HGSOC Cell Research Articles

Targeting estrogen metabolism in high-grade serous ovarian cancer shows promise to overcome platinum resistance

The high mortality rate due to chemoresistance in patients with high-grade ovarian cancer (HGSOC) emphasizes the urgent need to determine optimal treatment strategies for advanced and recurrent cases. Our study investigates the interplay between estrogens and chemoresistance in HGSOC and shows clear differences between platinum-sensitive and -resistant tumors. Through comprehensive transcriptome analyzes, we uncover differences in the expression of genes of estrogen biosynthesis, metabolism, transport and action underlying platinum resistance in different tissues of HGSOC subtypes and in six HGSOC cell lines. Furthermore, we identify genes involved in estrogen biosynthesis and metabolism as prognostic biomarkers for HGSOC. Additionally, our study elucidates different patterns of estrogen formation/metabolism and their effects on cell proliferation between six HGSOC cell lines with different platinum sensitivity. These results emphasize the dynamic interplay between estrogens and HGSOC chemoresistance. In particular, targeting the activity of steroid sulfatase (STS) proves to be a promising therapeutic approach with potential efficacy in limiting estrogen-driven cell proliferation. Our study reveals potential prognostic markers as well as identifies novel therapeutic targets that show promise for overcoming resistance and improving treatment outcomes in HGSOC.

Abstract 7347: A transcriptome correlation network analysis of the high grade serous ovarian tumor immune microenvironment

Abstract High Grade Serous Ovarian Cancer (HGSOC) affects many women and is the most common and lethal histopathological type of ovarian cancer. The molecular subtypes delineated by TCGA–mesenchymal, proliferative, immunoreactive, and differentiated subtypes, offer a sophisticated and innovative framework for HGSOC classification. These subtypes not only provide a detailed molecular characterization but also contribute to understanding the HGSOC Tumor Immune Microenvironment (TIME), comprised of both immune and tumor cells. The interplay within the TIME significantly influences pro- or anti-tumor responses, creating an important link between molecular landscape and dynamic immune-tumor interactions. Currently, there is a lack of understanding for the key drivers of clinical outcomes, molecular phenotypes, and diagnostic biomarkers for HGSOC. Here, we answer the question, does immune cell type, and proportion, drive clinical and molecular subtyping of primary HGSOC? Primary ovarian tumor samples from The Cancer Genome Atlas, TCGA (n=358) samples were collected and RNA-sequenced. FPKM values were used to construct the Weighted Gene Correlation Network Analysis (WGCNA), Gene Ontology (GO) and Proportion Estimations of Immune and Cancer cells (EPIC). Enrichment of HGSOC network module overlaps using a Fischer Exact Test were used to identify novel components of HGSOC progression that were significantly associated with clusters of highly correlated gene expression profiles, HGSOC stage, grade, and molecular phenotypes. We show genes associated with T cell proliferation (CD84) and B cell regulation (CD37), epithelial to mesenchymal transition (DOCK2), adaptor proteins present in T cells and myeloid cells (LCP2), inflammatory and metabolic signaling modulators (AIF1,CD53, SASH3), immunosuppressors of T lymphocytes and dendritic cells (FGL2), cell proliferation (PI3K) and relevant tertiary lymphoid structure gene (TNFAIP8L2) significantly correlated with HGSOC progression in both estimated tumor and proportions of immune cells. Future studies aim to confirm phosphorylation cascades associated with the aggressive molecular phenotypes of HGSOC and immune cell pathobiology. Citation Format: Kaylin M. Carey, Eric B. Dammer, Corey Young, Rajesh Singh, Ti'ara Griffen, James W. Lillard. A transcriptome correlation network analysis of the high grade serous ovarian tumor immune microenvironment [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 7347.

Abstract 631: Functional genetics of APR-246 rescuable TP53 mutants

Abstract TP53, often referred to as the guardian of the genome, encodes for the p53 tumor suppressor protein, and is one of the most frequently mutated genes in human cancers. Some cancer sub-types, such as non-small cell lung carcinoma (NSCLC) and high grade serous carcinoma (HGSC), have high TP53 mutation rates reaching up to 56% and 96%, respectively. Given its multi-faceted role in human cancers, there have been several emerging therapeutic approaches targeting mutant p53. APR-246 is a small molecule drug currently in clinical trials for the functional rescue of p53 missense mutants, the most common type of p53 mutant protein, across various cancer types. APR-246 is proposed to bind mutant p53 and promote thermodynamic stabilization of the protein allowing it to regain wild-type-like conformation and consequentially regain WT-like functionality. However, there has yet to be a functional genomic screening of p53 mutants to systematically catalog which mutants are rescuable by APR-246. To address this critical gap, we have performed functional genetic screenings using a TP53 mutagenesis library. This pooled lentiviral library contains ~8,000 amino acid variants, spanning the entirety of the TP53 gene. Following stable transduction of this library into p53-null NSCLC and HGSOC cell lines, we performed functional screenings against sublethal doses of APR-246. Next-generation sequencing of the untreated and treated cell population allows for comparison of variant representation and determination of APR-246-sensitivity of p53 mutants. Preliminary statistical analysis of our screens has identified a pool of 117 “hit” mutants which are significantly depleted following treatment, indicative of APR-246-sensitivity. Two of these mutants, C242F and G266V, are non-functional, damaging mutations which occur in patients. Identifying p53 mutants that can be rescued by APR-246 will help identify which patients may benefit most from treatment with the drug. Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chein. Functional genetics of APR-246 rescuable TP53 mutants [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 631.

Abstract 5432: The role of nuclear factorκB-inducing kinase in ovarian cancer stem cells, chemoresistance, and relapse

Abstract Ovarian cancer is the most lethal gynecological cancer in the United States, largely due to its high relapse rates. Research suggests these high relapse rates are due to cancer stem-like cells (CSCs), a subpopulation of tumor cells that are resistant to chemotherapy and have enhanced tumor-initiation capacity. Post-treatment, CSCs self-renew to re-establish new tumors, leading to recurrent disease that is now resistant to chemotherapy. Given that over-expression of NF-κB has been linked to CSCs, cancer progression, and chemoresistance in various tumors, including ovarian, studies uncovering the mechanisms of this pathway are needed to develop novel therapeutics for ovarian cancer treatment. The upstream activator of the alternative NF-κB pathway, Nuclear Factor κB-inducing Kinase (NIK), could be an enticing target for eliminating CSCs, however its function in these cells is not established. In our current studies, we show that HGSOC cells grown as CSC-enriching 3D spheroids have increased NIK expression and activity relative to monolayer cultured cells. Moreover, cells expressing CSC markers (CD117+/CD133+), have increased NIK gene expression, further supporting an association between NIK and CSC maintenance. Upon treatment with chemotherapies carboplatin and paclitaxel, NIK gene expression increased in the surviving population, indicating a role in chemoresistance. Knockdown of NIK using siRNA led to changes in alternative NF-kB transcriptional activity and higher sensitivity to carboplatin and paclitaxel relative to vehicle controls. Current studies are evaluating the role of NIK in tumor-initiation capacity, asymmetric division, proliferation, and changes in expression of stemness genes. Lastly, an in vivo relapse model will test the feasibility of using a NIK inhibitor following chemotherapy to prevent relapse and prolong survival. Findings from this work will further define the role of NF-kB pathway components in facilitating CSCs survival and re-establishment of tumors and identify a new potential target for eliminating CSCs in ovarian cancer. Citation Format: Cassidy C. Lucht, Mikella Robinson, Samuel F. Gilbert, Omar Lujano Olazaba, Carrie D. House. The role of nuclear factorκB-inducing kinase in ovarian cancer stem cells, chemoresistance, and relapse [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 5432.

Abstract 7209: Overcoming platinum resistance of high-grade serous ovarian cancer targeting the activated JAK/STAT pathways via extracellular vesicles

Abstract High-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer. While HGSOC usually responds to initial treatment, most cases eventually relapse and develop platinum-resistant ovarian cancer (PROC). The molecular definition of PROC is still unclear, and overcoming PROC has been the biggest challenges. Extracellular vesicles (EVs) have an essential role in cell-to-cell communication within both the primary and distant microenvironments. In this study. we aimed to identify molecular mechanisms of PROC via EVs to suggest novel therapeutic strategies. Platinum responses were categorized by primary treatment-free interval of platinum less than 6 months as PROC, and more than 36 months as platinum good response. Ascites and tumor tissues have been collected prior to initial chemotherapy in patients with HGSOC for analysis according to platinum response. mRNA sequencing in tumor tissues identified significantly upregulated JAK/STAT pathways in PROC. The JAK/STAT family was highly expressed in PROC than the good response group, and also increased than adjusted normal ovaries and fallopian tubes. In addition, PROC cell lines and PROC patient-derived xenograft model tumor tissues showed high JAK expression. Small RNA sequencing for HGSOC tissues and ascites-EVs revealed that miR-135a-5p and miR-221-5p were highly expressed in both PROC tissues and ascites, and the expression of the two miRNAs had a positive correlation between tissues and ascites. Overexpression of the two miRNAs in HGSOC cell lines resulted in higher JAK family expression and resistance to cisplatin. JAK inhibitors (JAKi) had equivalent efficacy for platinum-sensitive/resistant HGSOC cell lines and JAKi highly suppress tumor growth in the PROC mouse model. In addition, suppression of JAK family expression in PROC cell lines significantly increased sensitivity to cisplatin. JAKi also had a synergistic effect with cisplatin for PROC cell lines. IPA analyses in mRNA sequencing in tumor tissues, TGF-β was highly expressed in upstream regulators in PROC, and TGF-β stimulation resulted in the upregulation of the two miRNAs in mesothelial cells, but not in HGSOC cell lines and fibroblasts. In summary, we here demonstrate novel molecular mechanisms of PROC via EVs and provided the rationale for JAK-targeted therapy for PROC. EV-miRNAs can regulate the PROC activated JAK/STAT pathway and can be diagnostic and therapeutic biomarkers for PROC definition or JAKi indications, suggesting that patient stratification in precision medicine for initial treatment of HGSOC. Citation Format: Kazuhiro Suzuki, Akira Yokoi, Kousuke Yoshida, Yusuke Yamamoto, Hiroaki Kajiyama. Overcoming platinum resistance of high-grade serous ovarian cancer targeting the activated JAK/STAT pathways via extracellular vesicles [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 7209.

Abstract 4473: Targeting matrix metalloproteinase-3 (MMP-3) in high-grade serous ovarian cancer

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most aggressive and lethal gynecological malignancy. Surgery and chemotherapy (with platinum compounds) are the main treatments. Many patients relapse and develop chemotherapy resistance, limiting the therapeutic utility of these therapies. This persistent resistance makes HGSOC treatment difficult. Cisplatin-resistant ovarian cancer cells upregulate Matrix Metalloproteinase-3. MMP3 is an enzyme involved in active tissue remodeling and extracellular matrix protein breakdown during dynamic physiological processes. The structure of MMP3 includes the prodomain, the catalytic domain and the hemopexin domain. We showed that MMP3 protein levels are higher in cisplatin resistant as compared with cisplatin sensitive HGSOC cells. Our research team showed that small interference RNA (siRNA)-mediated MMP3 knockdown reduced cisplatin-resistant ovarian cancer cell growth and invasion. This data indicates that MMP3 may be a promising HGSOC treatment target. Targeting MMP3 with small chemical inhibitors (SCI) could represent a better option than siRNA because SCI are more stable and easier to deliver. The purpose of this project is to design SCI of MMP3 in the propeptide, catalytic, and hemopexin domains of MMP3. We used in silico methods to identify MMP3 inhibitors in these domains. The ZINC20 database of commercially compounds was used for virtual screening using Python Prescription (PyRx) software. We identified 25 ligands out of 370,000 with the greatest affinity for each MMP3 functional domains. SwissADME was then used to evaluate in silico absorption, distribution, metabolism, pharmacokinetics, drug-likeliness, and medicinal chemistry. This research lays the groundwork for HGSOC therapeutic strategies to improve treatment efficacy, especially for chemotherapy-resistant patients. Citation Format: Víctor G. Reyes-Burgos, Eliud Hernández-O'Farrill, Luis E. Vazquez Quiñones, Marienid Flores-Colón, Fatima Valiyeva, Pablo E. Vivas-Mejía. Targeting matrix metalloproteinase-3 (MMP-3) in high-grade serous 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 4473.

Abstract A013: Auranofin induces lethality driven by reactive oxygen species in high-grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) accounts for 70% of ovarian cancer cases in the clinical setting; the survival rate for this disease remains remarkably low due to the lack of a long-term consolidation therapy following standard platinum-based chemotherapy. There is minimal improvement in the survival rate of this disease due to the development of resistance to platinum drugs as the disease recurs. The purpose of this study was to develop a novel treatment modality against HGSOC using the gold complex auranofin (AF). AF primarily functions as a pro-oxidant agent through the inhibition of thioredoxin reductase (TrxR), an antioxidant enzyme that is overexpressed in ovarian cancer. We explored the effect of AF on TrxR activity and various mechanisms of cytotoxicity using HGSOC cells that are clinically sensitive (PEO1) or resistant (PEO4) to platinum. We treated PEO1 cells and PEO4 cells with AF for 72 hours and analyzed its short-term toxicity by assessing cell viability and DNA content by flow cytometry, and the long-term anti-proliferative capacity using colony formation assays. We also assessed the mechanisms of cell death induced by AF including the activation of mechanisms of apoptosis, activation of executor caspases -3 and -7, cleavage of poly-(ADP-ribose) polymerase (PARP), changes in the mitochondrial membrane potential (MMP), and DNA damage. In addition, we studied the interaction between AF and another pro-oxidant, L-buthionine sulfoximine (L-BSO), an anti-glutathione (GSH) agent. We demonstrate that AF potently inhibits the activity of TrxR at non-cytotoxic concentrations while reducing the viability of HGSOC cells regardless of their sensitivities to platinum. We showed that AF induces accumulation of reactive oxygen species (ROS), triggers the depolarization of the mitochondrial membrane, and kills HGSOC cells by inducing apoptosis. AF-induced cell death and morphological effects were abrogated by the ROS scavenger, N-acetyl cysteine (NAC). In addition, the lethality of AF in HGSOC was associated with the activation of caspase-3/7, cleavage of PARP, and the generation of DNA damage, effects that were prevented by the ROS scavenger NAC. Finally, when AF and L-BSO were combined, we observed a synergistic lethality against HGSOC cells, which was mediated by a further increase in ROS with a decrease in the antioxidant, GSH. In summary, our results support the concept that AF can be used alone to kill HGSOC cells by causing ROS-mediated apoptosis. Furthermore, we demonstrate that a large depletion of antioxidants caused by combining AF and L-BSO is highly efficient in synergistically killing HGSOC cells. These findings suggest that the combination of AF and L-BSO is a potential option to develop a consolidation therapy against HGSOC, which can overcome the problem of platinum resistance during recurrent stages of the disease. Citation Format: Farah H. Abdalbari, Elvis M. Jaramillo, Benjamin N. Forgie, Estelle Tran, Alicia A. Goyeneche, Carlos M. Telleria. Auranofin induces lethality driven by reactive oxygen species in high-grade serous ovarian cancer cells [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 A013.

Abstract B109: Uncovering novel vulnerabilities in ovarian cancer by studying cell death in neoplastic transformation

Abstract B109: Uncovering novel vulnerabilities in ovarian cancer by studying cell death in neoplastic transformation

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [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 B068.

Abstract A066: The cytotoxic effects of oxaliplatin and 5-fluorouracil against low-grade serous ovarian cancer cells

Abstract A066: The cytotoxic effects of oxaliplatin and 5-fluorouracil against low-grade serous ovarian cancer cells

Abstract B016: Repurposing nelfinavir for re-sensitizing platinum-resistant high grade serous ovarian cancer cells

Abstract B016: Repurposing nelfinavir for re-sensitizing platinum-resistant high grade serous ovarian cancer cells

Abstract C041: Drug repurposing to target TGF-β in chemoresistant high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) has low 5-year survival rate of 48%, due to chemotherapy resistance. Effective new therapies are required to improve the survival rate of HGSOC. TGF-β is a family of proteins associated with epithelial-mesenchymal transition and have been implicated in ovarian cancer progression and chemoresistance. Drugs that target TGF-β may have potential to be used to treat chemoresistant HGSOC and control tumour growth and progression. Drug repurposing is a strategy to identify new uses for approved drugs outside of their original indication, and ensure timely access to efficacious and cost-effective therapy for chemoresistant HGSOC. The aims of the study is to determine the effect of the repurposed drug teniposide on HGSOC cell growth, death, and apoptosis. A ligand-based screening was performed by Cresset Discovery for approved drugs that bind to the TGF-β receptor complex and ligand. Teniposide was shortlisted according to its dosing, lipid solubility, pharmacokinetics, and side effects. MTT assays were performed on eight molecularly-characterised HGSOC cell lines to obtain the IC50 of teniposide. The effect of the IC50 dose of teniposide on HGSOC cell growth, death, and apoptosis were monitored on the IncuCyte live-cell imager. Western blotting was performed to determine the expression of TGF-β signalling pathway proteins after treatment with teniposide. The average IC50 of teniposide in HGSOC cell lines is 10µM, within the range of the maximum plasma concentration (61µM). HGSOC cell lines treated with 10µM teniposide showed decreased cell growth, and increased cell death and apoptosis, similar to HGSOC cell lines treated with clinically-relevant dose of carboplatin (30µM). HGSOC cell lines treated with 10µM teniposide showed significantly higher cell death and apoptosis compared to treatment with 30µM carboplatin. Our preliminary results show that teniposide significantly reduced proliferation of HGSOC and has potential to be repurposed for treatment of chemoresistant HGSOC. Citation Format: Michelle WY Wong-Brown, Zlata Johnson, Saad Salem, Bayley G Matthews, Caroline E Ford, Deborah J Marsh, Nikola A Bowden. Drug repurposing to target TGF-β in chemoresistant high-grade serous ovarian cancer [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 C041.

Dual inhibition of CDK12 and CDK13 uncovers actionable vulnerabilities in patient-derived ovarian cancer organoids

BackgroundHigh grade serous ovarian cancer (HGSOC) is highly lethal, partly due to chemotherapy resistance and limited availability of targeted approaches. Cyclin dependent kinases 12 and 13 (CDK12/13) are promising therapeutic targets in human cancers, including HGSOC. Nevertheless, the effects of their inhibition in HGSOC and the potential synergy with other drugs are poorly known.MethodsWe analyzed the effects of the CDK12/13 inhibitor THZ531 in HGSOC cells and patient-derived organoids (PDOs). RNA sequencing and quantitative PCR analyses were performed to identify the genome-wide effects of short-term CDK12/13 inhibition on the transcriptome of HGSOC cells. Viability assays with HGSOC cells and PDOs were performed to assess the efficacy of THZ531 as single agent or in combination with clinically relevant drugs.ResultsThe CDK12 and CDK13 genes are deregulated in HGSOC and their concomitant up-regulation with the oncogene MYC predicts poor prognosis. HGSOC cells and PDOs display high sensitivity to CDK12/13 inhibition, which synergizes with drugs in clinical use for HGSOC. Transcriptome analyses revealed cancer-relevant genes whose expression is repressed by dual CDK12/13 inhibition through impaired splicing. Combined treatment with THZ531 and inhibitors of pathways regulated by these cancer relevant genes (EGFR, RPTOR, ATRIP) exerted synergic effects on HGSOC PDO viability.ConclusionsCDK12 and CDK13 represent valuable therapeutic targets for HGSOC. We uncovered a wide spectrum of CDK12/13 targets as potential therapeutic vulnerabilities for HGSOC. Moreover, our study indicates that CDK12/13 inhibition enhances the efficacy of approved drugs that are already in use for HGSOC or other human cancers.

Abstract 5512: TIC10/ONC201 in combination with ceralasertib exhibits potent synergy in high grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant form of ovarian cancer. Although patients with HGSOC are likely to respond to treatment, it is common for the cancer to recur and become resistant to treatment, therefore there is a need to develop new targets and novel combinations to increase patient survival rates. ATRi’s (Ataxia telangiectasia and Rad3 related inhibitors) have shown efficacy in HGSOC. Considering ATRi’s have shown moderate efficacy as a single treatment in HGSOC patients, we hypothesized that combining ATRi’s with ONC201 would enhance efficacy. ONC201 is a pro-apoptotic TRAIL-inducing compound that activates the integrated stress response and inactivates the Akt pathway which is upregulated in over 70% of HGSOC. Inactivation of the Akt pathway may result in a synergistic effect that could be translated for use in patients with HGSOC. Three HGSOC cell lines were treated with the novel drug combination of ceralasertib (ATRi) and ONC201 and CellTiterGLO viability assays were performed after 72 hours to evaluate the synergistic effect of combination treatment. Western blots were performed to investigate the synergistic effect of combination treatment on the Akt pathway as well as expression of the cellular metabolic stress protein ATF4. Cytokine profiling using the Luminex 200 technology was performed to study treatment-induced changes in the tumor microenvironment. Cell viability after ceralasertib and ONC201 combination treatment showed synergy in HGSOC cell lines (OVCAR3, KURAMOCHI, TOV21G). In TOV21G cell line, combination studies of ONC201 and ceralasertib calculated by compusyn software demonstrated a synergistic effect of both drugs, Combination indexes below one were observed at concentration of 0.008-2 μM of ceralasertib with 0.31-5 μM of ONC201 with the best combination index of 0.65. We similarly observed synergy in the two other cell line between ONC201 and ceralasertib. Western blotting showed reduction in total Akt and Erk expression and an increase in ATF4, consistent with the observed synergetic effect of ceralasertib and ONC201 combination. Our ongoing studies are exploring potential mechanisms of synergy between ceralasertib and ONC201 and effects on immune–mediated killing of HGSOC cells. Our results suggest a rational and potentially effective novel therapy combination of ATRi ceralasertib and ONC201 that maybe further developed for HGSOC. Citation Format: Maryam Ghandali, Kadir Mert Selvi, Kelsey E. Huntington, Andrew George, Anna Ochsner, Benedito A. Carneiro, Donn S. Dizon, Wafik S. El-Deiry. TIC10/ONC201 in combination with ceralasertib exhibits potent synergy in high grade serous ovarian cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5512.

ATF6-Mediated Signaling Contributes to PARP Inhibitor Resistance in Ovarian Cancer.

This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.

Single-cell analysis of a high-grade serous ovarian cancer cell line reveals transcriptomic changes and cell subpopulations sensitive to epigenetic combination treatment.

Ovarian cancer (OC) is a lethal gynecological malignancy with a five-year survival rate of only 46%. Development of resistance to platinum-based chemotherapy is a common cause of high mortality rates among OC patients. Tumor and transcriptomic heterogeneity are drivers of platinum resistance in OC. Platinum-based chemotherapy enriches for ovarian cancer stem cells (OCSCs) that are chemoresistant and contribute to disease recurrence and relapse. Studies examining the effect of different treatments on subpopulations of HGSOC cell lines are limited. Having previously demonstrated that combined treatment with an enhancer of zeste homolog 2 inhibitor (EZH2i) and a RAC1 GTPase inhibitor (RAC1i) inhibited survival of OCSCs, we investigated EZH2i and RAC1i combination effects on HGSOC heterogeneity using single cell RNA sequencing. We demonstrated that RAC1i reduced expression of stemness and early secretory marker genes, increased expression of an intermediate secretory marker gene and induced inflammatory gene expression. Importantly, RAC1i alone and in combination with EZH2i significantly reduced oxidative phosphorylation and upregulated Sirtuin signaling pathways. Altogether, we demonstrated that combining a RAC1i with an EZH2i promoted differentiation of subpopulations of HGSOC cells, supporting the future development of epigenetic drug combinations as therapeutic approaches in OC.

Abstract 1839: Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition

Abstract High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, the rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. Western blot analysis of underlying molecular mechanisms revealed activation by NFV of the three signaling arms of the unfolded protein response (UPR): PERK, IRE1alpha and ATF6 - in a similar manner to the classical endoplasmic reticulum (ER) stressor tunicamycin. NFV-mediated modulation of the UPR was accompanied by the inhibition of global protein synthesis as analyzed through a non-radioactive method by labelling the nascent polypeptides with puromycin. NFV maintained a proapoptotic environment through the UPR, evidenced by a dose- and time-dependent enhanced expression of ATF4, CHOP, increased Bax/Bcl2 ratio, and cleavage of executioner caspase-7. Enhanced gamma-H2AX suggested NFV-mediated induction of DNA damage, which was associated with decreased proliferation signals driven by the AKT and ERK pathways. NFV increased the level of autophagosome-associated protein LC3II in HGSOC cells of varying platinum sensitivities; however, the autophagic flux was not increased during co-treatment with NFV and the lysosomal inhibitor bafilomycin A1, suggesting a likely impairment of the lysosomes by NFV impeding autophagosome clearance. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Prisca Bustamante, Michael A. Lisio, Julia V. Burnier, Carlos M. Telleria. Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition [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 1839.

Abstract 4066: Anticancer effects of the gold complex auranofin on high-grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) is the 11th most common cancer-causing death in women. HGSOC is treated with platinum (Pt)-based chemotherapy, to which patients develop resistance over time, leaving them with very limited therapeutic options. Thus, new treatments are needed for these patients. In the present work, we provide evidence for the in vitro anti-HGSOC efficacy of the gold complex auranofin (AUF), a compound approved in the clinic in the 1980’s as an anti-rheumatic agent. AUF targets thioredoxin reductase 1 (TrxR1), which is overexpressed in many cancers, including ovarian cancer. Using the Kaplan-Meir plotter database we analyzed that high expression of TrxR1 predicted poor overall survival, suggesting that TrxR1 plays an important role in ovarian cancer progression. We show that AUF impairs the growth of HGSOC cells regardless of Pt sensitivity. HGSOC cells obtained from the same patient when it was clinically sensitive (PEO1) or resistant (PEO4) to Pt-based chemotherapy, were both equally sensitive to the growth inhibition induced by the gold complex in a concentration-dependent manner when using an MTT reduction assay. The toxicity of AUF against HGSOC cells was further studied exposing PEO1 cells to the compound and assessing cell number, cell viability, and cell cycle traverse. Results showed that AUF blocked cell proliferation, reduced viability, and caused accumulation of cells with hypo-diploid DNA content, suggesting a likely apoptosis-mediated cell death mechanism. We confirmed that AUF-triggered lethality was driven by apoptosis by measuring the accumulation of Annexin V positive/7-AAD negative cells via flow cytometry. We also found out that the lethality of AUF required the induction of oxidative stress because the effect was prevented by the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine. We further show that AUF-induced ROS production is associated with the generation of DNA damage as denoted by the increase in the phosphorylation of the Ser-139 residue of the histone variant H2AX by western blotting thus signaling a genotoxic endpoint. Finally, we generated evidence that the combination of AUF and the Pt agent cisplatin (CDDP) is more potent than either drug alone in inducing lethality in PEO4 cells isolated from a patient when Pt-resistant. In summary, we provide proof-of-principle that the TrxR1 inhibitor AUF impairs the survival capacity of HGSOC cells regardless of their Pt sensitivities, triggering a program of apoptosis induced by oxidative stress that is associated with DNA damage; such toxicity is enhanced when AUF is combined with CDDP. The novelty of this work for cancer research in general and HGSOC in particular is that, as AUF is clinically approved to treat arthritis-related conditions, the compound can be rapidly repositioned as a consolidation therapy following Pt-based standard of care and/or overcome Pt resistant disease. Citation Format: Farah H. Abdalbari, Alicia A. Goyeneche, Elvis M. Jaramillo, Siham Sabri, Carlos M. Telleria. Anticancer effects of the gold complex auranofin on high-grade serous ovarian cancer cells [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 4066.

Abstract 693: TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer

Abstract We have assessed the utility of 3-dimensional (3D) in vitro human cell models to understand barriers to chimeric antigen receptor (CAR)-T cell activity in high-grade serous ovarian cancer, (HGSOC) a disease with a poor response to immunotherapy. We defined mucin-1 (MUC1) as a potential target in HGSOC biopsies and the HGSOC cell lines, OvCAR3 and G164. We then generated CAR-T cells against MUC1 and tested them in spheroid and collagen gel cultures. In spheroids, although CAR-T cells killed OvCAR3 cells, G164 cells failed to induce CAR-T cell activation or cytotoxicity. However, when we added primary omental fibroblasts from ovarian cancer patients to G164 spheroids, CAR-T cells were activated and cytotoxic. Fibroblast conditioned medium also activated CAR-T cells to kill G164 cells in spheroids and this was due to their production of C-C motif chemokine ligand 2 (CCL2). Further experiments revealed that CCL2 produced by fibroblast stimulated CCR2/4 positive CAR-T cells to a higher state of activation, which enhanced the cytotoxicity of CAR-T cells against G164 cells. We then investigated CAR-T cell activity in co-cultures of OvCAR3 or G164 cells and primary fibroblasts embedded in collagen. CAR-T cells migrated into OvCAR3 gels and killed the malignant cells during a three-day period. However, CAR-T cells failed to migrate into gels with G164 cells and there was no malignant cell killing. Gels containing G164 cells had denser extracellular matrix (ECM) than OvCAR3 gels, as measured by staining for collagens and fibronectin. Previously, we showed that transforming growth factor-beta (TGFβ) secreted by HGSOC cells acted on fibroblasts to induce the production of ECM in collagen gels.1 Treating G164 gels with the TGFβ receptor inhibitor SB431542 reduced ECM density, stimulated CAR-T cell migration and restored CAR-T cell cytotoxicity against G164 cells. Using these different human 3D models we have demonstrated that malignant cell intrinsic factors can cause resistance to CAR-T cells. Sensitivity to CAR-T cell killing can be modulated both positively and negatively by fibroblasts. Targeting ECM along with CAR-T cell therapy might improve the efficiency of CAR-T cells in solid tumors. 1Delaine-Smith et al, iScience, 2021 Citation Format: Joash Dominic Joy, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Calleja, Sara Brett, Takis Athanasopoulos, Sadfer Ali, Peter Emery-Billcliff, Ida Ricciardelli, Charlotte Kay, Jayne Colebrook, Magda Ali, Katherine Strong, Frances Balkwill. TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer [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 693.

Abstract 5081: Proteomic analysis of ascites- and cancer cell-derived EVs for identifying ovarian cancer diagnostic biomarkers

Abstract Ovarian cancer is the most lethal gynecologic cancer in the world. Most patients are diagnosed at advanced stages with the formation of ascites. Extracellular vesicles (EVs) have been elucidated to play a pivotal role in cancer development and progression, also emerging as promising resources for clinical biomarkers in liquid biopsy. In this study, we aim to identify cancer-specific protein biomarkers in ascites-derived EVs for ovarian cancer diagnosis. Malignant ascites from high grade ovarian cancer patients (HGSOC) and benign peritoneal fluids from female patients with benign gynecologic diseases were collected with informed consent. EVs were isolated from these biofluids using the Exoquick kit. Meanwhile, HGSOC cell lines and normal cell lines were cultured in conditioned media supplemented with EVs-depleted FBS. EVs were isolated from the culture media. Proteins were extracted from EVs and fragmented into peptides. Tandem mass tag (TMT) hyper multiplex quantitation assay was carried out to detect the EV proteomic profiling. EVs isolated from biofluids and cell culture media were identified as double-membrane nanoparticles sizing ranging from 30nm to 200nm. Proteomic analysis of biofluids-derived EVs showed that malignant ascites-derived EVs displayed different proteomic profiling compared to benign peritoneal fluids-derived EVs. The differently expressed proteins between malignant ascites-derived EVs and benign peritoneal fluids-derived EVs were defined as candidate EV protein biomarkers. In addition, proteins that were expressed in at least one cancer cell line-derived EVs but not expressed in normal cell line-derived EVs were also identified as ovarian cancer-specific proteins. A diagnostic model constructed based on the integrative analysis of candidate EV protein biomarkers and ovarian cancer-specific proteins yielded a good discrimination ability between benign and cancer patients.In conclusion, this study demonstrates the distinct proteomic profiling in ascites- and cancer cell-derived EVs in ovarian cancer, which sheds new light on implementing EV proteins as a potential diagnostic strategy in clinical settings. Citation Format: Wenyu Wang, Dohyun Han, Yong Sang Song. Proteomic analysis of ascites- and cancer cell-derived EVs for identifying ovarian cancer diagnostic biomarkers [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 5081.