Platinum-resistant High-grade Serous Ovarian Cancer Research Articles

FANCD2 expression affects platinum response and further characteristics of high grade serous ovarian cancer in cells with different genetic backgrounds

High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.

Single-cell and spatial transcriptome sequencing uncover a platinum-resistant cluster overexpressed TACSTD2 in high-grade serous ovarian cancer.

Purpose: Platinum-based chemotherapy is effective but limited by resistance in high-grade serous ovarian cancer (HGSOC). Single-cell RNA sequencing (scRNA-seq) can reveal tumour cell heterogeneity and subclonal differentiation. We aimed to analyze resistance mechanisms and potential targets in HGSOC using scRNA-seq. Methods: We performed 10× genomics scRNA-seq sequencing on tumour tissues from 3 platinum-sensitive and 3 platinum-resistant HGSOC patients. We analyzed cell subcluster communication networks and spatial distribution using cellchat. We performed RNA-seq analysis on TACSTD2, a representative resistance gene in the E0 subcluster, to explore its molecular mechanism. Results: Epithelial cells, characterized by distinct chemotherapy resistance traits and highest gene copy number variations, revealed a specific cisplatin-resistant cluster (E0) associated with poor prognosis. E0 exhibited malignant features related to resistance, fostering growth through communication with fibroblasts and endothelial cells. Spatially, E0 promoted fibroblasts to protect tumour cells and impede immune cells infiltration. Furthermore, TACSTD2 was identified as a representative gene of the E0 subcluster, elucidating its role in platinum resistance through the Rap1/PI3K/AKT pathway. Conclusions: Our study reveals a platinum-resistant epithelial cell subcluster E0 and its association with TACSTD2 in HGSOC, uncovers new insights and evidence for the platinum resistance mechanism, and provides new ideas and targets for the development of therapeutic strategies against TACSTD2+ epithelial cancer cells.

Characteristics of Extracellular Vesicles from a High-Grade Serous Ovarian Cancer Cell Line Derived from a Platinum-Resistant Patient as a Potential Tool for Aiding the Prediction of Responses to Chemotherapy.

Platinum-resistant high-grade serous ovarian cancer (HGSOC) is invariably a fatal disease. A central goal of ovarian cancer research is therefore to develop new strategies to overcome platinum resistance. Treatment is thus moving towards personalized therapy. However, validated molecular biomarkers that predict patients' risk of developing platinum resistance are still lacking. Extracellular vesicles (EVs) are promising candidate biomarkers. EpCAM-specific EVs are largely unexplored biomarkers for predicting chemoresistance. Using transmission electron microscopy, nanoparticle tracking analysis and flow cytometry, we compared the characteristics of EVs released from a cell line derived from a clinically confirmed cisplatin-resistant patient (OAW28) and EVs released from two cell lines from tumors sensitive to platinum-based chemotherapy (PEO1 and OAW42). We demonstrated that EVs released from the HGSOC cell line of chemoresistant patients exhibited greater size heterogeneity, a larger proportion of medium/large (>200 nm) Evs and a higher number of released EpCAM-positive EVs of different sizes, although the expression of EpCAM was predominant in EVs larger than 400 nm. We also found a strong positive correlation between the concentration of EpCAM-positive EVs and the expression of cellular EpCAM. These results may contribute to the prediction of platinum resistance in the future, although they should first be validated in clinical samples.

The role of innate immune system in modulating CHK1 inhibitor (CHK1i) response in BRCA wild-type (BRCAwt), platinum-resistant high-grade serous ovarian cancer (PR-HGSOC): Exploratory analysis from a phase II study of CHK1i prexasertib.

5557 Background: Immune checkpoint blockade, e.g., anti-PD-1/PD-L1, demonstrated limited efficacy in HGSOC despite high levels of tumor-infiltrating lymphocytes, suggesting the innate, rather than adaptive, immune system may play a prominent role in anticancer response. Preclinical studies suggest CHK1i modulates the innate immune milieu. We previously showed clinical activity of prexasertib in BRCAwt, PR-HGSOC patients (pts). Here, we investigated the association of innate immunogenic features with CHK1i response. Methods: Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. Baseline (BL) fresh core biopsies were collected from pts enrolled in the biopsy (Bi) cohort for RNA sequencing. Computational immunogenomic analysis (CIBERSORT) was used for immune cell fractions quantification. Blood samples (BS) were obtained at BL and cycle 1 day 15 (C1D15) from pts enrolled in both Bi and non-Bi (nBi) cohorts for multiparametric flow cytometry (FC) analysis to evaluate dynamic changes in immune cell subsets. Mann-Whitney and Wilcoxon tests were used to compare unpaired and paired data, respectively. Results: 49 BRCAwt, PR-HGSOC pts were enrolled in the Bi cohort (n=25) and nBi cohort (n=24). 39 were evaluable per RECIST v1.1. 18/39 pts (46.2%) had a clinical benefit (CB), defined as progression-free survival≥6 months. Transcriptomic profiles of BL biopsies (n=15) met the quality control and assurance (Gene Similarity Index, Principal Component Analysis) for further in silico analysis. CIBERSORT analysis did not exhibit any differences in the immune cell fractions between CB and non-CB (NCB) groups at BL. FC analysis on paired BS (BL and C1D15) showed an increase of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs, p<0.001) and classical monocytes (CM, p=0.003) in the NCB group (18 paired BS). In the CB group (19 paired BS), CD141+ and CD1c+ dendritic cells (DCs) express higher rates of CD83, a marker of maturity and functionality (p<0.001 and p<0.001, respectively), despite no increase in the absolute number of DCs. At C1D15, a higher rate of M-MDSCs (median 7.8% vs. 5.52%; p=0.03) and CM (median 40.7% vs. 31.7%; p=0.03) were found in the NCB group (n=18) compared to the CB group (n=19); also, CM showed lower expression of HLA-DR in the NCB group (median 19.25% vs. 27.8%; p=0.02). Conclusions: Collectively, our data suggest the possible involvement of innate immunity in modulating CHK1i response. Increasing peripheral immunosuppressive cells and lower immunocompetence of the innate immune system may contribute to CHK1i resistance, whereas the functionality of DCs may be involved in the response. Enhancing innate immunity may represent a strategy to increase CHK1i response in this hard-to-treat population. Clinical trial information: NCT02203513 .

Randomized phase 2 study of gemcitabine with or without ATR inhibitor berzosertib in platinum-resistant ovarian cancer: Final overall survival (OS) and biomarker analyses.

5512 Background: The multicenter, open-label, randomized phase 2 NCI-9944 study (NCT02595892) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (HR = 0.57, one-sided log-rank p = 0.044, which met the one-sided significance level of 0.1 used for sample size calculation). Final overall survival (OS) and corresponding biomarker analyses are reported here. Methods: Patients (pts) with platinum-resistant high-grade serous ovarian cancer and unlimited previous lines of cytotoxic therapy in the platinum-sensitive setting but no more than one line of cytotoxic therapy in the platinum-resistant setting, were randomized 1:1 to gemcitabine/berzosertib versus gemcitabine alone. Randomization was stratified based on platinum free interval (PFI), PFI≤3 months versus > 3 months. Crossover from gemcitabine to gemcitabine/berzosertib was allowed upon disease progression by RECIST 1.1. OS was a secondary endpoint while preplanned exploratory correlative studies included assessment of DNA repair pathways and replication stress (RS) alterations by targeted gene sequencing and/or immunohistochemistry (IHC). Results: Seventy pts were randomly assigned to treatment with gemcitabine/berzosertib (34 pts) or gemcitabine alone (36 pts); 15 pts crossed over from gemcitabine to gemcitabine/berzosertib. At the final OS analysis (92.9% maturity), median follow-up was 53.2 weeks in the gemcitabine/berzosertib and 43 weeks in the gemcitabine alone groups. Median OS in the intent-to-treat (ITT) population was 59.4 weeks in the gemcitabine/berzosertib group versus 43.0 weeks in the gemcitabine alone group (HR 0.79, 90% CI 0.52-1.2, one-sided p = 0.18). However, when patients who crossed over to gemcitabine/berzosertib were excluded from analysis, a significant OS benefit was observed with gemcitabine/berzosertib (HR 0.60, 90%CI 0.37−0.97); HR was 0.26 (90% CI 0.1−0.7) in pts with PFI≤3 months and 0.89 (90%CI 0.50−1.59) in pts with PFI > 3 months. Furthermore, significant OS benefit was observed in pts with RS-low tumors (HR 0.39, 90%CI 0.17−0.91, defined as tumors harboring no genomic RS alterations related to loss of RB pathway regulation and/or oncogene-induced RS) but not in pts with RS-high tumors (HR 0.74, 90%CI 0.35−1.56). Additional targeted gene sequencing and IHC analyses as well as analyses adjusting for patient crossover will be reported at the meeting. Conclusions: In the ITT population, gemcitabine/berzosertib did not significantly improve OS versus gemcitabine alone. Pts with PFI≤3 months and pts with RS-low tumors may derive a survival advantage from addition of berzosertib to gemcitabine in the platinum-resistant setting. Clinical trial information: NCT02595892 .

Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status.

ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.

Abstract CT113: A phase II study of prexasertib, a cell cycle checkpoint kinase 1 (CHK1) inhibitor, in platinum-resistant recurrent high-grade serous ovarian cancer (HGSOC) with BRCA wild-type (BRCAwt)

Abstract Background: Platinum-resistance is associated with a poor prognosis in HGSOC and has limited treatment options. Inhibition of CHK1 has shown activity in BRCAwt HGSOC. Here, we report the clinical activity and tolerability of the second generation CHK1 inhibitor, prexasertib, in platinum-resistant BRCAwt HGSOC (NCT02203513). Methods: Eligibility included platinum-resistant recurrent HGSOC women without germline or somatic BRCA mutation, ECOG PS 0-2, good end organ function, and measurable disease. The primary endpoint was response rate (RR). Secondary endpoints were progression-free survival (PFS) and safety. Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. CT scans were performed every 2 cycles for RECIST v1.1 evaluation, and safety evaluation using CTCAE v4.0 every cycle. CBC was performed on day 8 of cycle 1 for ANC nadir. Pretreatment clinical samples were collected for biomarker analysis including HR deficiency and circulating tumor cells (CTC). Results: Between January 2017 and November 2020, 49 patients (median age 64.1 years [IQR 56.8 - 69.9]) received at least one dose. 12 (24.5%) patients were primary platinum-resistant, and 37 (75.5%) were secondary platinum-resistant. All were heavily pretreated (median 4 prior systemic therapies [IQR 3 - 7]), including bevacizumab (79%), PARP inhibitors (44%), and both (2%). 39 patients were evaluable by RECIST and RR was 30.7%. The median PFS was 5.8 months (range 1.7-26.4 months). The median duration of response among PRs was 5.5 months (range 1-19.8 months). The clinical benefit rate (PR+CR+SD >4 months) was 84.6%. 10 patients were inevaluable due to: withdrawal of consent (n=2), intercurrent illness related to disease (tumor invasion of mainstem bronchus (n=1) or vaginal wall (n=1), small bowel obstruction (n=2), infection (n=2), failure to thrive (n=1), and pneumothorax secondary to thoracentesis (n=1) during cycle 1. The common (>10%) grade 3 or 4 adverse events (AEs) included neutropenia (85%, 42/49) based on the cycle 1 day 8 CBC at the time of ANC nadir, lymphocytopenia (46%, 23/49), thrombocytopenia (40%, 20/49), anemia (30%, 15/49), and febrile neutropenia (12%, 5/49). 83% (41/49) of patients received growth factors support to avoid subsequent treatment delays. There were no deaths on the study. No significant difference of RECIST response or PFS was observed in patients with high (≥2) vs low (<2) CTC at baseline. Conclusions: Prexasertib monotherapy resulted in clinical benefit in subgroups of heavily pretreated BRCAwt platinum-resistant HGSOC. Prexasertib was well tolerated with manageable grade 3/4 AEs. Further studies on predictive biomarkers are ongoing. Citation Format: Grant Zurcher, Ann McCoy, Brooke Solarz, Jay Nair, Min-Jung Lee, Jane B. Trepel, Christina M. Annunziata, Jung-Min Lee. A phase II study of prexasertib, a cell cycle checkpoint kinase 1 (CHK1) inhibitor, in platinum-resistant recurrent high-grade serous ovarian cancer (HGSOC) with BRCA wild-type (BRCAwt) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT113.

Abstract 5408: Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in the management of high-grade serous ovarian cancer (HGSOC). However, most HGSOC ultimately develops resistance to PARPi, leading to unmet medical needs for better therapeutic strategies. ATR-mediated G2/M cell cycle checkpoints are important for cell survival and DNA repair, especially in HGSOC due to its dysfunctional p53-mediated G1/S checkpoint, making them an attractive therapeutic target. However, only modest monotherapy activity of ATR inhibitors (ATRis) has been reported in patients with HGSOC, highlighting the need for combination treatment. We conducted a high-throughput drug combination screen of ATRi (ceralasertib) with 1,912 drugs in two PARPi-resistant BRCA2-mutant PEO1 (PEO1/OlaR and PEO1/OlaJR) HGSOC cell lines. In this screen, 234 drugs showed synergistic cytotoxicity with ATRi, including 31 (13.2%) cell cycle checkpoint inhibitors, 27 (11.5%) chemotherapeutic drugs, and 13 (5.6%) PI3K/AKT pathway inhibitors. We prioritized the PI3K/AKT pathway inhibitors for ATRi combination because the PI3K/AKT pathway is upregulated in >70% of HGSOC and associated with an aggressive phenotype. Moreover, PI3K/AKT signaling is found to be activated in HGSOC cells with PARPi resistance. We separately validated that an ATRi (ceralasertib < 1 μM) in combination with an AKT inhibitor (AKTi; capivasertib < 10 μM) yielded additive/synergistic cytotoxic effects (combination index < 1) in various HGSOC cell lines, including acquired and de novo PARPi-resistant (PEO1/OlaR, PEO1/OlaJR, and PEO4) and platinum-resistant HGSOC cell lines (OVCAR3), by XTT and colony formation assays. With clinically attainable concentrations of ATRi (1 μM) and AKTi (10 μM), the combination treatment increased cell apoptosis compared to ATRi (increased 2.1 to 95.2-fold; P < 0.001) or AKTi alone (increased 3.3 to 92.2-fold; P < 0.001) in both PARPi-sensitive and -resistant HGSOC cells. ATRi+AKTi treatment also showed greater DNA damage as evidenced by increased percentage of cells with ≥ 5 γH2AX foci relative to ATRi (increased 1.1 to 7.6-fold; not significant in PEO1 and P < 0.001 in PARPi-resistant cells) or AKTi (increased 2.3 to 6.2-fold; P < 0.001) in both PARPi-sensitive and -resistant cells. Furthermore, ATRi+AKTi treatment augmented replication stress as measured by increased phospho-RPA+/γH2AX+ populations compared with ATRi (increased 1.7 to 4.7-fold; P < 0.001) or AKTi (increased 3.7 to 12.0-fold; P < 0.001). Overall, our results suggest that dual inhibition of ATR and AKT pathways results in greater cell death by increasing DNA damage and replication stress in HGSOC cells with PARPi resistance. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Nitasha Gupta, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5408.

Sequential azacitidine and carboplatin induces immune activation in platinum-resistant high-grade serous ovarian cancer cell lines and primes for checkpoint inhibitor immunotherapy

BackgroundPlatinum chemoresistance results in high-grade serous ovarian cancer (HGSOC) disease recurrence. Recent treatment advances using checkpoint inhibitor immunotherapy has not benefited platinum-resistant HGSOC. In ovarian cancer, DNA methyltransferase inhibitors (DNMTi) block methylation and allow expression of silenced genes, primarily affecting immune reactivation pathways. We aimed to determine the epigenome and transcriptome response to sequential treatment with DNMTi and carboplatin in HGSOC.MethodsIn vitro studies with azacitidine or carboplatin alone and in sequential combination. Response was determined by cell growth, death and apoptosis. Genome-wide DNA methylation levels and transcript expression were compared between untreated and azacitidine and carboplatin sequential treatment.ResultsSequential azacitidine and carboplatin significantly slowed cell growth in 50% of cell lines compared to carboplatin alone. The combination resulted in significantly higher cell death in 25% of cell lines, and significantly higher cell apoptosis in 37.5% of cell lines, than carboplatin alone. Pathway analysis of upregulated transcripts showed that the majority of changes were in immune-related pathways, including those regulating response to checkpoint inhibitors.ConclusionsSequential azacitidine and carboplatin treatment slows cell growth, and demethylate and upregulate pathways involved in immune response, suggesting that this combination may be used to increase HGSOC response to immune checkpoint inhibitors in platinum-resistant patients who have exhausted all currently-approved avenues of treatment.

Combination ATR and PARP Inhibitor (CAPRI): A phase 2 study of ceralasertib plus olaparib in patients with recurrent, platinum-resistant epithelial ovarian cancer

ObjectivePlatinum-resistant, high-grade serous ovarian cancer (HGSOC) has limited treatment options. Preclinical data suggest that poly(ADP-ribose) polymerase inhibitors (PARPi) and ataxia telangiectasia and Rad3-related kinase inhibitors (ATRi) are synergistic. CAPRI (NCT03462342) is an investigator-initiated study of olaparib plus ceralasertib in recurrent HGSOC. Herein, we present results from the platinum-resistant cohort. MethodsA Simon 2-stage design was utilized. Platinum-resistant HGSOC patients received ceralasertib 160 mg orally daily, days 1–7 and olaparib 300 mg orally twice daily, days 1–28 of a 28-day cycle until toxicity or progression. Primary endpoints were toxicity and efficacy including objective response rate (ORR) by RECIST. Secondary endpoint was progression-free survival (PFS). The null hypothesis (≤5% ORR) would be rejected if there were ≥ 1 responses in 12 patients. ResultsFourteen PARPi-naïve patients were evaluable for toxicity; 12 were evaluable for response. Three had BRCA1 mutations (1 germline, 2 somatic). Adverse events possibly related to treatment were primarily grade (G) 1/2. G3 toxicities included nausea (14.3%), fatigue (7.1%), anorexia (7.1%), and anemia (7.1%). No objective responses occurred. Best response was stable disease in 9 patients and progressive disease in three. Five patients had a ≥ 20% to <30% reduction in disease burden, including 3 with BRCA1 mutations. Three of 11 patients (27%; 2 with BRCA1 mutations) evaluable by Gynecologic Cancer Intergroup criteria had >50% CA-125 decline, including 2 with CA-125 normalization. Median PFS was 4.2 months overall (90% CI:3.5–8.2) and 8.2 months (3.6 months–not determined) for patients with BRCA1 mutations. ConclusionsOlaparib plus ceralasertib is well-tolerated. No objective responses occurred, though a signal of activity was seen particularly in disease associated with BRCA1. Further evaluation of this combination should include alternate dosing strategies in genomically-selected populations.

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

<h3>Objectives:</h3> The purpose of this study was to identify genes and pathways differentially expressed in platinum resistant high grade serous ovarian cancer (HGSOC) when compared to sensitive HGSOC. <h3>Methods:</h3> A total of 37 patients with HGSOC tissue samples underwent RNA sequencing performed by TEMPUS (N=37, 21 platinum sensitive, 16 resistant; 85% Stage III-IV; 58% received neoadjuvant chemotherapy). RNA gene expression data and significantly impacted pathways were analyzed using Advaita Bio's iPathwayGuide. Differentially expressed (DE) genes were identified using FDR of 0.05 and fold-change of 1.5. Genes from several impacted canonical metabolic pathways were validated by PCR against external data sets in a separate ovarian cancer sample group (n=15), platinum resistant ovarian cancer mouse tumor model, and wild-type sensitive and platinum resistant ovarian cancer cell lines. Relative gene expression was calculated using the comparative Ct method, also referred to as the "2 DDCT", using L27 as internal control gene. <h3>Results:</h3> We identified 177 differentially expressed (DE) genes out of a total of 16,607 genes (1.1%) with measured expression. 15 pathways were found to be significantly impacted. Of the 15 canonical pathways, all were up regulated in the resistant HGSOC and the majority of the most significantly altered (5/10) were related to metabolism (Retinol metabolism (p-value = 0.002); Tyrosine Metabolism (p-value = 0.005); Tryptophan Metabolism (p-value = 0.009); and Phenylalanine Metabolism (p-value = 0.012); CYP Drug Metabolism (p-value = 0.022)). A total of 3 separate genes from the CYP family and two from the Dopa Decarboxylase family of genes were validated against an external data set of human ovarian tissue samples, cell lines, mouse ovarian tumor model, and found to have similarly increased gene expression in the genes tested in the platinum resistant groups. Compilation of KEGG analysis and the common network genes revealed pathways associated with amino acid metabolism to be most significantly altered. <h3>Conclusions:</h3> We describe the identification of a unique transcriptomic profile associated with platinum resistance. Interestingly, the main pathways identified are related to metabolism, suggesting that the survival to chemotherapy demands a major metabolic adaptation. These findings also represent a first step towards the identification of biomarkers for the detection of chemo-resistant disease and metabolism-based drug targets specific for chemo-resistant tumors. Further validation of this model is required in order to determine its clinical value.

Promoter Methylation of the MGRN1 Gene Predicts Prognosis and Response to Chemotherapy of High-Grade Serous Ovarian Cancer Patients.

Aberrant DNA methylation is considered to play a critical role in the chemoresistance of epithelial ovarian cancer (EOC). In this study, we explored the relationship between hypermethylation of the Mahogunin Ring Finger 1 (MGRN1) gene promoter and primary chemoresistance and clinical outcomes in high-grade serous ovarian cancer (HGSOC) patients. The MALDI-TOF mass spectrometry assays revealed a strong association between hypermethylation of the MGRN1 upstream region and platinum resistance in HGSOC patients. Spearman’s correlation analysis revealed a significantly negative connection between the methylation level of MGRN1 and its expression in HGSOC. In vitro analysis demonstrated that knockdown of MGRN1 reduced the sensitivity of cells to cisplatin and that expression of EGR1 was significantly decreased in SKOV3 cells with low levels of MGRN1 expression. Similarly, EGR1 mRNA expression was lower in platinum-resistant HGSOC patients and was positively correlated with MGRN1 mRNA expression. Kaplan-Meier analyses showed that high methylation of the MGRN1 promoter region and low expression of MGRN1 were associated with worse survival of HGSOC patients. In multivariable models, low MGRN1 expression was an independent factor predicting poor outcome. Furthermore, low expression of EGR1 was also been confirmed to be significantly related to the poor prognosis of HGSOC patients by Kaplan-Meier. The hypermethylation of the MGRN1 promoter region and low expression of MGRN1 were associated with platinum resistance and poor outcomes in HGSOC patients, probably by altering EGR1 expression.

Methylomic Signatures of High Grade Serous Ovarian Cancer

ABSTRACT High-grade serous ovarian cancer (HGSOC) harbours aberrant epigenetic features, including DNA methylation. In this study we delineate pathways and networks altered by DNA methylation and associated with HGSOC initiation and progression to a platinum-resistant state. By including tumours from patients who had been treated with the hypomethylating agent (HMA) guadecitabine, we also addressed the role of HMAs in treatment of HGSOC. Tumours from patients with primary (platinum-naïve) HGSOC (n = 20) were compared to patients with recurrent platinum-resistant HGSOC and enrolled in a recently completed clinical trial (NCT01696032). Human ovarian surface epithelial cells (HOSE; n = 5 samples) served as normal controls. Genome-wide methylation profiles were determined. DNA methyltransferase (DNMT) expression levels were examined by immunohistochemistry and correlated with clinical outcomes. Cancer-related and tumorigenesis networks were enriched among differentially methylated genes (DMGs) in primary OC vs. HOSE. When comparing platinum-resistant and primary tumours, 452 CpG island (CGI)-containing gene promoters acquired DNA methylation; of those loci, decreased (P < 0.01) methylation after HMA treatment was observed in 42% (n = 189 CGI). Stem cell pluripotency and cytokine networks were enriched in recurrent platinum-resistant OC tumours, while drug metabolism and transport-related networks were downregulated in tumours from HMA-treated patients compared to HOSE. Lower DNMT1 and 3B protein levels in pre-treatment tumours were associated with improved progression-free survival. The findings provide important insight into the DNA methylation landscape of HGSOC tumorigenesis, platinum resistance and epigenetic resensitization. Epigenetic reprogramming plays an important role in HGSOC aetiology and contributes to clinical outcomes.

846P DRDscore can predict platinum-resistance in advanced high-grade serous ovarian cancer

High grade serous ovarian cancer (HGSOC) is conventionally treated with surgery and platinum chemotherapy. The initial response rate is 60-80%, but eventually the majority of patients develop resistance to platinum with subsequent recurrence. Currently, some biomarkers of immune changes, epigenetics, genomic and DNA repair, which are associated with platinum-resistant HGSOC, have been reported, but there is no relatively accurate algorithm to predict whether patients will be resistant to platinum-based therapy. We performed whole exome sequencing of a total of 111 tissue samples from 40 HGSOC patients as training cohort and 71 HGSOC patients as validation cohort. In the training cohort, the DNA Repair Deficiency score (DRDscore) algorithm was established using LASSO regression with Homologous Recombination Deficiency (HRD) Score, Tumor mutational burden (TMB) and Microhomology insertion deletion (MHID) as the basic parameters, and the accuracy of the DRD model was tested in the validation cohort. According to the consensus statement of Gynecologic Cancer Intergroup (GCOG), we classified Platinum response:(1) Platinum-resistant: progression-free interval since last line of platinum of less than 6 months; (2) Platinum sensitive: progression-free interval since last line of platinum of more than 6 months.Analysis of the accuracy of BRCA1/2 mutation, HRD score and DRD score in estimating platinum-sensitivity chemotherapy. The platinum-sensitivity rate was 68.57% (24/35) in patients with positive BRCA1/2 mutations, and the platinum-resistance rate was 36.11% (13/36) in patients with negative BRCA1/2 mutations. The platinum-sensitivity rate was 77.5% (31/40) in patients with positive HRDscore, and the platinum-resistance rate was 58.06% (18/31) in patients with negative HRDscore. The platinum-sensitivity rate was 86.53% (45/52) in patients with positive DRDscore, and the platinum-resistance rate was 84.21% (16/19) in patients with negative DRDscore. DRDscore is a robust prognostic indicator of the risk of platinum-resistance in advanced HGSOC patients. This assessment algorithm for platinum-sensitivity can be used to predict whether HGSOC patients are suitable for platinum-based therapy.

Abstract A56: Novel L-sugar linked glycosylated antitumor ether lipids for killing platinum-resistant human epithelial ovarian cancer cells

Abstract 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.

Abstract A72: Combination ATR and PARP inhibitor (CAPRI) for recurrent, platinum-resistant ovarian cancer

Abstract Background: Platinum-resistant (PR) ovarian cancer (OC) is a lethal disease for which effective therapies are limited. Preclinical data suggest that inhibitors of poly(ADP-ribose) polymerase (PARP) and ataxia telangiectasia and Rad3-related kinase (ATR) have synergistic antitumor activity in these tumors. We hypothesize that targeting two unique DNA repair pathways with combination therapy may increase response rates, durability of response, and lower off-target toxicities compared to standard treatments. This clinical trial examines PARP and ATR inhibition (PARPi-ATRi) in patients (pts) with recurrent OC (NCT03462342). Data from the PR arm of this trial are presented. Methods: Twelve patients were enrolled with PROC in the first stage of a Simon optimal two-stage design with alpha=0.10 and beta=0.10. Eligible pts had recurrent, PR high-grade serous OC, measurable disease, and no prior treatment with a targeted inhibitor of DNA repair. Pts may or may not have a somatic or germline mutation of BRCA1 or BRCA2. Pts received olaparib (O) 300mg orally twice daily on days 1-28 and AZD6738 (A) 160mg orally daily on days 1-7 of a 28-day cycle. Endpoints were safety (toxicity based on CTCAE v5.0), objective response rate (RECIST v1.1), and PFS (RECIST v1.1). If ≥1 patient of 12 treated has a partial or complete response, then 25 additional patients will be treated; if no responses are seen, the PR arm of this trial will be terminated. Results: Nine patients have been treated and 8 pts have had on-study imaging thus far. Median (M) age is 63 years (53-73); M ECOG, 0 (0-1); M prior lines of chemotherapy, 2.5 (1-4); M prior therapies after acquiring platinum resistance, 0.5 (0-2). BRCA1/2m status (positive/negative/unknown) is: gBRCA1 (1/5/4); gBRCA2 (0/6/4); sBRCA1 (1/7/2); sBRCA2 (0/8/2). No complete or partial responses were seen. Six of 8 patients achieved SD with a mean duration on study of 6.7 cycles (range 4-10 cycles), including 1 pt with gBRCA1m. Three of these 6 pts demonstrated 20-27% tumor regression of target lesions. Two patients had disease progression. Toxicities occurring in ≥50% of pts that were at least possibly related to combination therapy were nausea (grade (G) 1/2: 6, G3: 1); anorexia (G1/2: 5, G3: 1); fatigue (G1/2: 4, G3: 1), anemia (G1/2: 4, G3: 1). No G4 toxicities were observed and no pts discontinued therapy due to toxicity. Conclusions: Combination O and A is tolerable with mostly low-grade toxicity similar to that of olaparib single-agent. 75% of response-evaluable pts had stable disease, half of whom had substantial tumor regression. Duration of disease stability was clinically meaningful in this PR, largely g/sBRCA1/2 wild-type OC population. Biopsy samples will be evaluated by genomics and proteomics. Updated response data will be presented. Citation Format: Payal D. Shah, Haley Zarrin, Stephanie Wethington, Nawar Latif, Lainie Martin, Diego Rodriguez, Katie Elkins, Robert Giuntoli, Robert Burger, Janos Tanyi, Mark Morgan, Susan M. Domchek, Stephanie Gailliard, Deborah K. Armstrong, Fiona Simpkins. Combination ATR and PARP inhibitor (CAPRI) for recurrent, platinum-resistant ovarian cancer [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 A72.

LBA60 - Randomized phase II (RP2) study of ATR inhibitor M6620 in combination with gemcitabine versus gemcitabine alone in platinum-resistant high grade serous ovarian cancer (HGSOC)

LBA60 - Randomized phase II (RP2) study of ATR inhibitor M6620 in combination with gemcitabine versus gemcitabine alone in platinum-resistant high grade serous ovarian cancer (HGSOC)

Abstract 1774: Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC)

Abstract Resistance to platinum-based chemotherapy is a major problem in the clinical management of recurrent HGSOC. While the use of poly ADP-ribose polymerase inhibitors (PARPi) have made an impact in BRCA1/2 mutated and homologous recombinant-deficient HGSOC, resistance and limited response-rates to PARPi persist. Alisertib (AL) an inhibitor of AAK can inhibit entry and progression of cells through mitosis as well as the interaction with N-myc and its degradation (Front. Oncol. doi:10.3389/fonc.2015.00189). In models of HGSOC from patients with recurrent platinum-resistant disease, cisplatin (CP) treatment does not lead to cell cycle traverse perturbations and significant accumulation of cells in G2-M. Since a defect in the G2-M DNA damage checkpoint is a potential mechanism of resistance to cisplatin, we sought to test the hypothesis that inhibition of AAK that is involved in centrosome maturation and entry into mitosis would enhance the anti-tumor activity of cisplatin in platinum-resistant HGSOC. To test this hypothesis, we treated HGSOC cell lines from patients with recurrent platinum-refractory disease with increasing doses of CP (1- 2.5 μM), of AL (0.05 μM) or the combination and evaluated effects on cell proliferation, apoptosis and generation of reactive oxygen species (ROS). The sequential treatment of four different HGSOC cell lines with CP for 3 hours followed by AL for 48 hours and recovery in drug-free medium for 96-120 hours led to a synergistic increase in apoptosis (p&amp;lt;0.01) and a synergistic decrease in cell survival (p&amp;lt;0.001) compared to treatment with either drug alone. The enhanced apoptosis and decreased cell survival with (CP→AL) treatment was accompanied by a significant increase (p&amp;lt;0.05) in ROS. In contrast, sequential treatment with AL followed by CP (AL→CP) led to an antagonistic response. The synergistic effect of (CP→AL) was accompanied by a modest increase in mRNA and protein levels of AAK. However, protein levels of N-myc protein, which interacts with AAK and is degraded, were not altered. Interestingly, (CP→AL) treatment did not elicit the synergistic cytotoxic response or increased ROS in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. In platinum-refractory HGSOC cells that are wild-type for BRCA1/2 and resistant to olaparib, treatment with the combination of 0.01 μM AL and 1 μM olaparib for 48 hours resulted in an additive cytotoxic response. Based on the mutation landscape of the HGSOC cell panel evaluated, the synergistic cytotoxicity with the CP→AL treatment was independent of BRCA1/2 or TP53 mutation status. In summary, CP resistance in HGSOC due to a defective G2-M DNA damage response, can be potentially circumvented by sequential treatment with CP followed by AL. Citation Format: Ram N. Ganapathi, Eric J. Norris, Ashley P. Sutker, Hala Soliman, Mahrukh K. Ganapathi. Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1774.

Abstract A54: Generation and characterization of a novel panel of platinum-resistant HGSOC models

Abstract Background: High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype. Although it is initially responsive to platinum-containing chemotherapy, the emergence of chemoresistant cells is inevitable. Most women with HGSOC die of platinum-resistant cancer, and the five-year survival for ovarian cancer has changed little in recent decades. There is therefore a need to develop chemotherapy-resistant preclinical models to discover novel therapies for women with these resistant cancers. Methods: Established chemo-sensitive HGSOC cell lines Ovcar4, COV318 and OVSAHO were passaged in vitro with increasing doses of either cisplatin or carboplatin. Doses of carboplatin and cisplatin were incremented by 0.1μM when cells reached confluence. Resistance was assessed by clonogenic assay. To reflect the extent of chemoresistance commonly found in patient tumor samples, dose escalation was stopped once a 5-10 fold difference in IC50 was achieved in resistant compared to sensitive parental cells. Cells were then grown in parallel in the presence or absence of chemotherapy drug to determine whether resistance was maintained. Resistant cell lines were characterized based on molecular markers, cell growth, migration, and spheroid formation. Cells were lentivirally transfected with fluorescent proteins and Firefly luciferase. Intraperitoneal tumor growth was assessed in female CD1 nude mice. Survival was compared and tumors were sectioned and analyzed using H&amp;E staining and IHC for p53, PAX8, and Ki67 expression. Results: All six platinum-resistant cell lines achieved a 5-10 fold increase in resistance based on IC50 values. Maintenance of resistance when drug was withdrawn varied between different cell lines. In vitro proliferation, migration, and spheroid formation varied between matched sensitive and resistant cells. The carboplatin-resistant cells, Ov4Carbo and OVSAHOCarbo, proliferated more rapidly compared to the parental cells from which they were derived (Ovcar4 and OVSAHO) and compared to their cisplatin-resistant counterparts (Ov4Cis and OVSAHOCis). Carboplatin-resistant Ovcar4 cells (Ov4Carbo) also formed spheroids more effectively and displayed greater in vitro migration than Ovcar4 and Ov4Cis. We have tested Ovcar4, Ov4Carbo, and Ov4Cis in vivo and found that all formed tumors in the peritoneum of nude mice. Sequential bioluminescent readings correlated with survival. In keeping with our in vitro findings, these three cells lines exhibited different behavior in vivo, with Ov4Carbo being the most aggressive. Median survival was 6 weeks (Ov4Carbo), 17.4 weeks (Ovcar4), and 20 weeks (Ov4Cis). At necropsy, Ov4Carbo cells were found to be locally invasive and produced large volumes of hemorrhagic ascites, while Ov4Cis metastasized exclusively to lymph nodes without production of ascites or local invasion. H&amp;E staining and IHC confirmed HGSOC histology in all 3 cell lines. Conclusion: We have generated and characterized six new platinum-resistant HGSOC cancer cell lines and demonstrated that they display a range of phenotypes in vitro and in vivo. This novel cell panel will provide a diverse resource to investigate chemotherapy resistance and is a new platform for the development of novel therapies targeting chemoresistant HGSOC cancers. Citation Format: Joseph I. Hoare, Jayeta Saxena, Helen Hockings, Jackie McDermott, Ashley Browne, Michelle Lockley. Generation and characterization of a novel panel of platinum-resistant HGSOC models. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A54.

Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment

BackgroundNovel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors.MethodsExpression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents.ResultsMean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth.ConclusionsThe findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.