Model Of High-grade Serous Ovarian Cancer Research Articles

Immune Mechanisms of Resistance to Cediranib in Ovarian Cancer.

This article investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer (HGSOC), and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment (TME). After 4 to 5 weeks treatment of established tumors, cediranib had antitumor activity with increased tumor T-cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density, and pSTAT3, with increased T-cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL6 JAK/STAT3 signaling in the TME but high programmed cell death protein 1 (PD-1) signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD-1 and Programmed death-ligand 1. Combining cediranib with an anti-PD-1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL6 or anti-PD-1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.

Phenethyl Isothiocyanate Enhances the Cytotoxic Effects of PARP Inhibitors in High-Grade Serous Ovarian Cancer Cells.

While PARP inhibitor (PARPi) therapies have shown promising results in the treatment of high-grade serous ovarian cancer (HGSOC) harboring homologous recombination deficiencies, primary resistance to PARPi frequently occurs and even initial responders may eventually become resistant. Therefore, the development of novel effective combinatorial strategies to treat HGSOC is urgently needed. Here, we report that H2O2-induced oxidative stress sensitized HGSOC cells to PARPi BMN 673. Furthermore, Phenethyl isothiocyanate (PEITC) as a ROS-inducing agent significantly enhanced the cytotoxic effects of BMN 673. Mechanistically, combined use of PEITC and BMN 673 resulted in ROS overproduction and accumulation, enhanced DNA damage, G2/M arrest and apoptosis, all of which were significantly reversed by the ROS scavenger N-Acetyl-L-cysteine. We also showed that while PEITC did not further enhance the ability of BMN 673 on PARP1 trapping in HGSOC cells, the therapeutic effects of the PEITC/BMN 673 combination were at least in part dependent on the presence of PARP1. Importantly, the PEITC/BMN 673 combination potently abrogated the growth of HGSOC tumor spheroids and patient-derived organoid models of HGSOC and cervical cancer. Our findings provide a basis for further investigation of the utility of PARPi combination regimen in HGSOC and cervical cancer through ROS-mediated mechanisms.

Chemotherapy Induces Tumor-Associated Macrophages that Aid Adaptive Immune Responses in Ovarian Cancer.

Neoadjuvant chemotherapy (NACT) may stimulate anticancer adaptive immune responses in high-grade serous ovarian cancer (HGSOC), but little is known about effects on innate immunity. Using omental biopsies from HGSOC, and omental tumors from orthotopic mouse HGSOC models that replicate the human tumor microenvironment, we studied the impact of platinum-based NACT on tumor-associated macrophages (TAM). We found that chemotherapy reduces markers associated with alternative macrophage activation while increasing expression of proinflammatory pathways, with evidence of inflammasome activation. Further evidence of a shift in TAM functions came from macrophage depletion via CSF1R inhibitors (CSF1Ri) in the mouse models. Although macrophage depletion in established disease had no impact on tumor weight or survival, CSF1Ri treatment after chemotherapy significantly decreased disease-free and overall survival. This decrease in survival was accompanied by significant inhibition of adaptive immune response pathways in the tumors. We conclude that chemotherapy skews the TAM population in HSGOC toward an antitumor phenotype that may aid adaptive immune responses, and therapies that enhance or sustain this during remission may delay relapse.

HDAC6 Degradation Inhibits the Growth of High-Grade Serous Ovarian Cancer Cells.

Simple SummaryThe objective of this study was, firstly, to investigate the relationship between Histone deacetylase 6 (HDAC6) expression and survival in patients with ovarian cancer and, secondly, to test the effects of histone deacetylase 6 (HDAC6) inhibition on ovarian cancer cells in vitro. A meta-analysis of the correlation between HDAC6 gene expression and survival was performed on 3573 ovarian tumors from 19 datasets showed that high HDAC6 gene expression was associated with a decreased risk of death. Knockdown of HDAC6 gene expression with small interfering RNA (siRNA) and protein expression with a HDAC6 targeting protein degrader decreased ovarian cell proliferation, migration, and viability. Conversely, the selective inhibition of HDAC6 catalytic activity did not produce a robust inhibition of HDAC6 protein function. In summary, we demonstrated, for the first time, that HDAC6 over-expression in ovarian cancers is a favorable prognostic marker. We provide evidence to suggest that inhibition of HDAC6 catalytic activity has limited efficacy as a monotherapy in ovarian cancers.Histone deacetylase 6 (HDAC6) is a unique histone deacetylating enzyme that resides in the cell cytoplasm and is linked to the modulation of several key cancer related responses, including cell proliferation and migration. The promising anti-cancer response of the first-generation HDAC6 catalytic inhibitors continues to be assessed in clinical trials, although its role in high grade serous ovarian cancer is unclear. This study investigated HDAC6 tumor expression by immunohistochemistry in high-grade serous ovarian cancer (HGSOC) tissue samples and a meta-analysis of HDAC6 gene expression in ovarian cancer from publicly available data. The pharmacological activity of HDAC6 inhibition was assessed in a patient-derived model of HGSOC. HDAC6 was found to be highly expressed in HGSOC tissue samples and in the patient-derived HGSOC cell lines where higher HDAC6 protein and gene expression was associated with a decreased risk of death (hazard ratio (HR) 0.38, (95% confidence interval (CI), 0.16–0.88; p = 0.02); HR = 0.88 (95% CI, 0.78–0.99; p = 0.04)). Similarly, the multivariate analysis of HDAC6 protein expression, adjusting for stage, grade, and cytoreduction/cytoreductive surgery was associated with a decreased risk of death (HR = 0.19 (95% CI, 0.06–0.55); p = 0.002). Knock-down of HDAC6 gene expression with siRNA and protein expression with a HDAC6 targeting protein degrader decreased HGSOC cell proliferation, migration, and viability. Conversely, the selective inhibition of HDAC6 with the catalytic domain inhibitor, Ricolinostat (ACY-1215), inhibited HDAC6 deacetylation of α-tubulin, resulting in a sustained accumulation of acetylated α-tubulin up to 24 h in HGSOC cells, did not produce a robust inhibition of HDAC6 protein function. Inhibition of HGSOC cell proliferation by ACY-1215 was only achieved with significantly higher and non-selective doses of ACY-1215. In summary, we demonstrated, for the first time, that HDAC6 over-expression in HGSOC and all ovarian cancers is a favorable prognostic marker. We provide evidence to suggest that inhibition of HDAC6 catalytic activity with first generation HDAC6 inhibitors has limited efficacy as a monotherapy in HGSOC.

KLF7: a new candidate biomarker and therapeutic target for high-grade serous ovarian cancer

BackgroundIn spite of great progress in the surgical and clinical management, until now no significant improvement in overall survival of High-Grade Serous Ovarian Cancer (HGSOC) patients has been achieved. Important aspects for disease control remain unresolved, including unclear pathogenesis, high heterogeneity and relapse resistance after chemotherapy. Therefore, further research on molecular mechanisms involved in cancer progression are needed to find new targets for disease management. The Krüppel-like factors (KLFs) are a family of transcriptional regulators controlling several basic cellular processes, including proliferation, differentiation and migration. They have been shown to play a role in various cancer-relevant processes, in a context-dependent way.MethodsTo investigate a possible role of KLF family members as prognostic biomarkers, we carried out a bioinformatic meta-analysis of ovarian transcriptome datasets in different cohorts of late-stage HGSOC patients. In vitro cellular models of HGSOC were used for functional studies exploring the role of KLF7 in disease development and progression. Finally, molecular modelling and virtual screening were performed to identify putative KLF7 inhibitors.ResultsBioinformatic analysis highlighted KLF7 as the most significant prognostic gene, among the 17 family members. Univariate and multivariate analyses identified KLF7 as an unfavourable prognostic marker for overall survival in late-stage TCGA-OV and GSE26712 HGSOC cohorts.Functional in vitro studies demonstrated that KLF7 can play a role as oncogene, driving tumour growth and dissemination. Mechanistic targets of KLF7 included genes involved in epithelial to mesenchymal transition, and in maintaining pluripotency and self-renewal characteristics of cancer stem cells. Finally, in silico analysis provided reliable information for drug-target interaction prediction.ConclusionsResults from the present study provide the first evidence for an oncogenic role of KLF7 in HGSOC, suggesting it as a promising prognostic marker and therapeutic target.

Epithelial/mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with miRNA let-7 levels and predicts tumor growth and metastasis.

Patient-derived samples present an advantage over current cell line models of high-grade serous ovarian cancer (HGSOC) that are not always reliable and phenotypically faithful models of invivo HGSOC. To improve upon cell line models of HGSOC, we set out to characterize a panel of patient-derived cells and determine their epithelial and mesenchymal characteristics. We analyzed RNA and protein expression levels in patient-derived xenograft (PDX) models of HGSOC, and functionally characterized these models using flow cytometry, wound healing assays, invasion assays, and spheroid cultures. Besides invitro work, we also evaluated the growth characteristics of PDX invivo (orthotopic PDX). We found that all samples had hybrid characteristics, covering a spectrum from an epithelial-to-mesenchymal state. Samples with a stronger epithelial phenotype were more active in self-renewal assays and more tumorigenic in orthotopic xenograft models as compared to samples with a stronger mesenchymal phenotype, which were more migratory and invasive. Additionally, we observed an inverse association between microRNA let-7 (lethal-7) expression and stemness, consistent with the loss of let-7 being an important component of the cancer stem cell phenotype. We observed that lower let-7 levels were associated with the epithelial state and a lower epithelial mesenchymal transition (EMT) score, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy. Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: Cells with lower let-7 levels were more tumorigenic, but less migratory, and with a lower EMT score, than those with higher let-7 levels. We conclude that let-7 expression and epithelial/mesenchymal state are valuable predictors of HGSOC proliferation, invitro self-renewal, and tumor burden invivo.

Abstract 1682: Epithelial-mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with let-7 levels and predicts tumor growth and metastasis

Abstract Objective: Patient-derived samples present an advantage over current cell line models of high grade serous ovarian cancer (HGSOC), which are flawed in terms of being reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we characterized a panel of patient-derived cells to determine their epithelial and mesenchymal characteristics, invasiveness, proliferation, stemness, and in vivo growth. Experimental procedures: Patient-derived xenograft (PDX) models of HGSOC were analyzed in vitro for phenotypic (RNA, let-7 miRNA, and protein expression, flow cytometry) and functional aspects including growth in spheroids, wound healing assays, invasion assays and in vivo (orthotopic PDX) growth characteristics. Results: Samples fell along the spectrum from epithelial to mesenchymal, and all had hybrid characteristics. Those toward the more epithelial end of the spectrum were most active in self-renewal assays, and grew most robustly in orthotopic xenograft models. Chemoresistance correlated both with the mesenchymal state and with BRCA2 wild type status. Loss of microRNA let-7 (lethal-7) is an important component of the cancer stem cell phenotype, and we observed an inverse association between let-7 expression and the epithelial state. We observed lower levels of let-7, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy in cells with the most epithelial phenotype. Conclusions: Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: epithelial cells (those with least let-7 expression) were less migratory, but more tumorigenic, than the mesenchymal cells with higher let-7 expression. We conclude that epithelial/mesenchymal state and let-7 expression are valuable predictors of HGSOC proliferation, in vitro self-renewal, and tumor burden in vivo. Citation Format: Juli Unternaehrer, Evgeny Chirshev, Nozomi Hojo, Antonella Bertucci, Linda Sanderman, Anthony Nguyen, Hanmin Wang, Tise Suzuki, Emmanuel Brito, Shannalee Martine, Christine Castañón, Saied Mirshahidi, Marcelo Vazquez, Kerby C. Oberg, Yevgeniya J. Ioffe. Epithelial-mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with let-7 levels and predicts tumor growth and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1682.

Abstract A30: Claudin-4-dependent mechanisms of high-grade serous ovarian cancer progression

Abstract In The Cancer Genome Atlas, Claudins-3, -4, and -7 are highly expressed in high-grade serous ovarian cancer (HGSOC). While claudins-3 and -7 are often expressed at tight junctions, claudin-4 (CLDN4) appears to be cytoplasmic, both in normal cells where it is expressed rarely and in a model of HGSOC, often used to study cellular interactions in this disease, OVCAR3 cells. CLDN4 expression reduces response to paclitaxel and conveys worse overall survival. We, therefore, wanted to further elucidate CLDN4-dependent mechanisms of cancer progression and therapeutic response. To address CLDN4 biology, we utilized immunohistochemical (IHC) analysis, loss-of-function studies, and several nonbiased approaches, including RNA-sequencing and proteomics. The IHC showed CLDN4 protein localized in the Golgi compartment with GM130 and around the cells at sites of cell-cell contact. Knocking down CLDN4 by 80% in OVCAR3 cells led them to undergo a transition from a more epithelial morphology to a mesenchymal phenotype (EMT). RNA-Seq analysis supports this morphologic interpretation by showing an increase in mRNA for genes coding for mesenchymal proteins such as fibronectin-1 (FN1), CD44, VIM, TNC, MMP2, and SNAI2 and a decrease in epithelial genes such as E-cadherin (CDH1), CD24, KRT18, and KRT19. RNA-Seq analysis of non-membrane protein kinases showed a 2-fold increase in PLAU, MSN, CAV1, CAV2, and EPHA2 and a 2-fold decrease in IGFBP2 and ERBB2, results that correlate well with the sensitivity of cancer cells to the BCR-ABL inhibitor, dasatinib. To determine the proteins that associate with CLDN4, we performed biotin ligase analysis (Bio-ID) fusing biotin protein ligase to CLDN4 and introducing the gene for the fusion protein into both OVCAR3 and OVCAR8 cells. Of the 36 proteins repeatedly more highly biotinylated in the presence of CLDN4-biotin ligase, 20 were identified as plasma membrane proteins and eight as Golgi localized by Gene Ontology analysis. Notably, we discovered a tyrosine protein kinase, YES1, to be biotinylated in both cell lines. YES1 is a poor prognostic indicator in HGSOC and promotes signal propagation following receptor tyrosine kinase activation, which contributes to a plethora of downstream outputs, including cell-cell adhesion and cell survival. These findings imply that CLDN4 expression is related to EMT and knockdown of CLDN4 could increase the sensitivity HGSOC to inhibitors such as dasatinib (BCR-ABL) or saracatinib (c-Yes). Citation Format: Patricia Webb, Margaret C. Neville, Heidi Baumgartner-Wilson, Miriam D. Post, Andrew Goodspeed, Benjamin G. Bitler. Claudin-4-dependent mechanisms of high-grade serous ovarian cancer progression [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 A30.

Targeting Fatty Acid Oxidation to Promote Anoikis and Inhibit Ovarian Cancer Progression

Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by cell detachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient deprivation. In this report, we examined the contribution of fatty acid oxidation (FAO) in supporting anoikis resistance. We examined expression Carnitine Palmitoyltransferase 1A (CPT1A) in a panel of HGSOC cell lines cultured in adherent and suspension conditions. With CPT1A knockdown cells, we evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, flow cytometry, and colony formation. We assessed CPT1A-dependent mitochondrial activity and tested the effect of exogenous oleic acid on anoikis and mitochondrial activity. In a patient-derived xenograft model, we administered etomoxir, an FAO inhibitor, and/or platinum-based chemotherapy. CPT1A is overexpressed in HGSOC, correlates with poor overall survival, and is upregulated in HGSOC cells cultured in suspension. CPT1A knockdown promoted anoikis and reduced viability of cells cultured in suspension. HGSOC cells in suspension culture are dependent on CPT1A for mitochondrial activity. In a patient-derived xenograft model of HGSOC, etomoxir significantly inhibited tumor progression. IMPLICATIONS: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable antitumor response and improve patient outcomes.

Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids

High-grade serous ovarian cancer (HG-SOC)—often referred to as a “silent killer”—is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear. Here, we establish organoid-based tumor progression models of HG-SOC from murine oviductal and OSE tissues. We use CRISPR-Cas9 genome editing to introduce mutations into genes commonly found mutated in HG-SOC, such as Trp53, Brca1, Nf1 and Pten. Our results support the dual origin hypothesis of HG-SOC, as we demonstrate that both epithelia can give rise to ovarian tumors with high-grade pathology. However, the mutated oviductal organoids expand much faster in vitro and more readily form malignant tumors upon transplantation. Furthermore, in vitro drug testing reveals distinct lineage-dependent sensitivities to the common drugs used to treat HG-SOC in patients.

Abstract C091: Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications

Abstract Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional control (e.g. MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g. RB1, CCNE1, CDKN2A alterations). SY-5609 is an oral, potent, and highly selective CDK7 inhibitor that is advancing through IND-enabling studies to support initiation of a planned Phase 1 oncology trial in early 2020. Here we report on the relationship between pharmacokinetics (PK), pharmacodynamics (PD), and tumor growth inhibition (TGI) in xenograft models of tumor types with transcriptional and/or cell cycle aberrations including high grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and estrogen receptor positive breast cancer (ER+BC). Methods: The relationship between SY-5609 PK, PD, and TGI was evaluated in xenograft models of HGSOC (OVCAR3) and TNBC (HCC70) using once daily (QD) or twice daily (BID) dosing via oral gavage. SY-5609 TGI was evaluated as a single agent (SA) QD in patient-derived xenograft (PDX) models of HGSOC (n=3), SCLC (n=4), TNBC (n=4), and in combination with once weekly fulvestrant in ER+BC PDX models selected in vivo for resistance to the CDK4/6 inhibitor palbociclib (n=1) or resistance to both palbociclib and fulvestrant (n=1). Results: SY-5609 plasma exposure was dose proportional and did not accumulate after repeated therapeutic doses. Dose-dependent transcriptional responses in xenograft tissue were observed within 4 hours of SY-5609 dosing and were sustained for 24 hours. TGI was dose-dependent, with tumor regressions observed at doses significantly below the maximum tolerated dose (MTD). Similar TGI was seen when the same daily dose was administered either QD or BID suggesting that the effect was driven by overall exposure or minimum concentration. In HGSOC, SCLC, and TNBC PDX models, SA SY-5609 induced >50% TGI in all models tested (11/11), with 7/11 (64%) demonstrating robust anti-tumor activity (≥90% TGI or regression): 3/3 HGSOC, 2/4 SCLC, and 2/4 TNBC. In a palbociclib-resistant ER+BC PDX model, the combination of SY-5609 and fulvestrant induced significant TGI (89%), with no evident tumor regrowth up to 21 days after dosing cessation, distinguishing the observed effects from SY-5609 SA or fulvestrant SA. In a palbociclib and fulvestrant double-resistant ER+ BC PDX model, SY-5609 SA resulted in 33% TGI and fulvestrant SA had no activity. In contrast, the combination of SY-5609 and fulvestrant demonstrated significant TGI (68%; p<0.0001 vs fulvestrant SA), suggesting re-sensitization to fulvestrant. Conclusions: We have characterized PK, PD, and anti-tumor activity of the CDK7 inhibitor SY-5609 in a series of xenograft models. Exposure in plasma is dose-proportional and does not accumulate at therapeutic dose levels. SY-5609 induces dose-dependent transcriptional responses in tumor xenograft tissue and shows robust TGI, including regressions, in PDX models derived from multiple solid tumor indications. These results highlight the broad potential for SY-5609 across a variety of solid tumor types, including treatment resistant ER+BC, and support the development of SY-5609 in early phase clinical trials. Citation Format: Liv H Johannessen, Shanhu Hu, Nan Ke, Anthony D'Ippolito, Nisha Rajagopal, Jason Marineau, Anneli Savinainen, William Zamboni, Graeme Hodgson. Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C091. doi:10.1158/1535-7163.TARG-19-C091

The CHK1 Inhibitor Prexasertib Exhibits Monotherapy Activity in High-Grade Serous Ovarian Cancer Models and Sensitizes to PARP Inhibition.

PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.

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<0.01) and a synergistic decrease in cell survival (p<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<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 1103: The effects of neo-adjuvant chemotherapy on myeloid cells in high-grade serous ovarian cancer metastases

Abstract Many tumours have abundant macrophage populations. Tumour-associated macrophages (TAMs) frequently have tumour promoting roles and are associated with poor clinical outcome. We hypothesise that targeting TAMs in high-grade serous ovarian cancer (HGSOC) may improve response to chemotherapy. We have assessed the effects of chemotherapy on TAM populations in human HGSOC obtained pre- and post-chemotherapy as well as in murine HGSOC models harbouring a relevant mutational profile. We find that chemotherapy treatment decreases TAM density within tumour areas. Furthermore, TAMs expressing markers known to associate with disease progression were decreased following chemotherapy. In vivo and in vitro we have demonstrated an up-regulation of inflammasome activation and TLR signalling in live myeloid cells following chemotherapy and have shown that macrophages are killed by chemotherapy at clinically relevant drug concentrations. These observations suggest a mechanism for TAM depletion and highlight chemotherapy induced activation of innate immunity in HGSOC. The majority of HGSOC patients respond well to first line chemotherapy but will relapse and succumb to treatment resistant disease. We have developed a murine model of HGSOC relapse after first-line chemotherapy, which has the potential to extend translational studies into this clinically important area. We have found that TAMs are re-established in tumours at relapse, suggesting a clinically defined window of opportunity to target TAMs in HGSOC following first-line chemotherapy. Overall, our results provide a rationale for targeted re-programming of TAMs in HGSOC after chemotherapy. Citation Format: Owen M. Heath, Eleni Maniati, Chiara Belato, Ganga Gopinathan, Laura Lecker, Anissa Lakhani, Colin Pegrum, Jacqueline McDermott, Michelle Lockley, Desmond P. Barton, Frances Balkwill. The effects of neo-adjuvant chemotherapy on myeloid cells in high-grade serous ovarian cancer metastases [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 1103.

Combined MEK and BCL-2/XL Inhibition Is Effective in High-Grade Serous Ovarian Cancer Patient-Derived Xenograft Models and BIM Levels Are Predictive of Responsiveness.

Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy in vitro in most models. In vivo, GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models-those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.

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&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&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.

Abstract 1322: A systems-level approach identifies novel links between iron and fatty acid metabolism in high-grade serous ovarian cancer

Abstract The low survival rate for high-grade serous ovarian cancer (HGSOC) motivates novel approaches to identify causative and therapeutically targetable factors involved in HGSOC initiation and progression. Cancer cells are known to sequester iron, which can potentiate cancer progression via mechanisms that have not yet been completely elucidated. In order to uncover novel, and potentially therapeutically tractable links, between iron and HGSOC, we developed an algorithm to identify perturbed regulatory pathways containing differentially expressed iron-related genes in microarray data from clinical sources and an experimental model of HGSOC. Used in tandem with over-representation analysis for iron-related genes, the algorithm led us to uncover an iron dependence of fatty acid import and synthesis pathways, which are upregulated in HGSOC and other cancers, and to develop a network synthesizing the relationship between iron, fatty acid metabolism, and HGSOC. We use the network to derive specific hypotheses of mechanisms by which iron impacts fatty acid metabolic pathways to promote tumorigenesis. We have thus shown that a systems-level approach to identifying novel regulatory links between iron and HGSOC has yielded a previously unappreciated association between iron and fatty acid metabolism that may be exploited for therapeutic potential. This work has been supported in part by NIH grants F32CA214030 to AK, R01CA188025 to SVT, and RO1CA171101 to FMT. Citation Format: Anna Konstorum, Miranda L. Lynch, Suzy V. Torti, Frank M. Torti, Reinhard C. Laubenbacher. A systems-level approach identifies novel links between iron and fatty acid metabolism in high-grade serous ovarian cancer [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 1322.

CDK4/6 inhibition as maintenance and combination therapy for high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is a disease with a high relapse rate and poor overall survival despite good initial responses to platinum-based therapy. Cell cycle inhibition with targeted CDK4/6 inhibitors is a new therapeutic approach showing promise as a maintenance therapy in cancer. As multiple genes in the CDK4/6 pathway are commonly mutated or dysregulated in ovarian cancer, we evaluated the efficacy of the CDK4/6 inhibitor Ribociclib alone, in combination with chemotherapy, and as maintenance therapy in several models of HGSOC. Ribociclib restricted cellular proliferation in multiple ovarian cancer cell lines. Restricted proliferation was associated with a pseudo-senescent cellular phenotype; Ribociclib-treated cells expressed markers of senescence, but could rapidly re-enter the cell cycle with discontinuation of therapy. Surprisingly, concurrent Ribociclib and cisplatin therapy followed by Ribociclib maintenance was synergistic. Evaluation of the cell cycle suggested that Ribociclib may also act at the G2/M check point via dephosphorylation of ATR and CHK1. Consistent with this mechanism, Ribociclib demonstrated clear activity in both platinum-resistant and platinum-sensitive tumor models in vivo. This work supports clinical trials using Ribociclib in combination with cisplatin and as a maintenance therapy in ovarian cancer.

A Genomically Characterized Collection of High-Grade Serous Ovarian Cancer Xenografts for Preclinical Testing

High-grade serous ovarian cancer (HGSC) is the leading cause of morbidity and mortality from gynecologic malignant tumors. Overall survival remains low because of the nearly ubiquitous emergence of platinum resistance and the paucity of effective next-line treatments. Current cell culture-based models show limited similarity to HGSC and are therefore unreliable predictive models for preclinical evaluation of investigational drugs. This deficiency could help explain the low overall rate of successful drug development and the decades of largely unchanged approaches to HGSC treatment. We used gene expression, copy number variation, and exome sequencing analyses to credential HGSC patient-derived xenografts (PDXs) as effective preclinical models that recapitulate the features of human HGSC. Mice bearing PDXs were also treated with standard-of-care carboplatin therapy. PDXs showed similar sensitivity to carboplatin as the patient's tumor at the time of sampling. PDXs also recapitulated the diversity of genomic alterations (copy number variation and mutation profiles) previously described in large data sets that profiled HGSC. Furthermore, mRNA profiling showed that the PDXs represent all HGSC subtypes with the exception of the immunoreactive group. Credentialing of PDX models of HGSC should aid progress in HGSC research by providing improved preclinical models of HGSC that can be used to test novel targets and more accurately evaluate their likelihood of success.

RNA-Peptide nanoplexes drug DNA damage pathways in high-grade serous ovarian tumors.

DNA damaging chemotherapy is a cornerstone of current front‐line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo‐resistant disease; therefore, adjuvant treatments that synergize with DNA‐damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide‐nucleic acid complex that facilitates tumor‐specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex‐mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high‐grade serous OC to cytotoxic chemotherapy by blocking p38/MK2‐dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.