OVCAR-3 Cells Research Articles

ESM1 enhances fatty acid synthesis and vascular mimicry in ovarian cancer by utilizing the PKM2-dependent warburg effect within the hypoxic tumor microenvironment

BackgroundThe hypoxic tumor microenvironment is a key factor that promotes metabolic reprogramming and vascular mimicry (VM) in ovarian cancer (OC) patients. ESM1, a secreted protein, plays an important role in promoting proliferation and angiogenesis in OC. However, the role of ESM1 in metabolic reprogramming and VM in the hypoxic microenvironment in OC patients has not been determined.MethodsLiquid chromatography coupled with tandem MS was used to analyze CAOV3 and OV90 cells. Interactions between ESM1, PKM2, UBA2, and SUMO1 were detected by GST pull-down, Co-IP, and molecular docking. The effects of the ESM1-PKM2 axis on cell glucose metabolism were analyzed based on an ECAR experiment. The biological effects of the signaling axis on OC cells were detected by tubule formation, transwell assay, RT‒PCR, Western blot, immunofluorescence, and in vivo xenograft tumor experiments.ResultsOur findings demonstrated that hypoxia induces the upregulation of ESM1 expression through the transcription of HIF-1α. ESM1 serves as a crucial mediator of the interaction between PKM2 and UBA2, facilitating the SUMOylation of PKM2 and the subsequent formation of PKM2 dimers. This process promotes the Warburg effect and facilitates the nuclear translocation of PKM2, ultimately leading to the phosphorylation of STAT3. These molecular events contribute to the promotion of ovarian cancer glycolysis and vasculogenic mimicry. Furthermore, our study revealed that Shikonin effectively inhibits the molecular interaction between ESM1 and PKM2, consequently preventing the formation of PKM2 dimers and thereby inhibiting ovarian cancer glycolysis, fatty acid synthesis and vasculogenic mimicry.ConclusionOur findings demonstrated that hypoxia increases ESM1 expression through the transcriptional regulation of HIF-1α to induce dimerization via PKM2 SUMOylation, which promotes the OC Warburg effect and VM.

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer

BackgroundOvarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown.MethodsThe expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols.ResultsMSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells.ConclusionsCollectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

A Multi-Faceted Analysis Showing CRNDE Transcripts and a Recently Confirmed Micropeptide as Important Players in Ovarian Carcinogenesis.

CRNDE is considered an oncogene expressed as long non-coding RNA. Our previous paper is the only one reporting CRNDE as a micropeptide-coding gene. The amino acid sequence of this micropeptide (CRNDEP) has recently been confirmed by other researchers. This study aimed at providing a mass spectrometry (MS)-based validation of the CRNDEP sequence and an investigation of how the differential expression of CRNDE(P) influences the metabolism and chemoresistance of ovarian cancer (OvCa) cells. We also assessed cellular localization changes of CRNDEP, looked for its protein partners, and bioinformatically evaluated its RNA-binding capacities. Herein, we detected most of the CRNDEP sequence by MS. Moreover, our results corroborated the oncogenic role of CRNDE, portraying it as the gene impacting carcinogenesis at the stages of DNA transcription and replication, affecting the RNA metabolism, and stimulating the cell cycle progression and proliferation, with CRNDEP being detected in the centrosomes of dividing cells. We also showed that CRNDEP is located in nucleoli and revealed interactions of this micropeptide with p54, an RNA helicase. Additionally, we proved that high CRNDE(P) expression increases the resistance of OvCa cells to treatment with microtubule-targeted cytostatics. Furthermore, altered CRNDE(P) expression affected the activity of the microtubular cytoskeleton and the formation of focal adhesion plaques. Finally, according to our in silico analyses, CRNDEP is likely capable of RNA binding. All these results contribute to a better understanding of the CRNDE(P) role in OvCa biology, which may potentially improve the screening, diagnosis, and treatment of this disease.

Abstract LB421: Entinostat alters cell cycle in ovarian cancer cells

Abstract Introduction: We have reported that Class I/II HDACi sensitize PARPi resistant tumors, creating a possibility to expand the use of PARPi beyond homologous recombination deficient tumors. However, the underlying mechanism is still unknown. Here, we report leads obtained from differential gene expression in ovarian cancer cells treated with entinostat and olaparib. Methods: OVCAR3 cells were treated with 0.5 µM entinostat and 10 µM olaparib (alone and combined) for 6h, followed by RNA Seq. Cell cycle was analyzed by flow cytometry in SKOV-3 cells treated with olaparib and entinostat (alone and combined) for 24h, 48h and 72h. Results: RNA-Seq analysis showed reduced expression of genes that regulate G2/M checkpoint progression with both entinostat and olaparib. This correlated with flow cytometry results in SKOV-3 cells, where we observed accumulation of G2/M phase cells when treated with entinostat and olaparib. At 24h post treatment, olaparib and entinostat treated cells had higher population of cells in G2/M phase compared to control (18.5% and 16.7% respectively compared to 14.7% in control). Olaparib combined with entinostat further increased G2/M population to 21.7%. At 48h olaparib treated cells had 17.4% cells in G2/M compared to 14% in control. Entinostat treated cells had 18% G2/M cells. Combination treated cells had 19.7% G2/M cells. At 72h, olaparib and control had similar number of G2/M cells (9.2% and 9.7% respectively). Entinostat treated cells had 12% G2/M cells and combination treated cells had 13.6% cells at G2/M cells. This indicates that entinostat and olaparib disrupt progression of cells from G2/M phase, potentially leading to cell death before they can enter mitosis/cell division. Cell cycle analysis in OVCAR3 cells is in progress. Conclusion: Entinostat and olaparib cause G2/M cell cycle arrest in ovarian cancer cells. Entinostat maintains G2/M cell cycle arrest longer than olaparib, potentially extending the window of efficacy of the combination therapy. Validation of G2/M markers and other gene targets/pathways is in progress. Citation Format: Vijayalaxmi G. Gupta, Heng Liu, Rugang Zhang, Michael Onken, Andrew Wilson, Fiona Yull, Marta Crispens, Mary Mullen, Dineo Khabele. Entinostat alters cell cycle in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB421.

Anti-cancer effects of alpha lipoic acid, cisplatin and paclitaxel combination in the OVCAR-3 ovarian adenocarcinoma cell line.

Ovarian cancer is the leading cause of gynecological cancer deaths. One of the major challenges in treating ovarian cancer with chemotherapy is managing the resistance developed by cancer cells to drugs, while also minimizing the side effects caused by these agents In the present study, we aimed to examine the effects of a combination of alpha lipoic acid (ALA), with cisplatin and paclitaxel in ovarian cancer(OVCAR-3). The cytotoxic effects of ALA, cisplatin and paclitaxel on OVCAR-3 cells were determined. Four groups were formed: Control, ALA, Cisplatin + Paclitaxel, ALA + Cisplatin + Paclitaxel. The effects of single and combined therapy on cell migration, invasion and colony formation were analyzed. Changes in the expression of genes related to apoptosis, cell adhesion and cell cycle were analyzed with Real-time polymerase chain reaction(RT-PCR). The oxidative stress index and The Annexin V test were performed. The reduction in rapamycin-insensitive companion of mTOR(RICTOR) expression in the ALA + Cisplatin + Paclitaxel group was found statistically significant(p < 0.05). The decrease in MMP-9 and - 11 expressions the ALA + Cisplatin + Paclitaxel group was statistically significant(p < 0.05). The lowest values for mitogen-activated protein kinase(MAPK) proteins were found in the ALA + Cisplatin + Paclitaxel group. No colony formation was observed in the Cisplatin + Paclitaxel and ALA + Cisplatin + Paclitaxel groups. The lowest wound healing at 24h was seen in the ALA + Cisplatin + Paclitaxel group. This study is the first one to investigate the combined treatment of ALA, Cisplatin, Paclitaxel on OVCAR-3. While ALA alone was not effective, combined therapy with ALA, has been found to reduce cell invasion, especially wound healing in the first 24h, along with tumor cell adhesion.

GLUT3 transcriptional activation by ZEB1 fuels the Warburg effect and promotes ovarian cancer progression

Ovarian cancer (OvCa) is characterized by early metastasis and high mortality rates, underscoring the need for deeper understanding of these aspects. This study explores the role of glucose transporter 3 (GLUT3) driven by zinc finger E-box-binding homeobox 1 (ZEB1) in OvCa progression and metastasis. Specifically, this study explored whether ZEB1 promotes glycolysis and assessed the potential involvement of GLUT3 in this process in OvCa cells. Our findings revealed that ZEB1 and GLUT3 were excessively expressed and closely correlated in OvCa. Mechanistically, ZEB1 activates the transcription of GLUT3 by binding to its promoter region. Increased expression of GLUT3 driven by ZEB1 dramatically enhances glycolysis, and thus fuels Warburg Effect to promote OvCa progression and metastasis. Consistently, elevated ZEB1 and GLUT3 expression in clinical OvCa is correlated with poor prognosis, reinforcing the profound contribution of ZEB1-GLUT3 axis to OvCa. These results suggest that activation of GLUT3 expression by ZEB1 is crucial for the proliferation and metastasis of OvCa via fueling glycolysis, shedding new light on OvCa treatment.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient...

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p&amp;lt;0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p&amp;lt;0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p&amp;lt;0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p&amp;lt;0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p&amp;lt;0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of 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 5909.

Abstract 6995: Functional characterization of the novel cell adhesion molecule MPZL3 in ovarian cancer

Abstract Ovarian cancer (OVCA) is the most lethal gynecological malignancy in the United States often detected at advanced stages. OVCA progression involves transcoelomic metastasis, where cells disseminate into the peritoneal fluid, adhere to form multicellular aggregates that promote anchorage-independent survival and facilitate metastatic colonization of the peritoneum. To meet the requirements of each stage of this detachment, aggregation, and re-attachment cycle, OVCA cells have been shown to dynamically regulate the expression of multiple cell adhesion molecules (CAMs) throughout their progression. Moreover, targeting cell-cell adhesion molecules has shown to be an effective method to slow/inhibit the progression of OVCA. In this study, we characterize the novel role of the understudied CAM MPZL3 in OVCA. Myelin protein zero-like 3 (MPZL3) is a transmembrane protein with homology to other immunoglobulin-like (Ig) family of CAMs. While it has been reported that altered Ig-CAM expression plays a role in ovarian cancer, the function of MPZL3 has not been investigated. Interestingly, TCGA data demonstrates that chromosomal loss of the MPZL3 locus (11q23.3) is frequently detected in several cancer types, including 22% of clear cell and 30% of high grade serous ovarian cancers (HGSOC), implying that loss of genes located in this area has tumorigenic consequences. On the other hand, few studies showed that MPZL3 contains an oncogenic potential in breast and MET-amplified cancer cell lines, suggesting that the role of MPZL3 in cancer is context dependent. To study the function of MPZL3 in OVCA, we used shRNA mediated MPZL3 knockdown in OVCAR4 human HGSOC cells and examined transcriptome-wide effects by RNA-sequencing. The subsequent Gene Set Enrichment Analysis indicated that pathways involved in regulating cell growth, death, adhesion, and epithelial-mesenchymal transition were significantly altered by loss of MPZL3. To validate the candidate pathways, we knocked down or over-expressed MPZL3 in OVCA cells to test its importance in regulating OVCA cell growth, apoptosis, adhesion, and migration. We found that loss of MPZL3 resulted in decreased cell growth with a concomitant resistance to both Cisplatin and Olaparib treatments, both of which are commonly used for treating OVCA. Moreover, we show that knockdown of MPZL3 alters the homotypic adhesive-capacity of OVCA cells. Ongoing studies are underway to further characterize the role played by MPLZ3 in the mentioned contexts. Additionally, to elucidate the underlying molecular mechanism and identify novel interactors of MPZL3, we will conduct co-immunoprecipitation experiments in OVCA cells expressing V5-tagged MPZL3. Understanding the novel role of MPZL3 will provide further insight into OVCA progression, thus generating opportunities of developing new treatments for patients with low MPZL3 expression as an approach for precision cancer medicine. Citation Format: Ya-Yun Cheng, Beth Worley, Zaineb Javed, Amal T. Elhaw, Priscilla W. Tang, Nadine Hempel. Functional characterization of the novel cell adhesion molecule MPZL3 in 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 6995.

Abstract 565: PDE7 modulates EMT, cells morphology and mitochondrial cristae in high grade serous ovarian cancer

Abstract Background: Due to the acquisition of chemoresistant phenotype by epithelial ovarian cancer (EOC) cells and, consequently, disease relapse, the identification of novel therapeutic strategies is urgent. Herein, we targeted phosphodiesterase 7A (PDE7A) in EOC cells, based on data derived from RNA-sequencing assays comparing high-grade serous ovarian carcinoma (HGSOC) and fallopian tube samples. Methods: To understand the role of PDE inhibition, we evaluated the effects of its inhibitor BRL50481 alone or in combination with paclitaxel (PTX) on untreated A2780 EOC cells and multi-resistant high grade serous ovarian cancer (HGSOC) OVCAR3 cells. To do so, we pursued with the: validation of data obtained by high throughput RNA sequencing assays using qRT-PCR; evaluation of PDE7 expression in normal and EOC cells, as well as in normal Fallopian tube cells by qRT-PCR; immunoblotting; metabolic cell viability (MCV) by MTT assays; cytokine dosage by qRT-PCR and ELISA; scanning electron microscopy; transmission electron microscopy; statistical analysis (GraphPad software). Results: Drug-sensitive A2780 cells, but not OVCAR3 cells, exhibited lower MCV when treated with the PDE7 inhibitor; however, the combinatory therapy decreased MCV in both cell lines. Of clinical relevance, pre-treatment of A2780 and OVCAR3 with the PDE7 inhibitor BRL50481 followed by PTX lowered its IC50 by 103- and 625-fold, respectively. While the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT)/mammalian target of rapamycin (mTOR) pathway was inhibited in both cell lines, the pro-apoptotic protein Bcl-2 Associated X-protein (BAX) was overexpressed in A2780 cells only. Analysis of mRNA and protein expression in OVCAR3 pretreated with BRL50481 showed a 2-fold increase in IL-6 cytokine expression. Pre-treatment of OVCAR3 with BRL50481 followed by the combination of BRL50481 and PTX inhibited the expression of vimentin and Octamer-binding transcription factor 4 (OCT4) by 2-fold. BRL50481 promoted changes in EOC cells morphology and mitochondrial cristae. Conclusions: Altogether, our data led us to postulate that inhibition of PDE7 in EOC cells, specifically in those presenting the chemoresistant phenotype, is a promising strategy to fight this yet highly lethal disease. Citation Format: Leticia B. A. Rangel, Nayara Gusmão Tessarollo, Isabella dos Santos Guimarães, Diandra Zipinotti dos Santos, Taciane Barbosa Henriques, Marcele Lorentz de Souza, Leide Laura Figueiredo Maciel, João Carlos Aquino Almeida, Tatiana Massariol Pimenta, Josiany Carlos de Souza, Ranna Batista Wanzeler, Barbara Silva Martins, Solenny Maria Silva Butzene, Jose Matheus Simões Padilha. PDE7 modulates EMT, cells morphology and mitochondrial cristae 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 565.

Abstract 7557: Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in ovarian cancer

Abstract Background: Ovarian cancer (OC) is a lethal disease due to limited treatment options and the development of multidrug resistance. Bispecific T-cell engager (BiTE) therapy represents a promising immunotherapeutic approach to OC. Our previous findings suggest that resistance to BiTE therapy in OC is a complex interplay of factors including loss of tumor-associated antigens and epithelial-mesenchymal transition (EMT). Since the peritoneum is a relatively hypoxic (HX) tumor microenvironment, we hypothesized that HX plays a role in the modulation of MUC16 and potential resistance to BiTE therapy. Methods: After informed consent, samples were collected from patients with recurrent OC undergoing treatment with MUC16 BiTEs. For HX studies, we used OVCAR3 tumor cell lines which express full-length MUC16. Tumor cells were cultured under normoxic (NX) or HX conditions. The effect of HX on cell death was analyzed using an apoptosis assay. Cells were evaluated for changes in cell surface MUC16 expression by flow cytometry. MUC16 protein expression and EMT markers were analyzed by western blot. Proteasome inhibitors were used to stabilize MUC16 expression under HX conditions. Antibody internalization assays were conducted to assess MUC16 endocytosis and recycling. Results: In patients who progressed on MUC16 BiTEs, we found decreased MUC16 expression on tumor samples isolated from ascites samples. Using OVCAR3 cells, we found significantly decreased MUC16 expression on ovarian cancer cells grown under HX conditions (Fold change: 0.4 vs NX, p&amp;lt;0.001). Flow cytometry analysis revealed a time-dependent loss of MUC16 expression on the cell surface under HX (64.22% after 9 days HX vs 88.26% NX), highlighting the susceptibility of MUC16 to HX-induced downregulation. Treatment with proteasome inhibitors partially rescued MUC16 expression under HX conditions (Fold change: 1.4 vs NX, p&amp;lt;0.001). Notably, HX did not induce necrotic cell death in these tumor cells. In addition to the loss of MUC16, tumor cells under HX conditions showed alterations in EMT markers. E-cadherin was downregulated with prolonged HX exposure (Fold change: 0.3 vs NX, p &amp;lt; 0.001), while Vimentin (Fold change: 7.1 vs NX, p &amp;lt; 0.001) and Snail (Fold change: 2.8 vs NX, p &amp;lt; 0.001) were upregulated, indicating a potential shift towards a more aggressive phenotype. Epidermal growth factor receptor (EGFR) was also upregulated (Fold change: 4.9 vs NX, p &amp;lt; 0.001), potentially contributing to enhanced survival and invasion of OC cells under HX stress. Finally, under HX conditions, MUC16 internalization was slower in response to antibody binding over time due to reduced cell antigen expression, corroborating the observed MUC16 loss. Conclusion: Understanding hypoxia-induced MUC16 downregulation and its downstream effects on EMT markers and CA125 expression provides crucial insight into the mechanisms of BiTE therapy resistance in ovarian cancer. Citation Format: Mengyao Xu, Syem K. Barakzai, Artem Kononenko, Raj Kumar, Irva Veillard, Eugene Kim, Timothy S. Bond, David R. Spriggs, Bo R. Rueda, Oladapo Yeku. Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in 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 7557.

Abstract 3337: Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors

Abstract KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX-21020, inhibits KIF18A ATPase activity with an IC50 of 14.5 nM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 &amp;gt; 10 µM) and EG5 (IC50 5.87 µM). ATX-21020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 53.3 nM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX-21020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX-21020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX-21020 treatment, and Annexin V, phospho-Histone H3, and gamma-H2AX are not induced. In sensitive cell lines, ATX-21020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX-21020 exhibit fragmented nuclei and malformed mitotic spindles, in contrast to A2780 cells where cell division is unaffected, suggesting that KIF18A is dispensable in CIN negative cells. This selective dependency is also observed in vivo, where administration of ATX-21020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX-21020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors [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 3337.

Abstract 443: Unsaturated fatty acids promote cell cycle progression and proliferation of platinum resistant ovarian cancer cells

Abstract Introduction: Ovarian cancer (OC) depends on fat as fuel for metastasis and growth. We have previously demonstrated that cisplatin resistant (Pt-R) OC cells uptake higher amounts of fatty acids (FAs) compared to sensitive (Pt-S) cells- a process which facilitates cancer cell survival under cisplatin-induced oxidative stress. Here we aimed to determine which class of FAs promotes the Pt-R phenotype and to identify the mechanism through which FAs support cancer survival. Materials and methods: We used two isogenic OC cell lines pairs, sensitive and resistant to cisplatin: OVCAR5 Pt-S (IC50 = 2.881μM) vs Pt-R (IC50 = 6.831 μM) and PEO1 (IC50 = 4.901 μM) vs PEO4 (IC50 = 8.285 μM). Cells were cultured under low serum conditions, supplemented with 50 µM oleic (OA) or 50 µM palmitic acid (PA), and used for viability assays, RNA-Seq, and cell cycle analysis. Quantitative RT-PCR measured expression of key FAs transporters in Pt-S and Pt-R cells. Results: A viability assay showed that Pt-R cells were less viable under serum depletion compared to Pt-S OC cells. OA (unsaturated), but not PA (saturated), promoted proliferation of both OC cell lines, and rescued Pt-R vs Pt-S cells; 268.4% vs 91.6% for OVCAR-5 and 38.1% for PEO4 vs 12.1% for PEO1, suggesting that Pt-R cells depend more on unsaturated FAs compared to Pt-S cells. Quantification of key FAs transporters demonstrated that Pt-R cell expressed higher levels of FABP4, FABP5 and CD36 compared to Pt-S cells. Treatment with an FABP inhibitor reduced the Pt IC50 in Pt-R cells (from 11.893 μM to 5.0415 μM in OVCAR5 and from 8.285 μM to 6.968 μM in PEO4). RNA-Seq analysis analysed transcriptomic changes induced by 24 hour treatment with OA in OVCAR5 cells. There were 923 differentially expressed protein-coding genes, with 300 upregulated and 623 downregulated transcripts (FDR&amp;lt;0.05). Pathway analysis revealed cell cycle related pathways like: cell cycle mitotic, cell cycle checkpoints, G2/M checkpoints among the top 10 most significantly upregulated pathways and E2F1 was identified as the main effector. Cell cycle analysis confirmed that OA induced increased percentages of cells in S- and G2/M phases - from 6.95% to 11.6% (p&amp;lt;0.05), and 13.6 to 17.6% (P&amp;lt;0.05), respectively. Treatment with an E2F1 inhibitor significantly reduced the OA-induced cell proliferation. Conclusion: Pt-R OC cells are dependent on unsaturated FAs compared to Pt-S cells and upregulate key transporters to allow increased uptake of FAs. OA supports proliferation of Pt-R cells and cell cycle progression from G1 to S- and G2/M phases. Ongoing experiments focus on key mechanisms activated by unsaturated FAs in Pt-R cells. Citation Format: Ana Maria Isac, Guangyuan Zhao, Horacio Cardenas, Daniela Matei. Unsaturated fatty acids promote cell cycle progression and proliferation of platinum resistant ovarian cancer cells [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 443.

Abstract 4671: Efficacy and mechanism of mebendazole for ovarian cancer therapy

Abstract Introduction: Ovarian cancer (OvCa) is the most lethal gynecological malignancy. Although most OvCa patients initially respond to frontline platinum-based therapies with approximately 80% of patients experiencing remission, approximately 75% of those women experience a recurrence with only approximately 50% of patients alive 5 years following diagnosis. There is a critical need to develop novel therapeutic agents or methods to overcome chemoresistance and toxicities experienced with conventional chemotherapy regimens used for OvCa patients. Repurposing drugs is a promising strategy to safely and more rapidly evaluate potentially effective treatment regimens. Over the past two decades, Mebendazole (MBZ), an anti-parasitic drug, has gained much attention in oncology due to its favorable biosafety profile and potent anti-cancerous activity seen in several human malignancies. Therefore, we examined the efficiency and mechanism of MBZ against OvCa. Methods: The in vitro efficacy of MBZ was evaluated in cisplatin-resistant (CPR) and parent (WT/sensitive) OVCAR-8 cell lines as well as in ascites samples collected from chemo-resistant OvCa patients by using 2D and/or 3D cell viability assay and invasion assay. The in vivo efficacy of MBZ was assessed in orthotopic animal models, utilizing CPR-OVCAR8 spheroids, and patient-derived xenograft (PDX) animal models with specimens from chemo-resistant ovarian cancer patients. Molecular mechanisms were investigated through western blot and immunofluorescence analysis. Result: Our results confirmed that MBZ efficiently decreased cell viability, spheroid size, and the invasion ability of OvCa cell lines and ascites samples. OvCa cells treated with MBZ demonstrated downregulation of proliferation markers and upregulation of apoptosis markers indicating inhibition of cell proliferation and induction of apoptosis. Mechanistically, MBZ inhibited the Wnt/β-catenin signaling pathway by downregulating the expression and nuclear localization of β-catenin and epithelial-mesenchymal transition (EMT) markers. Preclinical studies showed that MBZ at 50 mg/kg/day dose for 28 days significantly reduced tumor growth in PDXs and orthotopic tumor models without any evidence of toxicity. Conclusion: Collectively, our study strongly supports the therapeutic potential of MBZ against OvCa particularly, which warrants further clinical studies. Citation Format: Rajani Rai, Dhanamjai Penta, Debasish Dey, Doris M Benbrook, Lin Wang, Magdalena Bieniasz, Lauren E Dockery. Efficacy and mechanism of mebendazole for ovarian cancer therapy [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 4671.

Abstract 5906: A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells

Abstract Despite advances in immune and targeted therapies, chemotherapy remains the primary treatment for ovarian cancer (OvCa) patients. Unfortunately, most OvCa patients develop chemotherapy-resistant disease that is inevitably fatal. Quiescence is an important yet poorly understood mechanism underlying chemotherapy resistance. Therefore, we aimed to characterize quiescent ovarian cancer (qOvCa) cells to identify novel therapeutic targets to improve OvCa patient outcomes. Primary patient-derived qOvCa cells and proliferating cells were isolated using a single-cell microfluidics culture device and collected for single-cell RNA-seq. scRNA-seq of qOvCa cells identified (i) downregulation of MBD3 and CHD4, which are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, and (ii) upregulation of proteasome-associated genes. Knockdown of either MBD3 or CHD4 by shRNA, or NuRD complex pharmacologic inhibition with the HDAC inhibitors such as Vorinostat resulted in significant cell growth arrest without cell death. Standard and Fucci reporter-based cell cycle analysis further confirmed that inhibition of the NuRD complex with Vorinostat induced 94.47% cells in the G0 phase (p&amp;lt;0.0001), suggesting a dense quiescent state. Through cell cycle phase maps produced by iterative immunofluorescence of 30 core cell cycle regulators and manifold learning, we found Vorinostat-treated OvCa cells demonstrated a completely different cell cycle structure from control cells, with most of the cells in an extreme G0 phase. Bulk RNA-seq of Vorinostat-treated cells in three OvCa cell lines validated the downregulation of pathways relating to cell cycle, cell division, and DNA replication (LFC&amp;lt;-1, p&amp;lt;0.05), which is consistent with stem cells. Upregulation of several pathways involved in proteostasis were also noted. We therefore performed a targeted drug screen for combinatorial therapy approaches to eradicate qOvCa. We found that Vorinostat combined with the proteasome inhibitor Carfilzomib demonstrated the most profound synergistic cell death (Loewe index synergy score 23-73). Increased proteasome activity was also found in both primary and Vorinostat-induced qOvCa, suggesting the essential role of the proteasome in qOvCa. Tumor xenograft studies confirmed that Vorinostat alone can delay tumor growth (p&amp;lt;0.05) and combinatorial therapy of Vorinostat and Carfilzomib is more efficacious (p=0.0049). Taken together, NuRD complex inhibition induces quiescence. Combination therapy with Vorinostat to induce quiescence combined with proteasome inhibitors resulted in the profound death of therapy-resistant quiescent cells. Our work suggests this is a novel therapeutic approach to eradicate chemoresistant qOvCa cells and increase cure rates. Citation Format: Qi Jiang, Michelle Ertel, Santiago Panesso- Gómez, Sara Sannino, April Sagan, Alexander J. Cole, Stacy C. McGonigal, Betsy Ann Varghese, Wayne Stallaert, Jeffrey L. Brodsky, Hatice U. Osmanbeyoglu, Ronald J. Buckanovich. A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells [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 5906.

Abstract 2782: Characterization of quiescence-associated secretory profile (QuASP) in cancer: A novel defining feature and therapeutic target

Abstract Senescent cells have a unique senescence-associated secretory profile (SASP) and understanding SASP has contributed to numerous advances in knowledge on aging and cancer biology. Quiescence, which is an important yet poorly understood mechanism for chemoresistance, is related but distinct from senescence. Quiescence shares a non-proliferative feature with senescence but is a reversible state in which quiescent cells can reenter the cell cycle. We hypothesized that, analogous to senescent cells, quiescent cells have a distinct quiescence-associated secretory profile (QuASP) and that defining the QuASP will uncover new genes and pathways associated with cancer cell viability and therapeutic resistance. Indeed, suggesting quiescent cells have a unique and biologically important secretome, we found that tumor conditioned medium (TCM) from quiescent ovarian cancer cells (qOvCa) vs. TCM from proliferating cancer cells increased chemoresistance. Similarly, TCM from quiescent cancer cells vs. TCM from proliferating cancer cells suppressed T cell proliferation and INF-gamma/Granzyme B secretion, suggesting a QuASP immunosuppressive effect. RNASeq analysis of qOvCa cells revealed the secreted factor Follistatin (FST) is upregulated in qOvCa and acted as a paracrine pattern to induce chemoresistance. Furthermore, FST was found to be upregulated in peritumoral fluid following chemotherapy and FST levels in patients with OvCa predicted poor prognosis. To identify additional QuASP factors, we used an ER-BIOID labeled system and LC-MS/MS to characterize the secreted proteins from qOvCa induced by serum starvation compared with normal cells in three OvCa cell lines (PT412, PT340, HEY1). Quiescent OvCa cells indeed have a secretome that is highly unique from proliferating cells, with 137/577 upregulated proteins (LFC&amp;gt;1) and 65/577 downregulated proteins (LFC←1 or no detection in quiescence). Several proteins identified as upregulated have been linked to immunosuppression and/or chemoresistance, oncogenesis and cell cycle were selected for validation and further analysis (CLU, VGF, SERPINE1). In conclusion, we find quiescent cells have a unique quiescence associated secretory profile and that the QuASP induces chemoresistance and immunosuppression. As such, this study has important implications for cancer therapy. Citation Format: Qi Jiang, Santiago Panesso-Gómez, Divya Natesan, Matthew L. MacDonald, Ronald J. Buckanovich. Characterization of quiescence-associated secretory profile (QuASP) in cancer: A novel defining feature and therapeutic target [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 2782.

Abstract 488: Modulation of ENPP1 activity and 2'3'-cGAMP degradation in ovarian cancer cell lines via loadable pre-formed lipid nanoparticles

Abstract Ovarian cancer is a complex heterogeneous disease with various subtypes and diverse molecular mechanisms of pathogenesis. Among different forms, epithelial ovarian cancer is the most common, representing up to 90% of total reported cases. Due to the lack of early diagnostic tools, the prognosis is mainly poor, as most cases are typically identified in advanced stages. Thus, a more thorough understanding of the inherent complexities of the disease, in terms of its biochemical etiology, will allow for identification of specific molecular and genetic profiles that could lead to more effective treatment plans. Recent studies have shown that ENPP1 may play a role in cancer cell proliferation, migration, and invasion in ovarian and other types of cancer. ENPP1 is a glycosylated type II transmembrane protein acting as a pyrophosphatase and phosphodiesterase with broad specificity. ENPP1 substrates include mononucleotides and cyclic dinucleotides such as the second messenger, 2'3'-cGAMP, which binds to and activates STING. 2'3'-cGAMP is exported extracellularly by cancerous cells into the tumor microenvironment where uptake into neighboring cells can activate their STING-dependent type 1 interferon pathway and promote antitumoral immune responses. ENPP1 can hydrolyze extracellular 2'3'-cGAMP however, allowing cancer cells to evade such an immune response. In this study, we assessed the activity of ENPP1 in CaOV-3, SK-OV-3, and PA-1 ovarian cancer cell lines using a fluorometric cell-based assay. The effects of ENPP1 inhibition via a chemical inhibitor on extracellular 2'3'-cGAMP levels were characterized and compared with siRNA-mediated knockdown of ENPP1 via loadable pre-formed lipid nanoparticles, which enable small-scale encapsulation and transfection of nucleic acid cargo. In addition, we explored whether uptake of exogenous 2'3'-cGAMP into THP-1 and RAW immune cell lines can be enhanced through delivery via preformed loadable lipid nanoparticles. Corresponding immune cell activation was also assessed using immunoblotting and RT-PCR. Our results demonstrate how modulation of ENPP1 activity and 2'3'-cGAMP levels in ovarian cancer cell lines using unconventional, pre-formed loadable lipid nanoparticle transfection reagent can aid in elucidating mechanisms of immune evasion in cancer cells. Citation Format: David Taylor, Jiajia Ji, Phillip Rzeczycki, Margaret L. Collins, Lauren Clements, Aileen R. Ariosa. Modulation of ENPP1 activity and 2'3'-cGAMP degradation in ovarian cancer cell lines via loadable pre-formed lipid nanoparticles [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 488.

Abstract 3237: KDM1A/LSD1 inhibition enhances chemotherapy response in ovarian cancer

Abstract Background: Ovarian cancer (OCa) is the deadliest of all gynecological cancers. The standard treatment for OCa is platinum-based chemotherapy, such as carboplatin or cisplatin in combination with paclitaxel. Most patients are initially responsive to these treatments; however, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Recent studies have revealed that the epigenetic modifier lysine-specific histone demethylase (KDM1A/LSD1) is highly overexpressed in OCa. However, whether KDM1A plays a role in chemoresistance and whether its inhibition enhances chemotherapy response in OCa is unknown. In this study, we explored the hypothesis that KDM1A inhibition will sensitize OCa cells to standard-of-care chemotherapy. Methods: Transduction of KDM1A-specific shRNA was used to generate KDM1A knockdown (KD) cells. Cell viability, colony formation, and apoptosis assays were used to determine the impact of KDM1A KD on sensitization of SKOV3, OVCAR8, and OVCAR5 OCa cells to cisplatin, carboplatin, and paclitaxel. The combination effect of KDM1A inhibitors (NCD38 and SP2509) and chemotherapy drugs was further determined. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, and Western blotting. In vivo efficacy ofKDM1A knockdown with or without chemotherapy was determined using OCa orthotopic xenograft models. Results: Analysis of TCGA datasets revealed that KDM1A expression is negatively correlated with OCa survival, and its expression is high in patients who do not respond to chemotherapy. Further, Western blotting results show that treatment with chemotherapy drugs cisplatin, carboplatin, and paclitaxel increased KDM1A expression in OCa cells. KDM1A-KD or KDM1A inhibition sensitized OCa cells to chemotherapy drugs in reducing cell viability, survival, and enhancing apoptosis. Moreover, knockdown of KDM1A sensitized carboplatin-resistantA2780-CP70 cells to carboplatin treatment. RNA-seq studies revealed that KDM1A-KD and cisplatin-regulated genes showed negative correction with epithelial-mesenchymal transition (EMT) and positive correlation with apoptosis-related pathways. Importantly, KDM1A KD in combination with cisplatin significantly reduced the tumor growth in the orthotopic OCa xenograft model. Conclusion: These results suggest that the combination of KDM1A inhibitors with chemotherapy is a viable therapeutic approach for the treatment of OCa Citation Format: Jessica D. Johnson, Yihong Chen, Sridharan Jayamohan, Yi He, Diksha Jamwal, Neetesh Pandey, Edward Kost, Ratna K. Vadlamudi, Gangadhara R. Sareddy. KDM1A/LSD1 inhibition enhances chemotherapy response in 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 3237.

Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition

Abstract CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors. Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [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 5705.

Abstract 954: An evaluation of the combination of metformin and Y15 for the treatment of platinum-resistant ovarian cancer

Abstract Ovarian cancer is the fifth leading cause of cancer mortality among women. This high mortality rate is linked to the development of resistance to first-line chemotherapy with platinum compounds which has been attributed in part to increased activity of focal adhesion kinase (FAK). The anti-diabetic drug Metformin was previously shown to inhibit the proliferation and migration of ovarian cancer cells and thus, the combination of a FAK inhibitor, Y15, and Metformin may be a promising treatment for platinum-resistant ovarian cancer (PROC). The objective of this study was to evaluate the combination of Y15 and Metformin on PROC cell viability and the mechanism of cell death. An MTT assay was used to analyze cell viability in PROC OVCAR3 cells after 48 h of treatment by measuring the absorbance at 570 nm with a microplate reader. Western blot was used to determine the protein levels of the apoptosis marker cleaved PARP and the autophagy marker LC3B-II. The exposure of OVCAR3 platinum-resistant ovarian cancer cells to Metformin (4.5 mM) + Y15 (5.5 µM) resulted in a significantly enhanced cytotoxicity (32.6 ± 1.8%) compared to single drug treatment with either Metformin (4.5 mM) (65.0 ± 4.2%) or Y15 (5.5 µM) (66.0 ± 4.8%). A combination of Metformin (7.8 mM) + Y15 (5.5 µM) also resulted in a significantly enhanced cytotoxicity (22.8 ± 1.5%) compared to single drug treatment with either Metformin (7.8 mM) (50.7 ± 3.7%) or Y15 (5.5 µM) (66.0 ± 4.8%). Cells treated with the combination of Metformin and Y15 for 24, 48, and 72-hours showed an increase in cleaved PARP compared to the control, Metformin alone and Y15 alone. For Metformin alone, there was an increase in cleaved PARP compared to the control at all timepoints and increased from 24 hours to 72 hours. For Y15 alone, the amount of cleaved PARP was also higher compared to the control at all time points, however, decreased over time from 24 hours to 72 hours. Cells treated with Metformin, Y15, and a combination of Metformin and Y15 showed no conversion of LC3B-I to LC3B-II, which occurs during autophagy. Thus, it is concluded that the mechanism of cell death for both Metformin and Y15 is through apoptosis and not autophagy and apoptosis is enhanced with the combination of the drugs. The delivery of Metformin and Y15 can result in an additive effect on cell viability through apoptosis and can be further explored as a promising approach for the treatment of PROC. Citation Format: Manuel A. Duarte, Kristal Garcia, Marco A. Arriaga, Sue Anne Chew, Arkene S. Levy, Isabela Mendez. An evaluation of the combination of metformin and Y15 for the treatment of platinum-resistant 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 954.