Clinicopathological and functional evaluation of replication protein A in epithelial ovarian cancers: A target validation study.

Introduction. Drug resistance results in dismal outcomes for cancer patients. Carboplatin is the backbone of treatment for high grade serous ovarian cancer (HGSOC), augmented by PARP inhibitor (PARPi) maintenance therapy for patients with tumors deficient in homologous recombination repair (HRR). However, most tumors evolve resistance to both drugs and no other agents, including immunotherapies, have improved survival. Therapeutic strategies that acknowledge and target this evolution offer an opportunity to preserve drug sensitivity and extend progression-free survival. Adaptive Therapy (AT) is one such approach. AT is based on the premise that fitness costs are incurred as resistance evolves, so that competition between drug-sensitive and resistant cells favors sensitive cells when drug is absent. We have developed AT in HGSOC from proof-of-concept to a randomized phase 2 trial called ACTOv, (Adaptive ChemoTherapy in Ovarian cancer) comparing carboplatin AT to standard carboplatin dosing in patients with relapsed, platinum sensitive high grade serous and endometrioid ovarian cancer. We are now extending our work to explore the evolutionary dynamics between PARPi-sensitive and PARPi-resistant HGSOC populations. Methods. We evolved resistance to cisplatin and carboplatin in HGSOC cell lines in vitro and in vivo via carboplatin treatment of mice with intraperitoneal xenografts. Resistance to cisplatin, carboplatin and the PARPis niraparib and olaparib was quantified by live/dead assays in the entire cell panel. Co-cultures of sensitive and resistant cells were created in vitro in low resource conditions (low serum or low glucose) to expose fitness deficits. Population growth dynamics were quantified over time using the Sartorius Incucyte™ Live Cell Analysis System. Mechanisms underpinning these dynamics were explored by flow cytometry to analyze cell cycle and expression of the standard apoptotic maker, Annexin V. HRR status was determined functionally by immunofluorescence for Rad51 and γH2Ax co-localization and for micronuclei formation, while BRCA expression was examined by western blot. In vivo co-cultures of GFP-expressing sensitive cells and RFP-expressing resistant cells enabled tracking of sensitive/resistant cell growth over time by qPCR. Sensitive/resistant cells were further examined by immunohistochemistry (IHC) in excised tumors. The influence of drug therapy on in vitro co-cultures of sensitive (GFP) and resistant (RFP) HGSOC was quantified by flow cytometry. AT was compared to standard carboplatin dosing in mice with HGSOC xenografts and excised tumors were examined by IHC. Cumulative drug dose and murine survival were compared. Cell-free DNA (cfDNA) and tumor biopsies were obtained from HGSOC patients during carboplatin and PARPi treatment and subjected to low pass whole genome sequencing and analysis with our published bioinformatic pipeline (LiquidCNA) that estimates the size of the emergent resistant population. Finally, established patient datasets were examined to adapt our pre-clinical discoveries for clinical testing in the ACTOv trial. Results. Cell viability assays confirmed platinum resistance. One murine HGSOC cell line with evolved platinum resistance (60577R3) was also resistant to olaparib and niraparib in vitro, whereas there was no difference in PARPi sensitivity in the other sensitive/resistant cell pairs. Fitness deficits of resistance in the absence of drug, were exposed by low resource co-cultures in vitro in four out of five HGSOC cell lines with evolved platinum resistance. Conversely, the resistant subline, 60577R3, exhibited greater fitness than sensitive, ancestral cells (60577). Resistant populations with reduced fitness declined due to both reduced proliferation and increased apoptosis in vitro and in vivo when drug was absent. Rad51 foci formation in response to double strand breaks was observed in platinum and PARPi-resistant 60577R3 cells but not sensitive 60577 indicating that functional HRR had been restored in 60577R3 as resistance evolved. Consistent with this, 60577R3 cells also made fewer segregation errors during mitosis. Western blot confirmed expression of BRCA1 in 60577R3 but not 60577 cells. Platinum treatment of in vitro co-cultures demonstrated that the size of sensitive and resistant HGSOC populations fluctuated with drug therapy and that fitter, sensitive cells re-grew when drug was withdrawn. In vivo, AT significantly prolonged survival of murine ovarian cancer models compared to standard carboplatin dosing without increasing mean daily drug dose or drug-related toxicity. Escape from control by AT was associated with growth of resistant cells in our in vivo models. In HGSOC patients, LiqCNA analysis of cfDNA obtained longitudinally during treatment showed sub-clonal selection through therapy and demonstrated that LiqCNA correlates with increases in the serum tumor marker, CA125, a proxy for disease progression. Existing clinical datasets of CA125 during standard carboplatin treatment defined the parameters for dose adaptations in patients receiving AT in the ACTOv trial. Conclusions. Platinum resistance can be associated with fitness deficits in HGSOC, which can be exploited by AT to preserve platinum sensitivity over the long-term and prolong survival. BRCA reversion mutations are the most well-documented cause of acquired PARPi resistance. Our data imply that this may provide a fitness advantage for HGSOC cells with implications for PARPi maintenance treatment and response to subsequent lines of therapy. These discoveries paved the way for us to launch the ACTOv trial, which is currently recruiting patients in ten NHS hospitals throughout the UK. ACTOv patients will provide biopsies and very frequent blood samples for circulating tumor DNA (total ∼1,400 samples from 80 patients), enabling us to interrogate clonal evolution during standard and adaptive therapy and relate this to clinical outcome. LiqCNA could provide a biomarker for the emergence of drug resistance to direct AT in second generation clinical trials. Citation Format: Mohammed Ateeb Khan, Helen Hockings, Maximilian Mossner, Ann-Marie Baker, Hall Amy, Robert L. Hollis, Weini Huang, Eszter Lakatos, Charlie Gourley, Trevor A. Graham, Michelle Lockley. Translating adaptive therapy to ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr SY43-01.

Objective: There is a critical need for improved diagnostic markers to detect ovarian high grade serous ovarian cancer (HGSOC). MicroRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and present in circulating microvesicles such as exosomes. Recent studies have confirmed the potential use of miRNAs profiling as a novel non-invasive biomarker for diagnosis of HGSOCs. The aim of this study is to identify which miRNAs are highly produced from HGSOCs and analyze whether serum miRNA can discriminate patients with HGSOC from healthy controls. Methods: Secreted exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray was performed and several elevated miRNAs specific to HGSOCs were picked up. Among these, we focused on miR-1290. Serum from 71 pre-operative, 46 post-operative ovarian cancer patients and 13 healthy controls were gathered and its expression levels were detected by quantitative Real Time PCR. Results: In HGSOC patients, miR-1290 emerged overexpressed compared to healthy controls (3.52-fold). ROC analysis showed that at the cut-off of 1.61(healthy controls ; 1), the sensitivity and specificity were 63 % and 85 % respectively for detecting HGSOC (AUC = 0.71). The AUC of CA125 + miR-1290 combination was 1.00. Its expression singnificantly decreased after operation(5.87 → 1.17 ; P < 0.01). In advanced stage HGSOC patients, moreover, it expressed marginally higher than early stage ones(4.23 VS 1.58 ; P = 0.23). Conclusions: Serum miR-1290 can be a potential diagnostic biomarker for HGSOC. Citation Format: Masaki Kobayashi, Sawada Kenjiro, Yoshimura Akihiko, Miyamoto Mayuko, Nakatsuka Erika, Kodama Michiko, Hashimoto Kae, Mabuchi Seiji, Kimura Tadashi. Elevated level of serum miR-1290 is correlated with high-grade serous ovarian epithelial ovarian cancer and might be a potential biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5408.

Background: We examined the association of platinum resistance with genetic mutations in high grade serous ovarian cancer (HSOC) patients undergoing high-throughput genomic tumor sequencing. Methods: Snap-frozen and fresh frozen paraffin embedded tissue samples were collected from HSOC patients enrolled on UNCseq (NCT01457196). UNCSeq is an institutional protocol which uses next generation sequencing to detect genetic mutations in a wide array of malignancies. Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Mutations with a quality score <100 were filtered from the data set, and only mutations rated to have a moderate to high impact were retained. Medical record review determined platinum sensitivity or platinum resistance. Tumors were defined as platinum sensitive or resistant if patients were noted to have > or < 6 months of disease free interval following completion of induction therapy, respectively. Results: Overall 39 HSOC cases met inclusion criteria; 32 tumors met criteria for platinum sensitive and 7 platinum resistant. 308 mutations were noted in at least one individual across all patients. The top observed mutations in platinum sensitive HSOC were TP53 (41%, n= 13), GucylA2 (19%, n=6), MLL2 (19%, n=6) and MTOR (16%, n=5). The top observed mutations in platinum resistant tumors were TP53 (71%, n=5), TET1 (43%, n=3), NF1 (43%, n=3) and MLL3 (43%, n=3). There was a trend toward more p53 mutations in resistant tumors (71% versus 41%, p=0.21) There was no difference in the total number of mutations per tumor in platinum sensitive and resistant patients, (3 vs. 4, p=0.516). Conclusions: We did not detect a difference in the number of genetic mutations in HSOC according to platinum sensitivity. Platinum resistant tumors had a trend toward higher frequency of TP53 mutations than platinum sensitive HSOCs. Furthermore, we identified 3 frequently mutated genes in platinum resistant HSOC: TET1, an epigenetic regulator, NF1, a tumor suppressor gene and MLL3, a histone modifier gene. Ongoing tumor sequencing of HSOCs on UNCseq will help to confirm these results. Citation Format: Tara Castellano, Leslie H. Clark, Naim Rashid, Victoria Bae-Jump. Genetic variation in platinum-sensitive and platinum-resistant high-grade serous epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 412. doi:10.1158/1538-7445.AM2017-412

BackgroundOvarian cancer is a significant public health concern with a poor prognosis for epithelial ovarian cancer. To explore the potential of immunotherapy in treating epithelial ovarian cancer, we investigated the immune microenvironments of 52 patients with epithelial ovarian cancer, including 43 with high-grade serous ovarian cancer and 9 with endometrioid ovarian cancer.ResultsFresh tumor tissue was analyzed for genetic mutations and various parameters related to immune evasion and infiltration. The mean stromal score (stromal cell infiltration) in high-grade serous ovarian cancer was higher than in endometrioid ovarian cancer. The infiltration of CD8 T cells and exhausted CD8 T cells were found to be more extensive in high-grade serous ovarian cancer. Tumor Immune Dysfunction and Exclusion scores, T cell exclusion scores, and cancer-associated fibroblasts (CAF) scores were also higher in the high-grade serous ovarian cancer group, suggesting that the number of cytotoxic lymphocytes in the tumor microenvironment of high-grade serous ovarian cancer is likely lower compared to endometrioid ovarian cancer.ConclusionsThe high mean stromal score and more extensive infiltration and exhaustion of CD8 T cells in high-grade serous ovarian cancer indicate that high-grade serous ovarian cancer exhibits a higher level of cytotoxic T cell infiltration, yet these T cells tend to be in a dysfunctional state. Higher Tumor Immune Dysfunction and Exclusion scores, T cell exclusion scores, and CAF scores in high-grade serous ovarian cancers suggest that immune escape is more likely to occur in high-grade serous ovarian cancer, thus endometrioid ovarian cancer may be more conducive to immunotherapy. Therefore, it is crucial to design immunotherapy clinical trials for ovarian cancer to distinguish between high-grade serous and endometrioid ovarian cancer from the outset. This distinction will help optimize treatment strategies and improve outcomes for patients with different subtypes.

Differentially expressed genes in platinum-resistant high-grade serous ovarian cancer

Platinum resistance remains a major challenge in the chemotherapeutic management of ovarian cancer. The anti-diabetic drug metformin has been previously shown to induce cytotoxicity in platinum resistant ovarian cancer cells and overexpression and increased phosphorylation of a tyrosine kinase called Focal Adhesion Kinase (FAK) has been implicated in the development of this platinum resistance. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Metformin and the focal adhesion kinase inhibitor 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 ranging from 10-100 μM, and metformin from 10-100mM to determine 1C50 values. Subsequently, cells were treated with Y15 (80 μM) and metformin (26mM) alone and in combination. All treatments were triplicated with duration of 24hrs and control cells exposed to media only. The cytotoxic profile of each treatment was assessed using the automated trypan blue assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death and we further evaluated the expression of phosphorylated FAK, p53 and p21 in response to treatments using western blot. Y15 alone produced 48% cell death. In combination, Y15 significantly increased the cytotoxic efficacy of metformin by 22%, when compared to the metformin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, metformin, and metformin +Y15 treatment groups. The Metformin +Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control and this confirmed reduced FAK activity. Reduced FAK auto phosphorylation also correlated with increased expression of p53 AND p21 in metformin and Y15 treatment groups. Our findings show that Y15 significantly enhances the cytotoxic profile of metformin in platinum resistant OVCAR-3 cells. Furthermore, a FAK dependent apoptotic mechanism appears to underlie the cytotoxic effect of metformin as well as Y15 as both drugs significantly reduced the phosphorylation of FAK alone, and in combination. Reduced FAK activity also correlated with increased p53 and p21 expression. This study is the first to report a FAK dependent cytotoxic mechanism of metformin in ovarian cancer and in further work we will evaluate the mechanisms why which metformin cooperates with Y15 to inhibit FAK activity in platinum resistant ovarian cancer. Citation Format: Arkene S. Levy, Zara Khan, Samuel Batko, Keerthi Thallapureddy, Robert Smith, Thanigaivelan Kanagasabai, Julie Torruellas Garcia, Appu Rathinavelu. Evaluation of the cytotoxic profile of Metformin and Y15 in platinum resistant ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2090.

BackgroundEpithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases.MethodsWe explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial–mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan–Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME).ResultsOur proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFβ1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFβ1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME.ConclusionsOur research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.

The United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) recently reported a reduction in the average overall mortality among ovarian cancer patients screened with an annual sequential, multimodal strategy that tracked biomarker CA125 over time, where increasing serum CA125 levels prompted ultrasound. However, multiple cases were documented wherein serum CA125 levels were rising, but ultrasound screens were normal, thus delaying surgical intervention. A significant factor which could contribute to false negatives is that many aggressive ovarian cancers are believed to arise from epithelial cells on the fimbriae of the fallopian tubes, which are not readily imaged. Moreover, because only a fraction of metastatic tumors may reach a sonographically-detectable size before they metastasize, annual screening with ultrasound may fail to detect a large fraction of early-stage ovarian cancers. The ability to detect ovarian carcinomas before they metastasize is critical and future efforts towards improving screening should focus on identifying unique features specific to aggressive, early-stage tumors, as well as improving imaging sensitivity to allow for detection of tubal lesions. Implementation of a three-stage multimodal screening strategy in which a third modality is employed in cases where the first-line blood-based assay is positive and the second-line ultrasound exam is negative may also prove fruitful in detecting early-stage cases missed by ultrasound.

Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.

Glycosylated antitumor ether lipids (GAELs) are a promising class of investigational anticancer agents with potent antitumor activity against a range of cancers, including platinum-resistant high-grade serous ovarian cancer (HGSOC). We previously demonstrated that epithelial ovarian cancer (EOC) cell lines (HGSOC and endometrioid) and primary cells derived from patient ascites (HGSOC and clear cell) were sensitive to the cell-killing effects of D-glucosamine-derived GAELs via an apoptosis-independent mechanism. However, D-linked carbohydrates are metabolized by endogenous glucosidases in vivo, rendering the GAEL inactive. Thus, the aim of the current study was to synthesize a novel class of GAELs with L-linked carbohydrates and to evaluate their efficacy in EOC cell lines and patient samples. Out of seven novel compounds tested, L-rhamnose-GAEL was identified as the compound with the greatest efficacy for killing EOC cell lines and patient cells that were grown under adherent or nonadherent (3D) conditions. The drug-sensitive and drug-resistant syngeneic endometrioid EOC cell lines, A2780s and A2780cp, respectively, as well as the NIH:OVCAR-3 and COV362 HGSOC lines, were incubated with L-rhamnose-GAEL for 48 hours and the CC50 determined as 15 μM for A2780s, 22.5 μM for A2780cp, and 5 μM for NIH:OVCAR-3 and COV362 cells. Similar experiments were conducted with primary EOC cells, chemo-naïve EOC126 (clear-cell adenocarcinoma), and platinum-resistant EOC 183I (HGSOC). Cell viability decreased in a dose-dependent manner with CC50 of 12 μM for EOC126 and 22.5 μM for EOC183l. For comparison, when EOC126 and EOC183I were grown as 3D cultures the CC50for cisplatin was 20 μM and 40 μM, respectively. To evaluate the effect of using low-dose treatment of L-rhamnose-GAEL on primary EOC cells (chemo-naive and chemoresistant), cells were exposed to L-rhamnose-GAEL for 96 hours. These studies revealed that prolonged incubation led to low CC50 of 1-5 μM. These results showed that primary EOC cells derived from chemo-naïve and platinum-resistant patients were sensitive to the L-rhamnose-GAEL and offer a novel drug class capable of killing chemotherapy-resistant EOC cells. Citation Format: Mark W. Nachtigal, Frank Schweizer, Gilbert Arthur. Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A56.

Curcumin, a compound derived from turmeric (Curcuma longa), inhibits the proliferation of multiple cancer cell types, including ovarian, pulmonary, brain, and breast through various mechanisms. In advanced ovarian cancer, focal adhesion kinase (FAK), a tyrosine kinase, is overexpressed and contributes to platinum resistance. The compound Y15 (1, 2, 4, 5-benzene tetra amine tetrahydrochloride) inhibits FAK phosphorylation and subsequent activation. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Curcumin and Y15 in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 (10-100 μM), and curcumin (0-20µM) to determine IC10 values. Cells were subsequently treated with Y15 (20 μM) and curcumin (5 µM) alone and in combination. The cytotoxic profile of each treatment was assessed using the MTT assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death. We further evaluated the expression of total FAK, phosphorylated FAK, p53, p21, and BAX in response to treatments using western blot. Y15 significantly increased the cytotoxic efficacy of curcumin when compared to the curcumin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, curcumin, and curcumin plus Y15 treatment groups. The curcumin plus Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control. Reduced FAK phosphorylation also correlated with increased expression of P53, P21 and BAX in curcumin and Y15 treatment groups. These results indicate that Y15 significantly enhances the cytotoxic profile of curcumin in platinum resistant OVCAR-3 cells. This study is the first to report a FAK dependent cytotoxic mechanism of curcumin in ovarian cancer and in further work we will evaluate the signaling pathways involved. Citation Format: Arkene S. Levy, Appu Rathinavelu, Nicole Coelho, Amanda Ramnot, Thanigaivelan Kanagasabai, Olena Bracho, Robert Smith. Evaluation of the efficacy of curcumin and Y15 in platinum resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4924.

Platinum resistance seriously impacts on the survival outcomes of patients with ovarian cancers. Platinum-induced DNA damage is processed through DNA repair. NBS1 is a key DNA repair protein. Here, we evaluated the role of NBS1 in ovarian cancers. NBS1 expression was investigated in clinical cohorts (protein level (n = 331) and at the transcriptomic level (n = 1259)). Pre-clinically, sub-cellular localization of NBS1 at baseline and following cisplatin therapy was tested in platinum resistant (A2780cis, PEO4) and sensitive (A2780, PEO1) ovarian cancer cells. NBS1 was depleted and cisplatin sensitivity was investigated in A2780cis and PEO4 cells. Nuclear NBS1 overexpression was associated with platinum resistance (p = 0.0001). In univariate and multivariate analysis, nuclear NBS1 overexpression was associated with progression free survival (PFS) (p-values = 0.003 and 0.017, respectively) and overall survival (OS) (p-values = 0.035 and 0.009, respectively). NBS1 mRNA overexpression was linked with poor PFS (p = 0.011). Pre-clinically, following cisplatin treatment, we observed nuclear localization of NBS1 in A2780cis and PEO4 compared to A2780 and PEO1 cells. NBS1 depletion increased cisplatin cytotoxicity, which was associated with accumulation of double strand breaks (DSBs), S-phase cell cycle arrest, and increased apoptosis. NBS1 is a predictor of platinum sensitivity and could aid stratification of ovarian cancer therapy.

5117 Background: Primary therapy for women with advanced stage epithelial ovarian cancer includes primary surgical cytoreduction followed by platinum-based chemotherapy. Although nearly 70% of patients demonstrate a complete clinical response to primary therapy, the majority of patients will develop platinum-resistant recurrent disease. Our understanding of this chemo-resistance remains incomplete. The expression of genes such as c-myc have been shown to be associated with chemoresistance in several human cancers. Recently, using microarray expression analysis of 55 primary epithelial ovarian cancers, we demonstrated that members of the c-myc pathway are predictive of response to primary platinum therapy. We hypothesized that increased c-myc gene expression also underlies platinum resistance in ovarian cancer cell lines, and that it can be reduced by use of a c-myc-targeted small interfering RNA (si-cmyc) approach, thus reversing platinum resistance. Methods: Expression of the c-myc gene was measured in platinum-sensitive (OV2008) mother and platinum-resistant (C13) daughter ovarian cancer cell lines using quantitative real time polymerase chain reaction (QRTPCR). Platinum-resistant cells were then subject to c-myc-targeted small interfering RNA (50nM, 100nM and 150nM), and c-myc gene expression was evaluated using Western blot analysis. Platinum-resistant ovarian cancer cells, subject to successful c-myc expression silencing by siRNA, were then evaluated for platinum-response using the MMT assay. Results: Expression of the c-myc gene was 1.78-fold higher in platinum-resistant versus platinum-sensitive ovarian cancer cells. Western blot analysis demonstrated a reduction of c-myc gene expression by 95% in platinum-resistant ovarian cancer cells subject to c-myc-targeted siRNA at 150nM. Conclusions: These findings support the hypothesis that the c-myc gene pathway is an important determinant of platinum-response in ovarian cancer cells. Further, we have demonstrated that siRNA approaches can selectively silence genes that underlie platinum-response and may thus represent a novel targeted molecular therapeutic approach to patients with platinum-resistant ovarian cancer. No significant financial relationships to disclose.

Background: High-grade serous ovarian (HGSC) cancer is the most common and aggressive histotype of epithelial ovarian cancer in women, accounting for 75% of epithelial ovarian carcinomas. Most women present at advanced stages III/IV at diagnosis and 5-year survival for patients with advanced stage disease is approximately 20-30%. However, for patients with stage I disease, five-year survival rates exceed 90%. Ovarian cancer risk factors include nulliparity, early age of menarche, late age of menopause, hormone replacement, obesity and strong family history of hereditary breast and ovarian cancer (HBOC). It is estimated that about 1 in 500 Americans have a mutation in BRCA1 or BRCA2. In women with a germline mutation there is a lifetime risk of: 40%-60% (BRCA1) and 11%-27% (BRCA2), of developing ovarian cancer. Recent studies of different immigrant populations in the United States and in their respective countries of origin have identified pockets of women who bare a similarly high genetic burden as the Ashkenazi Jewish population. Women of Bahamian and Trinidadian heritage are estimated to have 27.1% of breast cancer cases due to BRCA founder mutations. The ovarian cancer burden in these isolated high-risk populations is still unclear, but likely to be as high as those women of Ashkenazi descent. South Florida has the highest Caribbean immigrant community in the US and nearly 4 out of 10 immigrants in Florida were born in the Caribbean. We sought to elucidate the patient demographic, pathologic, and outcome within a university health system between the safety net hospital - Jackson Memorial Hospital (JMH) and the Sylvester comprehensive cancer center (SCCC). Methods: We conducted a retrospective chart review study of women diagnosed with epithelial ovarian cancer with the high-grade serous carcinoma histotype at Sylvester Comprehensive Cancer Center/Jackson Memorial Hospital between 2011 and 2015. We collected data including date of diagnosis, age, body mass index (BMI), country of birth, ethnicity, germ-line and somatic genetic tests results, medical insurance, treatment and vital status. SPSS software was used to analyze using independent samples t-tests when parametric and using Wilcoxon-Mann-Whitney tests when non-parametric; and Chi-squared tests for categorical and binary data. Results: 374 patients were diagnosed and treated for high-grade serous ovarian cancer (HGSC). 50.8% were non-Hispanic, 47.9% Hispanic and 1.3% Unknown. 12.% of the patient population was Black with a significantly higher BMI (39.1 vs 27.9, p=0.004). Mean age was 58 (Hispanic white 57.7yrs (17-93), non-Hispanic white 58.5yrs (17-93) and Black (53.7yrs (18-78), p=0.019)) compared to US national average of 63. At least 32.3% of patients were born in the US while, 60% of patients were born in Latin America and the Caribbean. 72.3% of patients born outside the US were diagnosed and treated at JMH (p&amp;lt;0.001). In a subset analysis of 102 patients, 31% had genetic or somatic testing, of these, 2 patients were Black, not of Afro-Caribbean descent. Patients at JMH presented with 8% Stage I vs 74.7% Stage III/IV, compared to 16.3% Stage I vs 73% Stage IV at SCCC. They had more advanced stage V - 42.7% vs 16.3% at SCCC. JMH patients were more likely to be on Medicaid or uninsured (68% vs 14.2%, p&amp;lt;0.001). Median survival was 47.0 months (44.1-49.9) at SCCC compared to 45.0 months (37.6-52.3) for women at JMH (p=0.392). Conclusion: The majority of women sought care at the SCCC when diagnosed with HGSC had a higher likelihood of early stage diagnosis, were insured and born in the USA, there was no difference in outcome between patients treated at JMH versus SCCC. Genetic testing within the Black population was low compared to Hispanic and non-Hispanic white patients. This data indicate that the quality of care received in this university health system is similar at both sites. Further studies are indicated to explore uptake of genetic testing, disparities between safety net systems and comprehensive cancer centers, particularly with the shift toward targeted disease prevention and personalized therapy. Note: This abstract was not presented at the conference. Citation Format: Sophia HL George, Marilyn Huang, Leah Dodds, Raleigh Butler, Sean Oldak, Moraima Batista, Judith Hurley, Joseph Pearson, Erin Kobetz, Brian Slomovitz. Analysis of High-Grade Serous Ovarian Cancer in a Cohort of Women diagnosed Epithelial Ovarian Cancer at University of Miami/Jackson Memorial Hospital. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B59.

Background: Hypoxia is a feature of high grade serous ovarian cancer (HGSOC) microenvironment and contributes to platinum and PARP inhibitor resistance. Hypoxia induces activator protein-1 (AP-1) transcription factor (TF) activity leading to sustained proliferation, invasion, metastasis and angiogenesis. The role of AP-1 in DNA damage signaling and repair (DDR) and platinum/PARP resistance is unclear in HGSOC. Methods: PARP sensitive and resistant HGSOC cells (PEO1, PEO1R) were monitored for the activity of 48 different TFs using a luciferase-based reporter assay. AP-1 subunits including c-JUN, JUND, JUNB, cFOS and FOSL2 were profiled for protein expression under normoxia and hypoxia (1% O2). PEO1 and PEO1R tumor xenografts were immunohistochemically evaluated for JUNB, FOSL2, MRE11, CA-9 and CD-31 expression. CRISPR knock outs (KO) of JUNB and FOSL2 were generated and investigated for DNA repair gene expression (by DNA repair profiler PCR arrays), whole genome RNA sequencing, proliferation, invasion, cisplatin sensitivity (clonogenic and 3D-spheroids). Functional assays included DNA double strand break (DSB) accumulation, cell cycle progression, apoptosis, immunofluorescence, protein stability assay, co-immunoprecipitation and chromatin immunoprecipitation. The clinicopathological significance of FOSL2, JUNB and MRE11 expression was investigated in 331 clinical epithelial ovarian cancers. Results: In platinum/PARP resistant PEO1R cells, upregulation of AP-1 transcription activity was evident compared to PEO1. Overexpression of JUNB and FOSL2 proteins was observed in normoxia and hypoxia (1% O2). PEO1 and PEO1R tumor xenografts showed high levels of JUNB and FOSL2 in tumor hypoxic areas. Compared to controls, JUNB and FOLS2_KO cells were less proliferative and have increased sensitivity to cisplatin/PARPi (olaparib), which was associated with increased DSBs, G2/M cell cycle arrest and increased apoptosis. DNA repair profiling revealed down regulation of several DNA repair genes in KO cells including MRE11, a key DDR factor. JUNB/FOSL2 proteins physically interacted with MRE11 and promoted its stability. RNA sequencing revealed enrichment of pathways such as platinum response, oxidative phosphorylation, translation and others in KO cells compared to control. In clinical cohorts, high FOSL2, high JUNB and high MRE11 expression was significantly associated with shorter progression-free survival (PFS) and worse overall survival (OS). Conclusion: Our data provides evidence that JUNB and FOSL2 may operate at the hypoxia-DDR interface in HGSOC. JUNB and FOSL2 not only have predictive and prognostic significance but could also be attractive anti-cancer targets including in platinum/PARP resistant HGSOC. Citation Format: Shatha Alqahtani, Rinad Mahmoud, Mashael Algethami, Asmaa Ibrahim, Ahmed Shoqafi, Jennie N. Jeyapalan, Sophie Kellaway, Nigel P. Mongan, Emad A. Rakha, Alan McIntyre, Srinivasan Madhusudan. Unravelling the role of AP-1 transcription factor in DNA damage signaling and response (DDR), platinum and PARP inhibitor resistance in ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2911.