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.

Unraveling the role of ubiquitin C-terminal hydrolase L1 in high grade serous ovarian cancer pathogenesis.

e15078 Background: High grade serous ovarian cancer (HGSOC) is the most lethal of all gynecologic malignancies, due to the fact that the majority of patients are diagnosed at an advanced stage and eventually develop chemoresistant disease 12-18 months following completion of frontline therapy. Therefore, novel targeted therapies are urgently needed to combat chemoresistance and improve patient clinical outcomes. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that plays a vital role in protein homeostasis and is highly overexpressed in HGSOC. It was previously uncovered that targeting UCHL1 with small molecule inhibition reduced cell viability in a chemoresistant ovarian cancer cell line. Methods: Comparative-label free proteomic analysis was used to uncover significant changes in HGSOC cell line OVCAR8WT following treatment with UCHL1 small molecule inhibitor, LDN-5744 or corresponding DMSO control. Significant pathway changes were determined by KEGG analysis. Matched chemosensitive and resistant HGSOC cells PEA1 and PEA2 were treated in combination with LDN-5744 and carboplatin, with cell viability was determined by an MTS assay. Furthermore, western blot analysis was employed to compare common cell growth pathway changes as a result of UCHL1 inhibition in HGSOC cells. Results: Proteomic analysis revealed a significant (p<0.05) upregulation in methyl phosphate capping enzyme (MEPCE) (4.9-fold), asparagine synthetase (ASNS) (2.5-fold), Phosphoserine aminotransferase 1 (PSAT1) (1.7-fold), and downregulation in centrosomal protein 55 (CEP55) (-7.2-fold), These identified proteomic changes were confirmed by western blot. KEGG pathways analysis using all significantly (p<0.05) differentially expressed proteins showcased significant (p<0.05) enrichment in amino acid biosynthesis, and metabolism, and chemical carcinogenesis-reactive oxygen species. Chemoresistant PEA2 cells treated in combination with LDN-5744 and carboplatin demonstrated a 59% reduction in cell viability, significantly (p<0.001) lower than the 13% and 22%, that was observed from UCHL1 and carboplatin treatment alone, respectively. Conversely, PEA1, the chemosensitive counterpart to PEA2 demonstrated a significant (p<0.001) 37% increase in cell viability upon combinatorial treatment compared with carboplatin alone, indicating that UCHL1 inhibition rescues the cells from chemotherapy induced cell death. Finally, LDN-5744 treatment in PEA2 led to a reduction in levels of phospho-(p-)AKT, p-STAT3, and p-ERK, while conversely in PEA1 cells levels of these proteins were increase or unchanged. Conclusions: Our findings demonstrate that targeting UCHL1 results in prominent metabolic changes in HGSOC cells and that the efficacy of UCHL1 inhibition is heavily dependent upon platinum status. Overall, this study highlights UCHL1’s novel therapeutic role in the recurrent HGSOC.

Abstract A034: Combination of ATR inhibitor (ATRi) and AKT inhibitor (AKTi) reduces tumor growth and prolongs survival in BRCA2 mutant (BRCA2m) PARP inhibitor (PARPi)-resistant high-grade serous ovarian cancer (HGSC)

Abstract A034: Combination of ATR inhibitor (ATRi) and AKT inhibitor (AKTi) reduces tumor growth and prolongs survival in <i>BRCA2</i> mutant (BRCA2m) PARP inhibitor (PARPi)-resistant high-grade serous ovarian cancer (HGSC)

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Unveiling the epigenomic mechanisms of acquired platinum-resistance in high-grade serous ovarian cancer.

Resistance to platinum-based chemotherapy is the major cause of death from high-grade serous ovarian cancer (HGSOC). We hypothesize that detection of specific DNA methylation changes may predict platinum resistance in HGSOC. Using a publicly available "discovery" dataset we examined epigenomic and transcriptomic alterations between primary platinum-sensitive (n=32) and recurrent acquired drug resistant HGSOC (n=28) and identified several genes involved in immune and chemoresistance-related pathways. Validation via high-resolution melt analysis of these findings, in cell lines and HGSOC tumours, demonstrated the most consistent changes were observed in three of the genes: APOBEC3A, NKAPL and PDCD1. Plasma samples from an independent HGSOC cohort (n=17) were analysed using droplet digital PCR. Hypermethylation of NKAPL was detected in 46% and hypomethylation of APOBEC3A in 69% of plasma samples taken from women with relapsed HGSOC (n=13), with no alterations identified in disease-free patients (n=4). Following these results, and using a CRISPR-Cas9 approach, we were also able to demonstrate that in vitro NKAPL promoter demethylation increased platinum sensitivity by 15%. Overall, this study demonstrates the importance of aberrant methylation, especially of the NKAPL gene, in acquired platinum resistance in HGSOC. This article is protected by copyright. All rights reserved.

Sequential Targeting of PLK1 and PARP1 Reverses the Resistance to PARP Inhibitors and Enhances Platin-Based Chemotherapy in BRCA-Deficient High-Grade Serous Ovarian Cancer with KRAS Amplification.

Ovarian cancer (OC) accounts for approximately 4% of cancer deaths in women worldwide and is the deadliest gynecologic malignancy. High-grade serous ovarian cancer (HGSOC) is the most predominant ovarian cancer, in which BRCA1/2 gene mutation ranges from 3 to 27%. PARP inhibitors (PARPi) have shown promising results as a synthetically lethal therapeutic approach for BRCA mutant and recurrent OC in clinical use. However, emerging data indicate that BRCA-deficient cancers may be resistant to PARPi, and the mechanisms of this resistance remain elusive. We found that amplification of KRAS likely underlies PARPi resistance in BRCA2-deficient HGSOC. Our data suggest that PLK1 inhibition restores sensitivity to PARPi in HGSOC with KRAS amplification. The sequential combination of PLK1 inhibitor (PLK1i) and PARPi drastically reduces HGSOC cell survival and increases apoptosis. Furthermore, we were able to show that a sequential combination of PLK1i and PARPi enhanced the cellular apoptotic response to carboplatin-based chemotherapy in KRAS-amplified resistant HGSOC cells and 3D spheroids derived from recurrent ovarian cancer patients. Our results shed new light on the critical role of PLK1 in reversing PARPi resistance in KRAS-amplified HGSOC, and offer a new therapeutic strategy for this class of ovarian cancer patients where only limited options currently exist.

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 &amp;gt;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 &amp;lt; 1 μM) in combination with an AKT inhibitor (AKTi; capivasertib &amp;lt; 10 μM) yielded additive/synergistic cytotoxic effects (combination index &amp;lt; 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 &amp;lt; 0.001) or AKTi alone (increased 3.3 to 92.2-fold; P &amp;lt; 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 &amp;lt; 0.001 in PARPi-resistant cells) or AKTi (increased 2.3 to 6.2-fold; P &amp;lt; 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 &amp;lt; 0.001) or AKTi (increased 3.7 to 12.0-fold; P &amp;lt; 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.

Abstract PR015: Developing adaptive therapy to suppress the evolution of treatment resistance in high-grade serous ovarian cancer

Abstract Survival in high grade serous ovarian cancer (HGSOC) is impacted by the emergence of resistance to platinum chemotherapy. The traditional ethos of killing the greatest number of cancer cells with the maximum tolerated dose of chemotherapy may be flawed in evolutionary terms, facilitating the growth of resistant subclones. Adaptive therapy is a new model of cancer treatment that exploits the competitive interactions between subclones. Its aim is to maintain a stable tumor burden by keeping a sufficient population of sensitive cells which suppress the proliferation of the ‘less fit’ resistant cells. Our lab has generated unique panels of carboplatin- and cisplatin-resistant OVCAR4 and COV318 HGSOC cell lines to model acquired resistance to chemotherapy. We have demonstrated competition between platinum-sensitive and resistant cells in vitro in a resource-poor environment. The competitive effect was removed on administration of chemotherapy but restored upon re-growth of the sensitive cell population. Flow cytometry for annexin V revealed that resistant cells undergo more apoptosis when cultured with sensitive cells, which increased in proportion to the size of the sensitive population. Competitive growth was also demonstrated in vivo in subcutaneous and intraperitoneal co-culture murine models. Importantly, carboplatin adaptive therapy significantly extended tumor control compared to standard treatment (60mg/kg carboplatin every 4 days for 3 doses) in a subcutaneous co-culture model, maintaining a stable tumor volume for up to 6 months. A challenge in bringing adaptive therapy to the clinical setting is quantifying the relative proportions of therapy-sensitive and resistant disease to guide the personalized scheduling of treatment. In HGSOC, chemotherapy resistance is correlated with a higher burden of copy number aberrations (CNAs). Our novel method “liquidCNA” quantifies the relative proportion of chemotherapy-resistant disease using low pass whole genome sequencing and CNAs as clonal markers. The method was verified using samples of sensitive and resistant HGSOC cells of varying tumor purity and subclonal ratio (proportion of resistant cells), and subsequently applied to longitudinal human samples collected from 18 patients, using the copy number profile from a diagnostic biopsy as a baseline for sensitive disease. We have demonstrated competition between chemo-sensitive and resistant cells both in vitro and in vivo. We have shown that adaptive therapy controls tumor burden for longer than standard therapy in a murine HGSOC model. Our novel method liquidCNA can be applied to liquid biopsies to monitor the relative drug-sensitive/resistant composition of tumors and could potentially be used to guide personalized treatment scheduling in future adaptive therapy clinical trials. Based on this research, we have developed the UK’s first clinical trial of adaptive therapy (ACTOv: Adaptive ChemoTherapy for Ovarian cancer), which will commence recruitment in Q2 2022. Citation Format: Helen Hockings, Michelle Lockley, Trevor Graham, Eszter Lakatos, Weini Huang. Developing adaptive therapy to suppress the evolution of treatment resistance in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR015.

Multiomic analysis identifies CPT1A as a potential therapeutic target in platinum-refractory, high-grade serous ovarian cancer

SummaryResistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line).

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.

Abstract 1014: Repurposing the anti-rheumatic gold compound auranofin for high-grade serous ovarian cancer therapy

Abstract Auranofin (AF) is a gold compound approved in 1985 as a main treatment against rheumatoid arthritis. However, as new anti-rheumatoid agents displaced the use of AF, there have been studies aiming to repurpose the drug to treat other diseases, including cancer. In this study, we investigated the potentiality of AF to impair the growth of high-grade serous ovarian cancer (HGSOC) cells regardless of platinum (CDDP) sensitivity. Preliminary studies utilizing a colorimetric proliferation assay revealed that HGSOC cells that are clinically sensitive (PEO1) or resistant (PEO4) to CDDP-based chemotherapy are both equally sensitive to the growth inhibition induced by AF. The resistant factor estimated for AF-defined as the ratio between IC50 values calculated for the resistant cells and for the sensitive ones—was close to one, suggesting that CDDP-resistant PEO4 cells are equally sensitive to AF than CDDP-sensitive PEO1 cells. As expected, the resistant factor for CDDP was 12-fold higher in PEO4 compared to PEO1. The toxicity of AF against HGSOC cells was further studied exposing PEO1 to various concentrations of AF and assessing cell number, cell viability, and cell cycle traverse after 72 h. Results showed that AF, in a dose-dependent manner, blocked cell proliferation and caused accumulation of cells with hypo-diploid DNA content, suggesting that AF-induced cytotoxicity was associated not only with inhibition of cell proliferation but also with cell death likely driven via apoptosis. We further studied the long-term or residual toxicity of AF by exposing PEO1 cells that were alive, after 72 h treatment with AF, to a clonogenic survival assay. The results clearly demonstrated that even if not dying within 72 h, the cells were affected in a manner not compatible with long-term cell cycle progression as demonstrated by the formation of ‘abortive' colonies (i.e. colonies with few cells that show abnormal phenotypes). Finally, as AF is a pro-oxidant agent, we evaluated whether AF-induced cell death was mediated by oxidative stress by incubating PEO1 cells with AF in the presence or absence of the anti-oxidant N-acetyl-cysteine (NAC). PEO1 cells treated with AF and NAC showed no decrease in cell viability and cell number in comparison to PEO1 cells treated with AF alone. This data reveals that AF most likely induces cell death in a reactive oxygen species (ROS)-dependent manner. In conclusion, our results suggest that AF can equally impair the growth of CDDP-sensitive or -resistant HGSOC cells, and that its toxicity is likely mediated by oxidative stress. Citation Format: Farah H. Abdalbari, Alicia A. Goyeneche, Elvis Martinez-Jaramillo, Siham Sabri, Carlos M. Telleria. Repurposing the anti-rheumatic gold compound auranofin for high-grade serous ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1014.

Abstract 1013: Mechanistic role of nelfinavir as drug-repurposing strategy against high-grade serous ovarian cancer

Abstract Platinum-based therapy following tumor-debulking surgery has been the backbone of treatment for high-grade serous ovarian cancer (HGSOC) since the 1970s; however, high recurrence of platinum-resistant disease necessitates the development of improved alternative therapies. Repurposing market-available drugs as cancer therapeutics carries the prospect of reducing the timeframe and cost of drug development, posing potential benefits to drug-resistant as well as financially underprivileged patients. Nelfinavir (NFV), an orally available anti-HIV drug, has shown promising effects against diverse cancers as demonstrated through a myriad of pre-clinical studies and clinical trials; however, its remedial benefits against HGSOC are still unclear. In this study, we explored the therapeutic efficacy of NFV on HGSOC cells generated from patients when platinum sensitive or resistant. Acute drug toxicity was assessed by total cell count, percent viability, and the level of hypo-diploid DNA content following 72 h of treatment with NFV. Living cells that tolerated 72 h of NFV treatment were subjected to further drug-free re-incubation for 14-21 days, to assess the residual anti-clonogenic potential of the drug. NFV triggered a dose-depended reduction of total cell number and viability, with a parallel increased hypo-diploid DNA content in both platinum-sensitive and resistant HGSOC cells. A dose-dependent reduction in the number of colonies - originating from cells that evaded acute toxicity - suggested long-term residual toxicity of NFV. Western blot analysis of underlying molecular mechanisms revealed phosphorylation of γH2AX, which suggested NFV-mediated induction of DNA damage. NFV also inhibited proliferation signals by reducing the phosphorylation of Akt and ERK. Moreover, NFV stimulated endoplasmic reticulum (ER) stress in a dose-dependent manner, which was evident from the enhanced expression of GRP78, IRE1α, ATF4, CHOP, and phosphorylated eIF2α. A time-dependent steady increase of ATF4 and CHOP suggested a progressive temporal apoptotic shift during NFV treatment. Finally, the cleavage of caspases-3 and -7 and their downstream effector PARP, in association with increased pro-apoptotic protein BAX, indicated caspase-associated cell-death during NFV-mediated cytotoxicity. In summary, we demonstrated that HGSOC cells of differential platinum sensitivities could be therapeutically targeted by NFV via multipronged mechanistic approaches, encouraging its prospective repurposing benefit against HGSOC. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Carlos M. Telleria. Mechanistic role of nelfinavir as drug-repurposing strategy against high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1013.

Abstract A51: The role of ATF6-mediate AP-1 signaling in promoting PARP inhibitor-resistant ovarian cancer

Abstract The purpose of this study is to define the role of AP-1 mediated DNA repair in PARP inhibitor (PARPi)-resistant high-grade serous ovarian cancer (HGSOC). AP-1 is a dimeric transcription factor comprising Jun, Fos, and/or ATF proteins and its signaling is known to regulate multiple DNA repair genes, including PARP1. Wnt activation can promote AP-1 signaling and upregulates expression of AP-1 subunits. We previously published that hyperactive Wnt signaling in PARPi-resistant HGSOC cells promotes DNA repair and attenuates PARPi response. We therefore hypothesized that Wnt signaling drives PARPi resistance thorough increased AP1-signaling and subsequent enhanced DNA repair. We performed transcription factor analysis of RNA sequencing data from TP53/BRCA2-mutated olaparib-sensitive (PEO1) and -resistant (PEO1-OR) HGSOC cells. We observed increased AP-1 target genes in resistant cells and confirmed elevated AP-1 activity with a reporter assay. Quantitative PCR (qPCR) demonstrated that a majority of AP-1 subunits were upregulated in olaparib-resistant cells, namely JUN, ATF5, and ATF6. An shRNA screen was performed to determine which AP-1 subunits are necessary to convey olaparib resistance. Four out of five shRNAs targeting ATF6 effectively resensitized PARPi-resistant cells to olaparib. We confirmed that ATF6 protein was elevated in PEO1-OR cells. To further explore the role of ATF6 in AP-1 mediated PARPi resistance, ATF6 was stably knocked down in PEO1-OR cells. Colony formation assays confirmed that loss of ATF6 resensitized cells to olaparib. Knockdown of ATF6 also resulted in decreased transcription of PARP1, as measured by qPCR, and higher baseline levels of DNA damage, as measured by phosphorylated histone gamma-H2AX. Future studies will evaluate ATF6-dependent DNA repair in in vitro and in vivo models of PARPi-resistant HGSOC. In summary, these data indicate activation of AP-1 signaling through increased expression of ATF6 is promoting enhanced DNA repair capacity, which could be contributing to PARPi resistance. Thus, pharmacologic inhibition of AP-1 or ATF6 could provide a targetable approach to overcome resistance and improve patient outcomes. Citation Format: Alexandra N. McMellen, Benjamin G. Bitler. The role of ATF6-mediate AP-1 signaling in promoting PARP inhibitor-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 A51.

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to “platinum-sensitive”, exhibiting a decreased IC50 value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.

Considerations for Chemotherapy Treatment in Platinum Resistant High-Grade Serous Ovarian Cancer

Ovarian cancer is considered to be the most fatal type of any gynecological cancer. Prognosis for the disease is poor, with a median survival of only thirty-two months following diagnosis and a five-year survival rate of only 39%. Many of the most lethal ovarian cancer cases are classified as part of the high-grade serous ovarian cancer (HGSOC) subtype, which is the most aggressive form of the disease. The primary concern with regards to treatment is that nearly 30% of patients will develop a resistance to forms of platinum chemotherapy, which is the main method of treatment. This suggests that a one-size fits all approach cannot be taken to treat ovarian cancer, and that further research must be done to understand how to treat the patients who present with platinum resistance. This literature review examines the mutations within two susceptible loci, specifically, the p53 and BRCA1/2 genes, in order to understand how platinum resistance develops and why it is present in some patients. The objectives of this review are to characterize the underlying genetic mechanisms affecting platinum resistance, specify the biomarkers associated with those mechanisms, and describe alternative methods for approaching the treatment of ovarian cancer on an individual scale.

C-met mediates invasion and chemotherapy resistance in high grade serous ovarian cancer

Abstract Background High grade serous ovarian cancer (HGSOC) is characterised by progressive chemo-resistance. Survival in advanced HGSOC is poor and new therapies are urgently required. The receptor tyrosine kinase c-Met, through gene amplification or protein over-expression, has emerged as a major target and mediator of chemo-resistance in HGSOC. Methods Novel chemo-resistant HGSOC cell lines were investigated for c-Met expression, activation with exogenous HGF and downstream signalling via western blot analysis. c-Met internalisation was assessed with fluorescent confocal microscopy, following staining with c-Met and the endosomal marker EEA-1. Human samples from HGSOC patients were obtained via Barts/UCLH Gynae Tissue Bank. Tissue sections underwent immunohistochemical staining for c-Met. Cancer associated fibroblasts (CAFs) isolated from HGSOC patients’ omentum were assessed by ELISA to detect HGF secretion. The effect of c-Met inhibitors on HGSOC cell line c-Met activation, migration, survival and invasion was investigated via western blot, transwell migration assay, cell viability in anoikis (CellTiter-Glo) and organotypic cultures. Results Immunostaining detected c-Met in HGSOC patient samples, with strong intensity in the malignant epithelial cells, at the plasma membrane but also in the cytoplasm. c-Met expression is significantly increased in novel cisplatin and carboplatin resistant HGSOC cells, versus isogenic parental cells. c-Met activation in HGSOC cells leads to its rapid endocytosis. Impairing endocytosis pharmacologically reduces c-Met signalling. Chemo-resistant HGSOC cells are more migratory and invasive than parental cells. Primary CAFs secrete various levels of HGF as determined by ELISA. CAFs also increase HGSOC cells’ invasion in organotypic cultures. Targeting c-Met pharmacologically reduces migration, invasion and survival of chemo-resistant HGSOC cells. Conclusions This study confirms c-Met as a therapeutic target using in vitro models and human HGSOC tissues. c-Met endocytosis in HGSOC has been illustrated here, for the first time. Further work is ongoing to develop novel biomarkers for accurate patient selection, exploiting c-Met intracellular localisation. Legal entity responsible for the study The authors. Funding Barts Charity. Disclosure All authors have declared no conflicts of interest.

Low pass whole genome sequencing can be used to calculate the relative proportion of chemotherapy resistant disease in high grade serous ovarian cancer

Abstract Background Survival in high grade serous ovarian cancer (HGSOC) is impacted by the emergence of resistance to platinum chemotherapy. The traditional ethos of killing the greatest number of cancer cells with the maximum tolerated dose of chemotherapy may be flawed in evolutionary terms, facilitating the growth of resistant subclones and accelerating decreased treatment efficacy. Adaptive therapy is a new model of cancer treatment that exploits the competitive interactions between drug-sensitive and resistant subclones. The aim is to maintain a stable tumour burden by keeping a sufficient population of sensitive cells which suppress the proliferation of the ‘less fit’ resistant cells. A challenge in bringing adaptive therapy to the clinical setting is quantifying the relative proportions of sensitive and resistant disease to guide the personalised scheduling of treatment. In HGSOC, chemotherapy resistance is not associated with any common, measurable point mutations, but is correlated with a higher burden of copy number aberrations. We have utilised this to develop a method of quantifying the relative proportion of chemotherapy resistant disease. Methods An in silico dilution series was created using known copy number profiles of paired sensitive and resistant HGSOC OVCAR4 cells. Low pass whole genome sequencing (1×) was performed on cell mixtures of known ratios and bioinformatician-blinded ratios, combined with varied amounts of ‘normal’ DNA extracted from donor leucocytes. Results Our proof of principle experiment verified the method of quantifying copy number aberrations as a biomarker of the resistant population. The technique is now being applied to circulating tumour DNA isolated from serial plasma samples collected from 10 HGSOC patients, using the copy number profile from a diagnostic biopsy as a baseline for sensitive disease. Conclusions We have developed a method to monitor the relative drug-sensitive/resistant composition of tumours that can be applied to liquid biopsies. This will enable real-time serial tracking of tumour composition allowing for tumour heterogeneity, and guide personalised treatment scheduling in adaptive therapy. Legal entity responsible for the study The authors. Funding Cancer Research UK. Disclosure All authors have declared no conflicts of interest.

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 701: Mitochondrial mass is a critical determinant of cisPt-induced cell death in ovarian cancer

Abstract Platinum compounds are an inherent part of chemotherapy for patients with high grade serous ovarian cancer (HGSC). Platinum based therapeutics, such as cisPt, induce DNA-damage by directly binding to nuclear DNA. Interestingly, cisPt also critically affects mitochondrial DNA. Elucidating the role of mitochondria in cisplatin induced cell death of HGSC cells likely identifies new efficient therapy approaches to overcome resistance. Flow cytometric analysis of cisPt sensitive and resistant HGSC cell lines from the NCI60 panel revealed higher mitochondrial mass and higher levels of mitochondrial ROS (mtROS) in cells that are sensitive to cisPt-induced cell death. In clonal sub-lines derived from OVCAR3 the mitochondrial mass correlates with basal oxygen consumption rate and survival after cisPt exposure. The correlation of mitochondrial mass and sensitivity to cisPt-induced apoptosis is corroborated by data from the human protein atlas (www.proteinatlas.org) that indicates high expression of the mitochondrial transcription factor TFAM and the mitochondrial inner membrane protein TIMM23 as favorable prognostic factors for the survival of ovarian cancer patients. Together, this shows the importance of mitochondrial mass in cellular response to cisPt treatment. Furthermore, exposure to cisPt enhances TFAM expression and increases mitochondrial mass as well as mitochondrial ROS. Incubation with cisPt in the presence of the ROS scavenger glutathione (GSH) or pan-caspase inhibitor zVAD-fmk reduces cisPt induced apoptosis while mitochondrial mass is not affected. However, the oxygen consumption rate in cells exposed to cisPt and GSH shows that increased ROS levels induce mitochondrial dysfunction. In line with a sensitivity-determining role of mitochondrial mass, knock-down of key-regulators of mitochondrial biogenesis, i.e. PGC-1α or TFAM, blocks mitochondrial ROS and protects cells from cisPt-induced apoptosis. Mitochondrial ROS is also reduced in an autoregulatory feedback loop by so-called uncoupling proteins (UCPs). Inhibition of UCP2 by Genipin results in increased induction of mitochondrial ROS by cisPt and consequently results in enhanced apoptosis. A comparable sensitization to cisPt-induced apoptosis and ROS production is evident in cells incubated with the iron chelator VLX600.Mitochondria are a critical element in cell response to cisPt since mitochondrial mass correlates with induction of ROS and apoptosis. We show the relevance of cellular mitochondrial content by manipulation of mitochondrial mass and function. We propose the relative mitochondrial content as a biomarker that indicates the response of ovarian cancer cells to cisPt-induced apoptosis. Furthermore, our data provides evidence that increasing mitochondrial mass or induction of mtROS enhances sensitivity to cisPt-induced apoptosis and therefore is a reliable strategy to overcome resistance of ovarian cancer. Citation Format: Markus Kleih, Kathrin Böpple, Andrea Gaißler, Meng Dong, Walter E. Aulitzky, Frank Essmann. Mitochondrial mass is a critical determinant of cisPt-induced cell death in ovarian cancer [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 701.

Abstract 412: Genetic variation in platinum-sensitive and platinum-resistant high-grade serous epithelial ovarian cancer

Abstract 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 &amp;lt;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 &amp;gt; or &amp;lt; 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