Multiple Ovarian Cancer Research Articles

Abstract B07: Targeting DAB2IP in ovarian cancer stem cells

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancies. Failure to eliminate ovarian cancer stem cells (OCSCs) by the end of conventional chemotherapy contributes to tumor relapse and metastasis. Understanding the molecular features of OCSCs may allow for effective targeting and eradicating these cells and tumor remission. We reported tumor-initiation and expansion capacity by ALDH+ OCSCs after platinum-based treatment of ovarian cancer xenografts. In addition, loss of DAB2IP in prostate and colon cancer promoted CSC-like features, suggesting a critical role in modulating CSC maintenance. DAB2IP, a novel member of Ras GTPase-activating (GAP) protein family, was identified as a potent tumor suppressor that interfered with many aspects of cancer progression and was frequently downregulated in tumors. In the current study, we aimed to elucidate the tumor-suppressor role of DAB2IP in ovarian cancer in the context of OCSCs and explore epigenetic approaches to upregulate its expression. OCSCs and non-CSCs subpopulations were isolated from multiple high-grade serous ovarian cancer cell lines (Kuramochi, OVCAR3, and COV362). Reduced (p<0.05) DAB2IP expression in OCSCs compared to non-CSC counterparts was observed, suggesting that DAB2IP suppresses OCSC maintenance. Overexpression of DAB2IP in OCSCs derived from Kuramochi decreased (p<0.05) OCSC population, and DAB2IP overexpression inhibited stemness-related phenotypes, including reduced (P<0.05) spheroid formation ability after 14-day incubation under stem cell conditions and decreased (P<0.05) colony formation. Decreased expression of stem cell markers (Oct4, Nanog, and Twist) in DAB2IP-overexpressing cells was also observed, indicating potential key effectors downstream of DAB2IP. Furthermore, elevated DAB2IP expression in OCSCs decreased (p<0.05) cisplatin IC50 and cell migration capacity in transwell assay, suggesting DAB2IP plays a role in OCSC function. Aberrant DNA promoter methylation and histone modifications were previously shown to be two major mechanisms of DAB2IP inactivation in cancer. We treated OCSCs with next-generation DNMT inhibitor guadecitabine and EZH2 inhibitor GSK-126, either alone or in combination. Single-agent guadecitabine or GSK-126 decreased ALDH expression with greater (p<0.05) effect in combination. Single GSK-126 was sufficient to increase DAB2IP expression (p<0.05) with additional increase when combined with guadecitabine, correlating with decreased ALDH expression. Collectively, our data suggested a possible combination epigenetic therapy to eliminate OCSCs, potentially through targeting DAB2IP, an important OCSC regulator. Citation Format: Xingyue Zong, Ali R. Ozes, Kenneth P. Nephew. Targeting DAB2IP in ovarian cancer stem cells. [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 B07.

Abstract A51: FOXM1 inhibition by thiostrepton synergizes with olaparib by attenuating adaptive response in ovarian cancer cells

Abstract Background: High-grade serous ovarian carcinoma (HGS-OvCa) is the most common subtype of epithelial ovarian cancer and accounts for around 70% of all ovarian cancers in the US. Cancer cells that show “BRCAness” are highly sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), including those with deleterious mutations in BRCA1/2 or defects in other components that are crucial for homologous recombination (HR) repair pathway. In HGS-OvCa, genes involved in HR are altered in about 50% of cases, making these cancers sensitive to PARPi. However, FOXM1 transcription factor network is activated in more than 84% of cases in HGS-OvCa, and activation of the FOXM1 pathway has been shown to upregulate genes involved in HR, which can lead to PARPi resistance. Therefore, FOXM1 becomes an attractive therapeutic target for exploring new combination therapies to overcome PARPi resistance and enhancing its therapeutic effects. Hypothesis: We hypothesize that FOXM1 inhibition induces “BRCAness” and sensitizes ovarian cancer cells to PARP inhibitors. Results: Olaparib increases the expression and nuclear localization of FOXM1 levels and FOXM1 target genes, such as BRCA1 and RAD51, suggesting that FOXM1 may play an important role in adaptive response after olaparib treatment. Furthermore, FOXM1 expression levels inversely correlates with olaparib sensitivity in multiple ovarian cancer cells. Downregulation of FOXM1 by siRNA sensitizes ovarian cancer cells to olaparib, while pharmacologic inhibition of FOXM1 by thiostrepton synergizes with olaparib as shown by colony formation assays. Thiostrepton downregulates DNA repair and anti-apoptotic genes, such as BRCC3, BRCA1/2, FANCF, and BCL2, and upregulates pro-apoptotic genes DDIT3, DDIT4, and PD-L1 in multiple ovarian cancer cell lines. Consistently, thiostrepton activates caspase 3 activity, induces apoptosis, and enhances DNA damage with combination of thiostrepton and olaparib in comet assay. Thiostrepton also increases PARP1 trapping onto chromatin in OVCA420 cells treated with olaparib. Finally, thiostrepton resensitizes rucaparib-resistant cells derived from MDA-MB-436 to rucaparib in cell viability assay and colony formation assay. Thiostrepton downregulates the stabilized mutant BRCA1, which has been shown to cause the resistance to PARPi in these cells, suggesting that FOXM1 may play a key role in acquired PARPi resistance, which can be overcome by inhibition of FOXM1. Conclusions: These data demonstrate that FOXM1 plays an important role in adaptive response induced by olaparib in ovarian cancer cells. Inhibition of FOXM1 by thiostrepton increases apoptosis and sensitizes ovarian cancer cells to olaparib by inducing “BRCAness” through downregulating HR genes. Interestingly, we also find that thiostrepton could increase PARP trapping onto chromatin and further enhance DNA damage. Moreover, FOXM1 inhibition by thiostrepton could resensitize acquired PARPi-resistant cells, probably through downregulating stabilized mutant BRCA1. Together, all these data suggest that FOXM1 inhibition represents an effective strategy to overcome PARPi resistance, and inhibition of adaptive response by FOXM1 may produce better therapeutic effects for PARP inhibitors. Citation Format: Pingping Fang, Jill Madden, Lisa Neums, Jeremy Chien. FOXM1 inhibition by thiostrepton synergizes with olaparib by attenuating adaptive response in ovarian cancer cells. [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 A51.

Tripeptide-Stabilized Oil-in-Water Nanoemulsion of an Oleic Acids-Platinum(II) Conjugate as an Anticancer Nanomedicine.

We report a nanoemulsion (NE) which is stabilized by self-assembling tripeptide lysine-tyrosine-phenylalanine (KYF) and encapsulates an oleic acids-platinum conjugate formed using simple Pt (II) coordination chemistry. The KYF-Pt-NE is evaluated both in cultured ovarian cancer cells and in an in vivo preclinical cancer model and shows pH dependent Pt (II) release, which is low at physiological pH and enhanced at tumoral pH. The biological activity of KYF-Pt-NE, evaluated in multiple ovarian cancer cell lines, is significantly higher when compared to the analogous Pt (II) complex used in the clinic. Concurrently, the KYF-Pt-NE platform shows good compatibility with the immune system. Preliminary in vivo testing of KYF-Pt-NE with tumor bearing mice indicates efficient Pt (II) delivery to the tumor. Together, these results demonstrate the potential of peptide-stabilized nanoemulsions, specifically KYF-Pt-NE as an effective nanomedicine against cancer.

Interleukin 8 is a biomarker of telomerase inhibition in cancer cells

BackgroundTelomerase activity is required for both initiation and maintenance of tumorigenesis and over 90% cancers overexpress telomerase. Therefore, telomerase targeting has emerged as a potential strategy for cancer treatment. In agreement with this, several telomerase inhibitors are being tested for cancer treatment and have shown some promise. However, because of the variability in response between the cancer patients, it is important to identify biomarkers that allow for distinguishing cancers that are responsive to telomerase inhibition from the cancers that are not. Therefore, in this study we performed experiments to identify a biomarker that can be used to predict telomerase inhibition induced tumor growth inhibition.MethodsIn our study, we have performed transcriptome-wide gene expression analysis on multiple ovarian and colon cancer cell lines that were treated with telomerase inhibitor imetelstat and were responsive to telomerase inhibition-induced tumor growth attenuation.ResultsWe demonstrate that telomerase inhibition by telomerase inhibitor imetelstat results in decreased expression of interleukin 8 (IL8) in all telomerase responsive cancer cell lines. This phenomenon is of general occurrence because we find that multiple ovarian and colon cell lines show decrease in IL8 mRNA and protein levels after telomerase inhibition. Additionally, we find loss of IL8 phenocopy Telomerase inhibition mediated growth inhibitory effect in cancer cells.ConclusionTaken together, our results show that IL8 is a biomarker that predict telomerase inhibition mediated growth attenuation of cancer cells and its loss phenocopy telomerase inhibition. Therefore, IL8 expression can be utilized as a biomarker for telomerase targeted cancer therapies to potentially predict therapeutic response.

Pterostilbene Suppresses Ovarian Cancer Growth via Induction of Apoptosis and Blockade of Cell Cycle Progression Involving Inhibition of the STAT3 Pathway.

A growing body of evidence has demonstrated the promising anti-tumor effects of resveratrol in ovarian cancer cells, including its inhibitory effects on STAT3 activation. Nonetheless, the low bioavailability of resveratrol has reduced its attractiveness as a potential anti-cancer treatment. In contrast, pterostilbene, a stilbenoid and resveratrol analog, has demonstrated superior bioavailability, while possessing significant antitumor activity in multiple solid tumors. In this study, the therapeutic potential of pterostilbene was evaluated in ovarian cancer cells. Pterostilbene reduces cell viability in several different ovarian cancer cell lines by suppressing cell cycle progression and inducing apoptosis. Further molecular study has shown that pterostilbene effectively suppressed phosphorylation of STAT3, as well as STAT3 downstream genes that regulate cell cycle and apoptosis, indicating that inhibition of STAT3 pathway may be involved in its anti-tumor activity. The addition of pterostilbene to the commonly used chemotherapy cisplatin demonstrated synergistic antiproliferative activity in several ovarian cancer cell lines. Pterostilbene additionally inhibited cell migration in multiple ovarian cancer cell lines. The above results suggest that pterostilbene facilitates significant anti-tumor activity in ovarian cancer via anti-proliferative and pro-apoptotic mechanisms, possibly via downregulation of JAK/STAT3 pathway. Pterostilbene thus presents as an attractive non-toxic alternative for potential adjuvant or maintenance chemotherapy in ovarian cancer.

Abstract 874: Pharmacologically induced RAS-GTP levels and CRAF-BRAF hetero-dimerization drive sensitization to Type II pan-RAF inhibitors in KRAS mutant cancer

Abstract Although effective in BRAF V600 mutant melanoma, Type 1.5 RAF inhibitors such as vemurafinib and dabrafenib have not proven to be successful in KRAS mutant cancers, neither as single agent nor in combination with MEK inhibitors. Through large-scale cellular compound combination screening we found that Type II RAF inhibitors such as AZ-628 do show synergistic activity with MEK inhibitors in multiple KRAS mutant indications, including NSCLC, colorectal cancer and ovarian cancer. The combination of Type II RAF inhibitors and MEK inhibitors demonstrates robust and durable abrogation of MAPK signaling both on canonical markers of MAPK activity such as pERK and pRSC as well as transcriptional output. We also observe synergistic in vivo tumor growth inhibition in two independent models of KRAS mutant cancer by this combination treatment. We found that treatment with MEK inhibitors alone drives the increase of active RAS-GTP levels and induces CRAF:BRAF hetero-dimerization. These induced dimers are active and able to phosphorylate MEK in vitro. This increased dimerization renders cells sensitive to Type 2 RAF inhibitors. We find that this effect is not limited to KRAS mutant cells, as a subset of KRAS wild-type cells show increased RAS-GTP levels upon MEK inhibitor treatment. These cells also show synergistic sensitivity to Type 2 RAF inhibition. Additionally, we observed significantly higher synergy and higher RAS-GTP levels in KRAS G13D mutant cells, which have intrinsically high GDP exchange and low intrinsic GTP hydrolysis. Finally, we show that GDC-0941 and GDC-0032, two broad PI3K inhibitors, also induce RAS-GTP levels in cells independent of PIK3CA or KRAS mutation status. We subsequently observed a synergistic sensitivity to Type 2 RAF inhibitors in these PI3K inhibitor-treated cells. Overall, we demonstrate that pharmacologic inhibition of MEK or PI3K increases RAS-GTP levels and drives increased CRAF:BRAF hetero-dimerization. This in turn sensitizes cells to Type 2 RAF inhibition, leading to a synergistic drug effect. Combination of these inhibitors may be a viable therapeutic approach in KRAS mutant cancer, and may be especially effective in KRAS G13D-carrying tumors. Citation Format: Christiaan N. Klijn, Ivana Yen, Frances Shanahan, Mark Merchant, Christine Orr, Thomas Hunsaker, Matthew Durk, Hank La, Xiaoling Zhang, Scott Martin, Eva Lin, John Chan, Yihong Yu, Dhara Amin, Amy Gustafson, Scott Foster, Joachim Rudolph, Shiva Malek. Pharmacologically induced RAS-GTP levels and CRAF-BRAF hetero-dimerization drive sensitization to Type II pan-RAF inhibitors in KRAS mutant 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 874.

Abstract 715: Expression of tumor cell-associated immune checkpoint molecules in multiple human solid tumors

Abstract Cancer immunotherapy using monoclonal antibodies targeting inhibitory immune checkpoint molecules is being established as a new paradigm in cancer treatment. This crucial strategy involves inhibition of the interaction between immune checkpoint receptors, expressed on the immune cells, and their respective ligands, expressed on antigen-presenting cells. While this receptor-ligand concept is well accepted in infection immunology, there have been numerous reports on the expression of immune checkpoint molecules in tumor cells. In addition to well-characterized tumor-intrinsic expression of PD11 and PD-L12, many other checkpoint molecules are associated with tumor cells3, conferring important impact on tumor biology and clinical consequences. In this study, we evaluated in situ single-cell expression profiles of immune checkpoint molecules in the tumor microenvironment (TME) of archived FFPE tissues from multiple solid cancers, including breast, kidney, ovarian, stomach, colorectal, and non-small cell lung cancer. Applying the RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, tissue microarrays consisting of 20-35 FFPE tumors per each cancer type were stained for key therapeutic targets including PD1, PD-L1, TIM3, and LAG3. We observed that, in addition to PD-L1, all evaluated immune checkpoint molecules were expressed in the tumor cells of multiple tumor types though at a lower level than in immune cells. While there was notable heterogeneity of expression in different tumor regions in some samples, the single-cell expression pattern of each checkpoint molecule across all tumor cells in each region suggested a potential tumor-intrinsic mechanism for the expression of these molecules. In selected lung and ovarian cancers, fluorescence multiplex ISH-IHC staining confirmed that multiple different checkpoint molecules were co-expressed together in individual tumor cells. These findings add to the growing evidence that immune checkpoint genes can be expressed in tumor cells in addition to immune cells, which will be missed by gene expression analysis techniques using bulk tissue. The RNAscope® ISH platform is especially well suited for this type of analysis since it allows for assessing cell type-specific expression of any gene within the native tissue architecture even when it is expressed at very low levels. The approach illustrated in this study may help to better understand the anti-tumor activities of immune checkpoint inhibitors and provide new insights into better predictive biomarkers and therapeutic strategies.

Abstract 3159: Rac1 and Cdc42 as drivers in ovarian cancer metastasis

Abstract Five-year ovarian cancer patient survival has not improved for decades, largely due to disease recurrence. Therefore, drivers of relapse need to be identified. We first reported that Rac1 and Cdc42 GTPases are highly overexpressed in serous ovarian cancer using immunohistochemistry. To correlate GTPase expression with survival outcomes, we analyzed gene expression data in The Cancer Genome Atlas, revealing that ovarian cancer patients with the highest RAC1 RNA levels had significantly worse age-adjusted survival than those with low RAC1. Quantitative analyses of Rac1b, a constitutively active Rac1 splice variant, show dramatic overexpression in tumor epithelia relative to stroma and normal epithelia. Since high Rac1 and Cdc42 levels drive cancer cell adhesion, invasion and metastasis, we used multiple assays to assess the dependence of ovarian cancer cell behaviors on Rac1 and Cdc42. Using Matrigel invasion chambers, treatment of multiple ovarian cancer cell lines with specific Rac1 and Cdc42 inhibitors resulted in significantly decreased cellular invasion. Conversely, with overexpression of Rac1, ovarian cancer cells demonstrated increased invasion. Furthermore, IP injection of ovarian cancer cells overexpressing Rac1 resulted in increased adhesion, invasion and metastasis in the omentum and peritoneum in vivo. These data support Rac1 and Cdc42 as important drivers of invasive and metastatic ovarian cancer and as novel high-value therapeutic targets with potential impact in reducing disease metastasis. Citation Format: Melanie Rivera, Dayna Dominguez, Christine Pauken, Elsa Romero, S. Ray Kenney, Yang Shi, Ji-Hyun Lee, Jennifer Gillette, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 and Cdc42 as drivers in ovarian cancer metastasis [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 3159.

Abstract 5474: Activity of the S1P pathway promotes ovarian cancer and serves as a novel metabolic target of metformin

Abstract Sphingosine-1-phosphate (S1P), a metabolite of the phospholipid sphingosine, is a bioactive lipid that directly interacts with specific G-protein-coupled receptors to transduce signals that regulate growth, proliferation and motility. Sphingosine kinases (SPHK1 and 2) catalyze the formation of S1P from sphingosine, thereby regulating S1P homeostasis and the sphingolipid rheostat. Recently, using TCGA data, we noted SPHK1 amplification in several gynecologic cancers and that high expression of SPHK1 was associated with increased mortality in high-grade serous ovarian cancer (HGSOC). The findings reported here show that exposure of HGSOC tumor cells to exogenous S1P, or overexpression of SPHK1, induced migration, proliferation and clonogenecity in multiple ovarian cancer cell lines in vitro. Likewise, in a xenograft mouse model of ovarian cancer, overexpression of SPHK1 markedly enhanced tumor growth. In prior pre-clinical studies, we have demonstrated a strong protective effect of the diabetes medication metformin in ovarian cancer. Interestingly, we found that patients with HGSOC that use metformin for diabetes have reduced serum S1P levels compared to controls, suggesting that the sphingolipid rheostat may be a novel metabolic target of metformin in ovarian cancer. Supporting this, we identified that SPHK1 expression in ovarian cancer cell lines is reduced by treatment with metformin, and further that the reduction of SPHK1 by metformin was mediated through inhibition of transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Finally, we show that overexpression of SPHK1 in HGSOC cell lines enhanced the cytotoxic effects of metformin. Taken together, our data indicates that hypoxia-induced SPHK1 expression and downstream S1P signaling promote tumor progression in HGSOC, and that tumors utilizing this pathway may be particularly vulnerable to the anti-cancer effects of metformin. Citation Format: Peter C. Hart, Tatsuyuki Chiyoda, Marion Curtis, Xiaojing Liu, Chun-Yi Chiang, Stephanie McGregor, Ricardo Lastra, Jason Locasale, Ernst Lengyel, Iris L. Romero. Activity of the S1P pathway promotes ovarian cancer and serves as a novel metabolic target of metformin [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 5474.

Abstract 1619: Personalized medicine: A CLIA-certified high-throughput drug screening platform for ovarian cancer

Abstract Background: Metastatic disease in ovarian cancer is difficult to treat and patients often exhaust standard-of-care regimens. To get a better understanding of potential treatments, genomic data is used in some cases. However, this only points to therapeutic options in a minority of cases highlighting an urgent need to develop assays to identify potential therapies. Current functional tests are limited in the number of tested drugs. Here we present the first CLIA certified high-throughput functional assay employing organoid cultures derived from primary patient specimens to directly aid oncologists for personalized treatment selection (P.A.R.I.S. Assay, SEngine Precision Medicine, Seattle, WA). Experimental Procedures: Organoids are treated for 6 days with a library of 123 clinically relevant drugs. Compounds are evaluated at a multi-dose response curve and ranked by SPM score, which weights both the sensitivity (degree of cell death) and specificity, which compares the patient's tumor cells response to the drug relative to all prior patients. The results are further integrated with genomic data and reported to the clinician to highlight treatment options. SEngine has performed >150 drug screens and established high reproducibility including multiple ovarian cancer cases from either ascites, surgical or core biopsies. Results: Here we present two n-of-one patient studies. Patient 1 is a 48 year old woman with positive family history who was diagnosed with late stage serous ovarian cancer. SEngine generated cancer organoids and performed high-throughput screening with a panel of 123 drugs. Sensitivities to PARP inhibitors prompted SEngine to advise for germline testing which revealed a BRCA1 and TP53 mutation. The cells were also uniquely sensitive to paclitaxel and the combination with carboplatin resulted in remission indicating concordance with clinical data. In addition, highlighting the importance of functional screening, a unique response to a sub-group of EGFR inhibitors was identified, of potential consideration in case of recurrence. The second case also has a family history of ovarian cancer, but no BRCA mutations were detected. Genomic data indicated FGF6 and FGF23 amplification which directly corresponded to a unique sensitivity to one FGFR inhibitor (AZD-4547). The organoids were also resistant to PARP inhibitors, consistent with the absence of BRCA mutations. Impact: We developed a robust ex vivo screening platform to objectively quantify patient specific sensitivity to a panel of 123 oncology drugs. SEngine is compiling a registry capturing clinical data, outcome following the P.A.R.I.S. test as well as genomic data. The power of high throughput technology and organoid isolation will enable the rapid selection of optimal individualized therapies as single agents or in combination. Citation Format: Hallie A. Swan, Rachele Rosati, Caroline Bridgwater, Michael J. Churchill, Roland M. Watt, Reid C. Shaw, Stephanie A. Murphy, Robert L. Diaz, Shalini C. Pereira, Franz X. Schaub, Carla Grandori. Personalized medicine: A CLIA-certified high-throughput drug screening platform for 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 1619.

EPIDEMIOLOGICAL ASPECTS OF OVARIAN CANCER IN THE STRUCTURE OF PRIMARY MULTIPLE MALIGNANT TUMORS OF THE FEMALE REPRODUCTIVE SYSTEM (ON THE EXAMPLE OF THE CHELYABINSK REGION)

The problem of primary multiplicity of malignant tumors remains relevant in oncology due to the growing number of patients with polyneoplasia, especially for tumors of the female reproductive system. The analysis of the frequency and combinations of polyneoplasias of the reproductive system in women on the example of primary multiple ovarian cancer according to the data of the Chelyabinsk Regional Clinical Center of Oncology and Nuclear Medicine for 15 years. The calculated indicators of the adjusted cumulative survival of patients with polyneoplasia and solitary ovarian cancer were calculated. The data obtained can be used in clinical practice, as the primary and secondary prevention of cancer in women.

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.

Phase I/II study of low-dose cyclophosphamide, tumor-associated peptide antigen-pulsed dendritic cell and low dose GM-CSF for solid and hematological tumors.

TPS216 Background: Despite advances in understanding the biology of hematologic malignancies (HM) and solid malignancies (SM), and the availability of new treatment options, many patients with HM and SM remain incurable. Since the majority of cancer patients display a defective immune response to tumor antigens, the ex-vivo activation of dendritic cells (DC), through their exposure to tumor associated antigens, is an attractive and active area of investigation. We and others have explored the potential immunotherapeutic targets included our DC vaccine, i.e., SP17, Ropporin, AKAP4, PTTG1, SpanxB, NY-ESO-1, MAGE-1, HM1.24, Her2/Neu. Notably, SP17-pulsed DC have been reported to induce a safe and clinically significant response in patients with multiple myeloma and ovarian cancer. Methods: We hypothesize that treatment of patients with HM or metastatic SM using autologous DC will result in antigen-specific CD4+ T-cell and/or CD8+ CTL responses without significant toxicities. We also hypothesize that the responses generated against specific antigens may translate into clinical anti-tumor activity. Primary Objective: Phase I (6 subjects). To determine safety of intradermal/subcutaneous DC vaccine therapy, low-dose cyclophosphamide and GM-CSF, in patients with metastatic SM or HM who demonstrate a response, or whose disease remains stable, after conventional first-line systemic therapy, or who have failed conventional systemic therapy. Secondary Objective: Phase II (up to 17 subjects). To determine immune responses associated with intradermal/subcutaneous DC vaccine therapy, low-dose cyclophosphamide and GM-CSF, in patients with metastatic SM or HM who demonstrate a response, or whose disease remains stable, after conventional first-line systemic therapy, or who have failed conventional systemic therapy. The study population is currently drawn from patients at various clinical institutions following contractual agreements. Clinical trial information: NCT02709993; NCT02705703; NCT02224599; NCT02223312.

Abstract A102: A comparative pharmacokinetic-pharmacodynamic-treatment study of PARP inhibitors demonstrates favorable properties for niraparib activity in preclinical tumor models

Abstract Introduction: Niraparib is an orally bioavailable and selective poly (ADP-ribose) polymerase (PARP)-1/-2 inhibitor approved for maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in a complete or partial response to platinum-based chemotherapy. This study examined the pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of niraparib as compared to two other approved PARP inhibitors, olaparib and rucaparib, in several ovarian cancer and triple-negative breast cancer xenograft models. Experimental Procedures: Niraparib, olaparib, and rucaparib were administered orally to mice at near maximum tolerated dose (MTD) for each drug in the BRCA1 deficient MDA-MB-436 cell line-derived triple-negative breast cancer model, BRCA wildtype A2780 cell line-derived ovarian cancer model, and two BRCA wildtype patient-derived ovarian cancer xenograft models. Tumor growth was monitored twice per week post randomization for the efficacy studies. Tumor growth inhibition was calculated after prolonged treatment at the end of study for each model. For PK and PD analysis, plasma, tumor, and brain tissue samples were collected at 7 different time points on the 5th day of continuous treatment. Results: Niraparib demonstrated potent antitumor activities in both the BRCA deficient and BRCA wildtype models tested. Among the 3 clinically approved PARP inhibitors, niraparib consistently demonstrated a high level of antitumor activity at a relatively lower dose. Niraparib showed high tumor exposure (AUC0-24h: 213,959 ng/g•hr) and high brain exposure (AUC0-24h: 18,721 ng/g•hr) as compared to rucaparib (tumor AUC0-24h: 74,338 ng/g•hr; brain AUC0-24h: 4,380 ng/g•hr) and olaparib (tumor AUC0-24h: 7,586 ng/g•hr; brain AUC0-24h: 336 ng/g•hr) in MDA-MB-436 xenograft model at steady state. A unique feature of niraparib was that its exposure in tumor was more than 3 times higher than that in plasma, which is consistent with its high volume of distribution in the animal models and the clinical setting. Pharmacodynamic analysis indicated that niraparib is able to deliver ~90% PARP inhibition for 24 hours at steady state. Conclusions: Niraparib treatment induces potent antitumor activities in multiple ovarian and breast cancer models regardless of BRCA status. The potent antitumor activity induced by niraparib is consistent with its high exposure and durable PARP inhibition in tumor. Our nonclinical findings suggest that niraparib exhibits unique pharmacologic properties within the PARP class of inhibitors. Citation Format: Kaiming Sun, Grace Poon, Sarah Wang, Paul Vancutsem, Sridhar Ramaswamy, Jeffrey Hanke, Jing Yu Wang. A comparative pharmacokinetic-pharmacodynamic-treatment study of PARP inhibitors demonstrates favorable properties for niraparib activity in preclinical tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A102.

Identification of a pyridine derivative inducing senescence in ovarian cancer cell lines via P21 activation.

Cellular senescence is a state of irreversible cell growth arrest. Increasing evidence suggests that cellular senescence contribute to tumour suppression in vivo. However, only a few anti-cancer drugs have been discovered to induce cellular senescence. Searching for new compounds which can inhibit cancer cell growth by inducing senescence is becoming one of the most attractive research fields. To test the effects of candidate compounds on cancer cell growth, cell proliferation assays, senescence-associated β-galactosidase (SA-β-gal) staining, and flow cytometry assay were performed. Immunofluorescence, western blot, and qRT-PCR experiments were used to further study the molecular mechanisms of the candidate compounds. We demonstrated that a pyridine derivative, 4-(4-fluorophenyl)-2-phenyl-5, 6, 7, 8-tetrahydroquinoline (FPTHQ), from a pool of 46 compounds can induce senescence of ovarian cancer cells in a dose-dependent manner. FPTHQ caused growth inhibition by inducing G0/G1 cell cycle arrest in A2780 cells. Increased activities of SA-β-gal were observed in FPTHQ-treated A2780, OVCAR-3 and SKOV-3 cell lines. In addition, FPTHQ treatment increased the protein levels of MMP3 and the mRNA levels of IL-6 and IL-8 in A2780 cells, indicating the appearance of senescence-associated secretary phenotype (SASP) in the cells. Furthermore, we found that p21 was up-regulated and DNA damage was accumulated in FPTHQ-treated ovarian cancer cells. So far, our data suggest that FPTHQ can induce senescence in multiple ovarian cancer cell lines through activation of p21 signalling pathway by causing excessive DNA damage.

Abstract 5084: Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action

Abstract ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein. Citation Format: Amaury E. Fernández-Montalván, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Díaz-Sáez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hübner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [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 5084. doi:10.1158/1538-7445.AM2017-5084

Abstract 2556: Loss of progesterone receptor through epigenetic regulation is associated with poor prognosis in multiple solid tumors

Abstract Introduction: As a tumor suppressor in the endometrium, progesterone and its synthesized analogue progestin have a long history as a treatment for endometrial cancer. Loss of progesterone receptor (PR) in endometrial cancer leads to therapeutic failure, and our group has identified several mechanisms underlying PR loss, most notably epigenetic silencing of PR transcription. Recently, a Finnish group demonstrated that progestin therapy in premenopausal women is associated with a lower incidence of not only endometrial cancer, but also ovarian, pancreatic and lung cancers. This unexpected protective function of progestin in organs outside of the reproductive system led us to hypothesize that tumor progression effects in endometrial, ovarian, pancreatic and lung cancer is occurs due to the loss of progesterone’s protective effects. Methods and results: Supporting a potential protective role of progesterone, we discovered that PR is downregulated in multiple tumors, including breast, endometrial, ovarian, cervical, pancreatic and lung cancers, and loss of PR was consistently associated with a poor prognosis based on analysis of the Oncomine database. Similarly, PR expression was low in multiple ovarian, pancreatic and lung cancer cells. Mechanistic studies revealed that PR downregulation was mediated by the polycomb-repressor complex and DNA methylation, though the precise mechanism differed among cell lines and cancer types. Treatment with epigenetic modulators, which are FDA-approved for multiple myeloma, restored functional PR expression at both the mRNA and protein level and promoted marked cell death through induction of apoptosis. . Moreover, overexpression of PR or treatment with epigenetic modulators in combination with progesterone enhanced this effect, indicating a key protective role for progesterone signaling through PR. Conclusion: These data suggest loss of PR may be a general theme for tumorigenesis or disease progression in multiple tumor types beyond the classically studied endometrial cancer. In addition, the epigenetic mechanisms contributing to PR downregulation in endometrial tumors were recapitulated in other tumor types, including ovarian, pancreatic and lung tumors. These studies collectively set the stage for use of progestin therapy in combination with epigenetic modulators, a concept we term “molecularly enhanced progestin therapy,” as a novel therapeutic approach for many cancers. This combinatorial strategy has the power to revitalize the use of epigenetic modulators, which have proved disappointing in solid tumors, as an approach to resensitize cancer cells to a tumor suppressor. Citation Format: Yiyang Li, Tamar Kavlashvili, Cheng Huang, Yuping Zhang, Xiangbing Meng, Kristina Thiel, Kimberly Leslie, Shujie Yang. Loss of progesterone receptor through epigenetic regulation is associated with poor prognosis in multiple solid tumors [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 2556. doi:10.1158/1538-7445.AM2017-2556

Abstract 1165: Plitidepsin targets the moonlighting functions of eEF1A2 in cancer cells

Abstract Plitidepsin, a cyclic depsipeptide of marine origin, has shown potent anticancer activity in preclinical assays and recently finished with positive results a pivotal phase III trial (clinicaltrials.gov identifier: NCT01102426) for the treatment of multiple myeloma patients. We have recently found that eukaryotic elongation factor 1A2 (eEF1A2), one of the two isoforms of the alpha subunit of eEF1, is the pharmacological target of plitidepsin. Although it shares 96% homology with eEF1A1 (the other isoform), they display an exclusive pattern of expression, being eEF1A2 solely expressed in brain and muscle in healthy individuals. However, it has been found that many tumors abnormally overexpress this protein, including multiple myeloma, prostate, pancreatic, ovarian, breast, lung and liver cancers. Furthermore, although eEF1A2 canonical function consists in the delivery of aminoacyl-tRNAs to the A site in the ribosome, it has been shown to have pro-oncogenic moonlighting activities, including inhibition of apoptosis, protein degradation by the proteasome, heat shock response, cytoskeleton organization and regulation of oxidative stress. We now investigated several of the pro-oncogenic activities of eEF1A2 to analyze the impact that plitidepsin could have preventing them. Indeed, we observed that plitidepsin interfered with the interaction between eEF1A2 and Peroxiredoxin 1 (PRDX1), a complex that allosterically enhances the enzymatic activity of PRDX1. This way, plitidepsin would diminish PRDX1 antioxidant activity, possibly originating the oxidative stress that has been described in the bibliography as one of the first effects triggered by the drug in cancer cells. PRDX1 only interacts with the GDP-bound form of eEF1A2, while plitidepsin exclusively binds to the GTP-bound form, most probably sequestering this protein from the pool that could interact with and activate PRDX1. Furthermore, we have confirmed that eEF1A2 interacts with Sphingosine kinase 1 (SPHK1), a complex that has been described in the bibliography as having enhanced SPHK1 activity. SPHK1 phosphorylates sphingosine producing sphingosine-1-P, second messenger that binds to its receptors in the cell membrane and conveys growth and survival signals to the cell. We could see that plitidepsin treatment reduced the production of sphingosine-1-P in HeLa cells, destabilizing the equilibrium towards the pro-apoptotic ceramide/sphingosine side and promoting cell death. Thus, through its binding to eEF1A2, plitidepsin derails a series of its moonlighting functions that are essential for the survival of tumor cells, driving them into apoptosis. Citation Format: Alejandro Losada, Maria Jose Muñoz, Juan F. Martínez-Leal, Juan M. Domínguez, Carlos M. Galmarini. Plitidepsin targets the moonlighting functions of eEF1A2 in cancer cells [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 1165. doi:10.1158/1538-7445.AM2017-1165

Abstract 5426: LPA stimulates glycolytic shift in ovarian cancer

Abstract Cancer cells resort to aerobic glycolysis to meet the increased energy consumption and growth requirement during active proliferation. While it has been observed that the cancer cells preferentially shift to aerobic glycolysis, the extraneous growth factors involved in this process is largely unknown. Since our previous studies have shown that lysophosphatidic acid (LPA) plays a critical role in the pathophysiology of ovarian cancers, we investigated the role of LPA in the metabolic reprogramming of ovarian cancer cells. Our results using XFe96 analyzer indicate that LPA stimulates aerobic glycolysis in multiple ovarian cancer cell lines as well as in patient-derived ovarian cancer cells. Physiological role for LPA in this process is demonstrated by the observation that the inhibitors of LPA-receptors attenuate the glycolytic shift stimulated by the ascitic fluid derived cancer patients. We further demonstrate that the silencing Gαi2, a downstream signaling mediator for LPA, inhibits LPA-stimulated glycolytic shift in ovarian cancer cells. Further analyses indicated that LPA-stimulated metabolic reprogramming involves the generation of reactive oxygen species (ROS). Treatment of cells with N-Acetyl Cysteine (NAC), a ROS-scavenger, inhibited the glycolytic shift stimulated by LPA. In addition, our studies point to a role for LPA-stimulated HIF1α activation in this process. These results indicate that LPA-stimulated metabolic reprogramming in ovarian cancer cells involve ROS-generation via LPA-Gαi2-HIF1α-dependent signaling pathway. Together with the findings that the glycolytic shift is crucially involved in tumor cell survival our studies point to LPA-Gαi2-HIF1α signaling axis as a potential target for therapy in ovarian cancer. Citation Format: Ji Hee Ha, Rangasudhagar Radhakrishnan, Jeremy D. Ward, Muralidharan Jayaraman, Danny N. Dhanasekaran. LPA stimulates glycolytic shift in 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 5426. doi:10.1158/1538-7445.AM2017-5426

Abstract MIP-057: TARGETING THE LONG–NONCODING RNA MALAT1 IN OVARIAN CANCER

Abstract PURPOSE: Ovarian cancer metastasizes via direct seeding, whereby cells must resist anoikis (detachment-induced cell death) in the peritoneal cavity before invading through the mesothelium to metastasize into peritoneal organs. The molecular mechanisms that facilitate these processes are not fully understood. Using an unbiased screen, we identified the long non-coding RNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) as being a potential regulator of anoikis resistance. We hypothesized that MALAT1 promotes progression through the early steps of metastasis, including anoikis resistance, peritoneal invasion, and metastatic outgrowth, and that therapeutic inhibition of MALAT1 will reduce ovarian tumor burden and metastatic spread. EXPERIMENTAL PROCEDURES: Modified anti-sense oligonucleotides (ASO) targeting MALAT1 were used to inhibit MALAT1 expression in ovarian cancer cell lines (OVCA420, HEY, OV1847). Transfections were performed using lipofectamine as well as novel lipid-based nanoparticles that are minimally toxic and have potential in vivo applications. Transcriptome profiling was performed on Affymetrix arrays. Migration and invasion assays were performed on an XCelligence Real Time Cell Analyzer, and proliferation was measured by crystal violet. Anoikis was measured using Caspase 3/7 assay. Knockdown of RBFOX2 and KIF1B-beta was performed using shRNAs. Gene expression was measured by RT-qPCR. Publicly available datasets including the Cancer Genome Atlas were analyzed for association of MALAT1 gene expression and DNA copy number with clinicopathologic variables. RESULTS: MALAT1 gene expression significantly increased after 24 hours in forced suspension culture versus attached culture in multiple anoikis-resistant ovarian cancer cell lines. MALAT1 knockdown via ASO resulted in decreased proliferation, migration, invasion, anchorage-independent growth, and increased anoikis. Lipid nanoparticle-mediated delivery of MALAT1-targeted ASO resulted in effective gene knockdown with minimal off-target toxicity. Transcriptome profiling of MALAT1-knockdown cells revealed significant changes in splicing patterns, including alternative splicing of the pro-apoptotic gene KIF1B (Kinesin Family Member 1B). MALAT1 knockdown resulted in decreased gene expression of the splicing factor RBFOX2 (RNA Binding Protein Fox-1, Homolog 2), which is associated with epithelial-to-mesenchymal transition (EMT). KIF1B contains RBFOX2 binding sites within spliced regions, suggesting that MALAT1 may control alternative splicing through regulation of RBFOX2. Indeed, RBFOX2 knockdown resulted in increased expression of the pro-apoptotic isoform of KIF1B (KIF1B-beta), as well as increased anoikis. Furthermore, knockdown of KIF1B-beta reversed the apoptotic effects of MALAT1-knockdown. In ovarian cancer patients, high MALAT1 gene expression is associated with higher stage disease, increased risk of recurrence, and reduced overall survival. Additionally, MALAT1 is amplified in 3% of ovarian cancers, and amplification is associated with increased MALAT1 gene expression and reduced overall survival. CONCLUSIONS: The lncRNA MALAT1 facilitates a pro-metastatic phenotype in ovarian cancer cells by promoting alternative splicing and differential expression of anti-apoptotic and EMT-related genes. In vivo studies to determine the efficacy of MALAT1-knockdown via lipid-mediated delivery to inhibit progression of ovarian cancer cell lines and patient derived xenografts are ongoing. Citation Format: Michael A Gordon, Dawn R Cochrane, Jamie Betker, Tom Anchordoquy, Jennifer K Richer. TARGETING THE LONG–NONCODING RNA MALAT1 IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-057.