administration in a syngeneic and orthotopic murine model, which may be optimal for delivery of this irreversible microtubule stabilizer. Citation Format: Samantha S.M. Yee, Lin Du, April L. Risinger. Evaluating the efficacy of a novel class of covalent microtubule stabilizers in taxane resistant ovarian cancer [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 3000.

Abstract A50: Lapatinib potentiates the antitumor effects of paclitaxel treatment in resistant ovarian cancer cells

Abstract Introduction: Frontline treatment regimens for ovarian cancer routinely consist of repeated cycles of paclitaxel/platinum doublets; however, chemotherapy resistance frequently leads to recurrent or refractory disease. Novel therapeutic strategies aimed at circumventing or preventing resistance are needed for improved disease control. Experimental Procedures: Multiple paclitaxel-resistant ovarian cancer cell line models were established via repeated cycles of paclitaxel treatment in vitro. Resistance to paclitaxel was assessed using dose response cell viability assays with CellTiter-Glo 2.0 and calculation of IC50 values using a 4-parameter logistic regression model. RNAseq and whole-exome sequencing were used to examine genomic variation distinguishing resistant lines from their sensitive counterparts. Recurrent expression differences associated with paclitaxel resistance were identified, a subset of which were selected for further validation. Candidate genes and pathways associated with paclitaxel resistance were manipulated using siRNA and/or small-molecule inhibitors and assessed for the ability to alter the resistant phenotype. Synergistic interactions for drug combinations were evaluated using the Loewe Additivity model. Results: Paclitaxel-resistant ovarian cancer cell lines were derived from TOV-21G, OVCAR-3, and UWB1.289 cells. Resistant cell lines exhibited increases in paclitaxel IC50 ranging from 6.5 to 94-fold over their parental control cells. RNAseq analysis identified the transcript encoding P-glycoprotein (P-gp), ABCB1, among the most highly upregulated genes in the resistant TOV-21G and OVCAR-3 cells while no such induction of expression was seen in resistant UWB1.289 cells. Previous reports have shown several different tyrosine kinase inhibitors (e.g., lapatinib, neratinib, dasatinib, etc.) have the ability to inhibit P-gp function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant P-gp-overexpressing ovarian cancer cells to paclitaxel. The most potent synergistic antiproliferative activity was observed when lapatinib was combined with paclitaxel treatment of resistant TOV-21G and OVCAR-3 cells; however, a similar interaction was not observed in resistant UWB1.289 cells that do not overexpress P-gp. The dependence of this synergistic effect on P-gp expression was confirmed by using siRNA to inhibit P-gp expression in paclitaxel-resistant TOV-21G and OVCAR-3 cells, significantly reducing paclitaxel IC50 by 20.7 and 6.2-fold, respectively, and eliminating cytotoxic synergy. Conclusions: Paclitaxel and lapatinib have synergistic anticancer properties when used to treat P-gp-overexpressing ovarian cancer cells. The addition of lapatinib to second-line dose-dense paclitaxel therapy could be an effective strategy for a subset of ovarian cancer patients with recurrent disease and warrants further clinical exploration. Citation Format: Rob McCorkle, Justin W. Gorski, Abigail Anderson, Jill M. Kolesar. Lapatinib potentiates the antitumor effects of paclitaxel treatment in resistant ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A50.

Increasing potency and safety of anticancer drugs through sortilin receptor-mediated cancer therapy: A new targeted approach for the treatment of ovarian cancer.

e17068 Background: The development of personalized therapies against ovarian cancer remains highly challenging in current modern oncology. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to specific peptide ligands that selectively target receptors abundantly and/or exclusively expressed on these cells. Increased expression of Sortilin, a scavenging receptor, has been clinically observed in invasive ovarian cancer biopsies, and correlated with tumor grades. In light of this, we developed a peptide conjugation platform and a new Sortilin receptor-mediated vectorization strategy to increase cell targeting selectivity and cell delivery efficacy of anticancer agents. Methods: As a proof-of-concept, Doxorubicin was conjugated to a Sortilin binding peptide (KA-peptide). In vitro, intracellular delivery of the Doxorubicin-KA-peptide conjugate (DoxKA) was assessed in ES-2 and SKOV-3 ovarian cancer cell line models using flow cytometry and fluorescent microscopy. Sortilin gene silencing was performed with specific siRNA. DoxKA efficacy and safety were evaluated in vivo using ES-2 (CD1 nude mice) and SKOV-3 (athymic mice) subcutaneous xenograft models. Results: Uptake of DoxKA was observed in both Sortilin-positive ovarian cancer cell lines tested and was reduced when Sortilin expression was specifically silenced or upon competition with the Sortilin ligands Neurotensin and Progranulin. Results indicate that the uptake of DoxKA occurs via Sortilin-mediated endocytosis in contrast to simple diffusion for Doxorubicin. DoxKA was found to bypass the P-glycoprotein (P-gp) efflux pump in MDCK-MDR1 cells overexpressing P-gp as the uptake of DoxKA was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, DoxKA showed lower potential side effects than Doxorubicin alone did, with decreased accumulation in healthy tissues such as heart and ovary. DoxKA caused a more potent inhibition of human ovarian tumor xenografts growth in mice and was better tolerated (absence of leukopenia and neutropenia) than the unconjugated Doxorubicin at an equivalent dose. Conclusions: These results strongly support the future clinical use of this platform to generate novel personalized therapeutics with specific targeting of Sortilin-positive tumors in the next stage of development in phase 1 clinical trial.

Novel synthesis of platinum complexes and their intracellular delivery to tumor cells by means of magnetic nanoparticles.

Platinum-based drugs are popular in clinics as chemotherapeutic agents to treat solid tumors. However, severe side effects such as nephro- and neurotoxicity impose strict dosage limitations that can lead to the development of drug resistance and tumor relapse. To overcome these issues Pt(iv) prodrugs and platinum delivery systems might represent the next generation of platinum-based drugs. In this study four novel Pt(ii) complexes (namely, PEG-Glu-Pt-EDA, PEG-Glu-Pt-DACH, PEG-Mal-Pt-EDA and PEG-Mal-Pt-DACH) were synthesized and a general strategy to covalently bind them to iron oxide nanoparticles was developed. The intracellular uptake and cell distribution studies of Pt-tethered magnetic nanoparticles on breast and ovarian cancer cell line models indicate that binding of the Pt complexes to the nanoparticles facilitates, for all the complexes, cellular internalization. Moreover, the magnetic nanoparticles (MNPs), as shown in a magnetofection experiment, enhance the uptake of MNP-Pt conjugates if a magnet is placed beneath the culture dish of tumor cells. As shown by a Pt release experiment, intranuclear platinum quantification and TEM analysis on cell sections, the presence of a pH-sensitive dicarboxylic group coordinating the Pt complex, triggers platinum dissociation from the NP surface. In addition, the triazole moiety facilitates endosomal swelling and the leakage of platinum from the endosomes with intranuclear localization of platinum release by the NPs. Finally, as assessed by MTT, caspase, calcein/ethidium bromide live/dead assays, among the four NP-Pt conjugates, the NP-Glu-Pt-EDA complex having a glutamate ring and ethylenediamine as a chelating amine group of the platinum showed higher cytotoxicity than the other three MNP-platinum conjugates.

Developing a genetic signature to predict drug response in ovarian cancer.

There is a lack of personalized treatment options for women with recurrent platinum-resistant ovarian cancer. Outside of bevacizumab and a group of poly ADP-ribose polymerase inhibitors, few options are available to women that relapse. We propose that efficacious drug combinations can be determined via molecular characterization of ovarian tumors along with pre-established pharmacogenomic profiles of repurposed compounds. To that end, we selectively performed multiple two-drug combination treatments in ovarian cancer cell lines that included reactive oxygen species inducers and HSP90 inhibitors. This allowed us to select cell lines that exhibit disparate phenotypes of proliferative inhibition to a specific drug combination of auranofin and AUY922. We profiled altered mechanistic responses from these agents in both reactive oxygen species and HSP90 pathways, as well as investigated PRKCI and lncRNA expression in ovarian cancer cell line models. Generation of dual multi-gene panels implicated in resistance or sensitivity to this drug combination was produced using RNA sequencing data and the validity of the resistant signature was examined using high-density RT-qPCR. Finally, data mining for the prevalence of these signatures in a large-scale clinical study alluded to the prevalence of resistant genes in ovarian tumor biology. Our results demonstrate that high-throughput viability screens paired with reliable in silico data can promote the discovery of effective, personalized therapeutic options for a currently untreatable disease.

Abstract 5146: Increasing penetration of anticancer drugs through sortilin receptor-mediated cancer therapy: A new targeted and personalized Approach in the treatment of ovarian cancer

Abstract The development of personalized therapies against ovarian cancer remains highly challenging in current modern oncology. One way to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to specific peptide ligands that will selectively target receptors abundantly and/or exclusively expressed on these cells. Increased expression of Sortilin, a scavenging receptor, has been clinically observed in several human cancers including breast, prostate, colon, pancreas, skin, and pituitary. In particular, Sortilin has also been reported to be overexpressed in ovarian cancers as compared to non-malignant ovarian tissue. In light of this, we developed a peptide conjugation platform using a new Sortilin receptor-mediated vectorization strategy to increase cell targeting selectivity and cell delivery efficacy of anticancer agents. As a proof-of-concept, Doxorubicin was conjugated to peptide sequences, termed Katana peptides (KA). In vitro, significantly increased Sortilin mediated intracellular delivery of the Doxorubicin-Katana peptide conjugate (DoxKA) was observed in SKOV3 and ES-2 ovarian cancer cell line models, with conserved efficient Doxorubicin cytotoxic mechanism. Uptake of DoxKA in ES-2 ovarian cancer cells was reduced when Sortilin expression was specifically silenced by using siRNA or upon competition with the Sortilin ligands neurotensin and progranulin. In addition, DoxKA was found to bypass the P-glycoprotein (P-gp) efflux pump since, in contrast to Doxorubicin uptake in MDCK-MDR1 cells overexpressing the P-gp efflux pump, the uptake of DoxKA was unaffected by the P-gp inhibitor Cyclosporin A. In vivo, DoxKA was better tolerated and caused a more potent inhibition of human ovarian tumor xenografts growth than did Doxorubicin alone. These results provide strong evidence for the potential of this drug development platform in the generation of novel personalized therapeutics with specific targeting of SORT1-positive cancer cells. Citation Format: Michel Demeule, Jean-Christophe Curie, Alain Larocque, Cyndia Charfi, Richard Béliveau, Claude Vezeau, Borhane Annabi. Increasing penetration of anticancer drugs through sortilin receptor-mediated cancer therapy: A new targeted and personalized Approach in the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5146. doi:10.1158/1538-7445.AM2017-5146

Abstract TMEM-038: THE CULTIVATION AND PARTITIONING OF INVASIVE OVARIAN CANCER CELLS AFTER THEIR ISOLATION FROM WHOLE BLOOD

Abstract Ovarian cancer patients suffer from high rates of recurrence and mortality. Circulating tumor cells (CTCs) can serve as a minimally invasive tool to improve the clinical management of such cancer patients. Hence there is growing interest in the isolation, characterization and in vitro culture of CTCs. To this end, the porous membrane cell separators developed by MetaCell s.r.o. provide a simple solution. Ovarian cancer cell line models OVCAR3 and SKOV3 were studied by gene expression profiling and immunostaining for the expression of EPCAM, EGFR, HER2, MET and certain other novel therapeutic targets. Cultivation of the cancer cells after their spike recovery from either cell culture medium or from whole blood was explored using MetaCellTM separators. The cells growing on the separator membranes housed in multi-well culture plates was conveniently visualized with vital cell stains. The SKOV3 cells, but not the OVCAR3 cells, were found to migrate through the membrane pores and populate the well bottoms of the culture plates. This can be attributed to the more plastic and invasive nature of SKOV3 cells as characterized by high Vimentin and low EPCAM expression levels. This is akin to the profile exhibited by highly invasive cancer cells that have undergone epithelial to mesenchymal transition (EMT) and that are implicated in the metastatic spread of cancer in patients. Furthermore, the invasive cells growing on the culture plate well bottoms were found to still stain positively for the therapeutic targets of interest. This cultivation device thus offers the ability to selectively interrogate the more invasive sub-populations within the highly heterogeneous CTCs that are typically seen in cancer patients. After the isolation and cultivation of CTCs, if the partitioned invasive cells remain addressable for the therapeutic target under consideration, the patients are more likely to respond to that therapy for the arrest of metastatic disease. In this context, the MetaCellTM separators have the potential to serve as very effective adjunctive aids for the selection and monitoring of ovarian cancer patients for treatment with targeted therapies. Citation Format: Uday K. Veeramallu, M.S. THE CULTIVATION AND PARTITIONING OF INVASIVE OVARIAN CANCER CELLS AFTER THEIR ISOLATION FROM WHOLE BLOOD [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-038.

Platinum-Based Chemotherapy Induces Methylation Changes in Blood DNA Associated with Overall Survival in Patients with Ovarian Cancer.

Purpose: DNA damage repair can lead to epigenetic changes. DNA mismatch repair proteins bind to platinum DNA adducts and at sites of DNA damage can recruit the DNA methylating enzyme DNMT1, resulting in aberrant methylation. We hypothesised that DNA damage repair during platinum-based chemotherapy may cause aberrant DNA methylation in normal tissues of patients such as blood.Experimental Design: We used Illumina 450k methylation arrays and bisulphite pyrosequencing to investigate methylation at presentation and relapse in blood DNA from patients with ovarian cancer enrolled in the SCOTROC1 trial (n = 247) and in a cohort of ovarian tumor DNA samples collected at first relapse (n = 46). We used an ovarian cancer cell line model to investigate the role of the DNA mismatch repair gene MLH1 in platinum-induced methylation changes.Results: Specific CpG methylation changes in blood at relapse are observed following platinum-based chemotherapy and are associated with patient survival, independent of other clinical factors [hazard ratio, 3.7; 95% confidence interval, 1.8-7.6, P = 2.8 × 10-4]. Similar changes occur in ovarian tumors at relapse, also associated with patient survival (hazard ratio, 2.6; 95% confidence interval, 1.0-6.8, P = 0.048). Using an ovarian cancer cell line model, we demonstrate that functional mismatch repair increases the frequency of platinum-induced methylation.Conclusions: DNA methylation in blood at relapse following chemotherapy, and not at presentation, is informative regarding survival of patients with ovarian cancer. Functional DNA mismatch repair increases the frequency of DNA methylation changes induced by platinum. DNA methylation in blood following chemotherapy could provide a noninvasive means of monitoring patients' epigenetic responses to treatment without requiring a tumor biopsy. Clin Cancer Res; 23(9); 2213-22. ©2016 AACR.