Human Ovarian Cancer Model Research Articles

Measurement of Tumor Antioxidant Capacity and Prediction of Chemotherapy Resistance in Preclinical Models of Ovarian Cancer by Positron Emission Tomography.

Drug resistance is a major obstacle for the effective treatment of patients with high-grade serous ovarian cancer (HGSOC). Currently, there is no satisfactory way to identify patients with HGSOC that are refractive to the standard of care. Here, we propose the system xc - radiotracer (4S)-4-(3-[18F]fluoropropyl)-l-glutamate ([18F]FSPG) as a non-invasive method to measure upregulated antioxidant pathways present in drug-resistant HGSOC. Using matched chemotherapy sensitive and resistant ovarian cancer cell lines, we assessed their antioxidant capacity and its relation to [18F]FSPG uptake, both in cells and in animal models of human ovarian cancer. We identified the mechanisms driving differential [18F]FSPG cell accumulation and evaluated [18F]FSPG tumor uptake as predictive marker of treatment response in drug-resistant tumors. High intracellular glutathione (GSH) and low reactive oxygen species corresponded to decreased [18F]FSPG cell accumulation in drug-resistant versus drug-sensitive cells. Decreased [18F]FSPG uptake in drug-resistant cells was a consequence of changes in intracellular cystine, a key precursor in GSH biosynthesis. In vivo, [18F]FSPG uptake was decreased nearly 80% in chemotherapy-resistant A2780 tumors compared with parental drug-sensitive tumors, with nonresponding tumors displaying high levels of oxidized-to-reduced GSH. Treatment of drug-resistant A2780 tumors with doxorubicin resulted in no detectable change in tumor volume, GSH, or [18F]FSPG uptake. This study demonstrates the ability of [18F]FSPG to detect upregulated antioxidant pathways present in drug-resistant cancer. [18F]FSPG may therefore enable the identification of patients with HGSOC that are refractory to standard of care, allowing the transferal of drug-resistant patients to alternative therapies, thereby improving outcomes in this disease.

Combination immunotherapy with IL-4 Pseudomonas exotoxin and IFN-α and IFN-γ mediate antitumor effects in vitro and in a mouse model of human ovarian cancer.

We have shown that IL-4 fused to Pseudomonas exotoxin (IL-4-PE) is cytotoxic to ovarian cancer cell lines. The antineoplastic properties of IFN-α, IFN-γ and IL-4-PE have been studied and showed some promise in the clinical trials. Here, we investigated whether the combination of IL-4-PE, IFN-α and IFN-γ will result in increased ovarian cancer cell death in vitro and in vivo. Ovarian cancer cells were tested in vitro to analyze the cytotoxic effects of IL-4-PE, IFN-α and IFN-γ, and the combination of all three. Tumor-bearing xenograft mice were treated with the combination of IL-4-PE, IFN-α and IFN-γ to monitor their overall survival. The JAK/STAT phosphorylation signaling pathways were studied to delineate the mechanism of synergistic antitumor activity. The combination of IL-4-PE with IFN-α and IFN-γ resulted in increased ovarian cancer cell death in vitro and in vivo. Mechanistically, the synergistic antitumor effect was dependent on interferon signaling, but not IL-4-PE signaling as determined by signaling specific chemical inhibitors. The combination therapy induced the activation of critical mediators of apoptosis. The combination of IL-4-PE with interferons increased overall survival of mice with human ovarian cancer xenograft. These data suggest that this novel combination could provide a unique approach to treating ovarian cancer.

Triply Loaded Nitroxide Brush-Arm Star Polymers Enable Metal-Free Millimetric Tumor Detection by Magnetic Resonance Imaging.

Nitroxides occupy a privileged position among plausible metal-free magnetic resonance imaging (MRI) contrast agents (CAs) due to their inherently low-toxicity profiles; nevertheless, their translational development has been hindered by a lack of appropriate contrast sensitivity. Nanostructured materials with high nitroxide densities, where each individual nitroxide within a macromolecular construct contributes to the image contrast, could address this limitation, but the synthesis of such materials remains challenging. Here, we report a modular and scalable synthetic approach to nitroxide-based brush-arm star polymer (BASP) organic radical CAs (ORCAs) with high nitroxide loadings. The optimized ∼30 nm diameter "BASP-ORCA3" displays outstanding T2 sensitivity with a very high molecular transverse relaxivity ( r2 > 1000 mM-1 s-1). BASP-ORCA3 further exhibits excellent stability in vivo, no acute toxicity, and highly desirable pharmacokinetic and biodistribution profiles for longitudinal detection of tumors by MRI. When injected intravenously into mice bearing subcutaneous plasmacytomas, BASP-ORCA3 affords distinct in vivo visualization of tumors on translationally relevant time scales. Leveraging its high sensitivity, BASP-ORCA3 enables efficient mapping of tumor necrosis, which is an important biomarker to predict therapeutic outcomes. Moreover, BASP-ORCA3 allows for detection of millimetric tumor implants in a disseminated murine model of advanced-stage human ovarian cancer that possess genetic, histological, and vascular characteristics that are similar to those seen in patients. This work establishes BASP-ORCA3 as a promising metal-free spin contrast agent for MRI.

Expanded CD56superbrightCD16+ NK Cells from Ovarian Cancer Patients Are Cytotoxic against Autologous Tumor in a Patient-Derived Xenograft Murine Model.

Natural killer (NK) cells are useful for cancer immunotherapy and have proven clinically effective against hematologic malignancies. However, immunotherapies for poor prognosis solid malignancies, including ovarian cancer, have not been as successful due to immunosuppression by solid tumors. Although rearming patients' own NK cells to treat cancer is an attractive option, success of that strategy is limited by the impaired function of NK cells from cancer patients and by inhibition by self-MHC. In this study, we show that expansion converts healthy donor and immunosuppressed ovarian cancer patient NK cells to a cytotoxic CD56superbrightCD16+ subset with activation state and antitumor functions that increase with CD56 brightness. We investigated whether these expanded NK cells may overcome the limitations of autologous NK cell therapy against solid tumors. Peripheral blood- and ascites-derived NK cells from ovarian cancer patients were expanded and then adoptively transferred into cell-line and autologous patient-derived xenograft models of human ovarian cancer. Expanded ovarian cancer patient NK cells reduced the burden of established tumors and prolonged survival. These results suggest that CD56bright NK cells harbor superior antitumor function compared with CD56dim cells. Thus, NK cell expansion may overcome limitations on autologous NK cell therapy by converting the patient's NK cells to a cytotoxic subset that exerts a therapeutic effect against autologous tumor. These findings suggest that the value of expanded autologous NK cell therapy for ovarian cancer and other solid malignancies should be clinically assessed. Cancer Immunol Res; 6(10); 1174-85. ©2018 AACR.

Covalent Rpn13-Binding Inhibitors for the Treatment of Ovarian Cancer.

Substitution of the m,p-chloro groups of bis-benzylidinepiperidone RA190 for p-nitro, generating RA183, enhanced covalent drug binding to Cys88 of RPN13. Treatment of cancer cell lines with RA183 inhibited ubiquitin-mediated protein degradation, resulting in rapid accumulation of high-molecular-weight polyubiquitinated proteins, blockade of NFκB signaling, endoplasmic reticulum stress, an unfolded protein response, production of reactive oxygen species, and apoptotic cell death. High-grade ovarian cancer, triple-negative breast cancer, and multiple myeloma cell lines were particularly vulnerable to RA183. RA183 stabilized a tetraubiquitin-linked firefly luciferase reporter protein in cancer cell lines and mice, demonstrating in vitro and in vivo proteasomal inhibition, respectively. However, RA183 was rapidly cleared from plasma, likely reflecting its rapid degradation to the active compound RA9, as seen in human liver microsomes. Intraperitoneal administration of RA183 inhibited proteasome function and orthotopic tumor growth in mice bearing human ovarian cancer model ES2-luc ascites or syngeneic ID8-luc tumor.

Anetumab ravtansine inhibits tumor growth and shows additive effect in combination with targeted agents and chemotherapy in mesothelin-expressing human ovarian cancer models.

Despite the recent advances in the treatment of ovarian cancer, it remains an area of high unmet medical need. Epithelial ovarian cancer is associated with high levels of mesothelin expression, and therefore, mesothelin is an attractive candidate target for the treatment of this disease. Herein, we investigated the antitumor efficacy of the mesothelin-targeting antibody-drug conjugate (ADC) anetumab ravtansine as a novel treatment option for ovarian cancer in monotherapy and in combination with the antitumor agents pegylated liposomal doxorubicin (PLD), carboplatin, copanlisib and bevacizumab. Anetumab ravtansine showed potent antitumor activity as a monotherapy in ovarian cancer models with high mesothelin expression. No activity was seen in mesothelin-negative models. The combination of anetumab ravtansine with PLD showed additive anti-proliferative activity in vitro, which translated into improved therapeutic in vivo efficacy in ovarian cancer cell line- and patient-derived xenograft (PDX) models compared to either agents as a monotherapy. The combination of anetumab ravtansine with the PI3Kα/δ inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell lines in vitro, showing increased apoptosis in response to the combination treatment. In vivo, the combination of anetumab ravtansine with copanlisib resulted in more potent antitumor activity than either of the treatments alone. Likewise, the combination of anetumab ravtansine with carboplatin or bevacizumab showed improved in vivo efficacy in the ST081 and OVCAR-3 models, respectively. All combinations were well-tolerated. Taken together, these data support the development of anetumab ravtansine for ovarian cancer treatment and highlight its suitability for combination therapy with PLD, carboplatin, copanlisib, or bevacizumab.

Abstract IA10: Deconstructing and reconstructing the ovarian cancer microenvironment

Abstract The tumor microenvironment (TME) is critical for cancer growth and spread. However, the relationships between molecular components of the TME and higher-order features, tissue stiffness, extent of disease, and cellularity are not known. Using clinical samples of metastatic high-grade serous ovarian cancer (HGSOC) that ranged from minimal to extensive disease, we have defined RNA and protein profiles that evolved with these higher-order features. We obtained a unique profile of the evolving metastatic microenvironment of HGSOC that includes data not only on gene expression and proteomics but also cytokine/chemokine expression, ECM organization, and biophysical properties. This “deconstruction” of a metastatic microenvironment provides a critical reference for our mouse model systems and a template for reconstruction of 3D multicellular human ovarian cancer models as will be detailed in this presentation. Although our work was conducted on omental metastases of HGSOC, we found evidence for a conserved mechanism that may be relevant to many human solid tumors: a pattern of matrix-associated gene expression that we termed the matrix index. In the HGSOC samples, the matrix index significantly associated with various leukocyte signaling pathways of cells associated with poor prognosis including Th2 cells and macrophage-associated genes and correlated with extent of disease and tissue stiffness. Interrogating transcriptional datasets from a wide range of human solid cancers, including HGSOC, we found that the matrix index had prognostic significance irrespective of patient age, stage of disease, or first response to therapy. This led us to suggest that there may be a common matrix response to human cancer. Citation Format: Frances R. Balkwill. Deconstructing and reconstructing the ovarian cancer microenvironment. [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 IA10.

Abstract 5826: Enhanced anti-tumor effects of selinexor and niraparib in preclinical models of ovarian cancer

Abstract Introduction: Selinexor (KPT-330) is a first-in-class oral exportin-1 (XPO1/ CRM1) inhibitor that induces cell cycle arrest and apoptosis in cancer cells through reactivation of tumor suppressor proteins and inhibition of DNA damage repair genes. Here, we studied selinexor in combination with niraparib, an inhibitor of the DNA damage repair proteins PARP1 and 2, in preclinical models of ovarian cancer. Given that both compounds can inhibit DNA damage repair responses, we hypothesized the combination of selinexor and niraparib would enhance cancer cell death by accumulation of DNA damage that cannot be resolved in ovarian cancer. Methods: Selinexor and niraparib alone and in combination were tested in vitro on the BRCA wildtype ovarian cancer cell line A2780. Total RNA and protein were extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous A2780 xenograft mouse model was treated with selinexor [10 mg/kg, once per week (M) for three weeks] or niraparib [37.5 mg/kg, once-daily for five days per week (M-F) for three weeks] as single agents or in combination. Tumor growth and body weights were measured for 21 days. Tumors were harvested at the end of the study and analyzed by immunohistochemistry (IHC). Results: Selinexor and niraparib as single agents inhibited A2780 cell proliferation (selinexor IC50: 200nM; niraparib IC50: 800nM). The combination of selinexor and niraparib showed synergistic cytotoxicity in A2780 cells. Increased expression of phospho (S139) H2A.X with the combination confirmed induction of DNA damage. In vivo, the combination enhanced tumor inhibition (51.0% in combination versus 24.3% and 29.6% in selinexor and niraparib respectively) and improved median survival compared to vehicle or each agent alone (18 days in combination group versus 11, 14 and 16 days in vehicle, selinexor, and niraparib groups, respectively). IHC analysis showed enhanced nuclear p53 and p21 staining in selinexor treated tumors as well as in tumors treated with the combination. Increased apoptosis was observed in tumor samples treated with both selinexor and niraparib as compared to vehicle control or each agent alone. Conclusions: Selinexor plus niraparib demonstrated enhanced anti-tumor activity in preclinical models of human ovarian cancer. Since both drugs could inhibit DNA damage repair pathway, the drug combination efficiently accumulated DNA damage, which was associated with reduced cell proliferation, and induced apoptosis. This combination therapy warrants further investigation as a treatment option for patients with ovarian cancer. Citation Format: Hua Chang, Trinayan Kashyap, Sophie Debler, Thaddeus J. Unger, Sarah Wang, Keith Mikule, Jing Wang, Mansoor R. Mirza, Sharon Shacham, Yosef Landesman. Enhanced anti-tumor effects of selinexor and niraparib in preclinical models of 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 5826.

Abstract 1780: Anetumab ravtansine has single-agent activity in mesothelin-expressing human ovarian cancer models and potentiates the activity of chemotherapeutics and targeted agents

Abstract Ovarian cancer remains an area of high unmet medical need, with 239,000 patients newly diagnosed per year. Here we describe the mesothelin-targeting antibody drug-conjugate anetumab ravtansine as a novel treatment option for ovarian cancer. Internalization of anetumab ravtansine and co-localization with lysosomal markers in ovarian cancer cells was accompanied by rapid resynthesis of mesothelin, which may allow consecutive anetumab ravtansine treatment cycles. The strong antitumor activity of anetumab ravtansine was preserved during repeated treatment cycles in the OVCAR-3 ovarian cancer in vivo model. Mechanistically, treatment with anetumab ravtansine caused mitotic arrest characterized by increased pHH3 signal and monopolar spindle structures. Interestingly, anetumab ravtansine monotherapy also induced DNA damage as indicated by focal γH2AX signals. Ultimately, the cells engaged in apoptotic cell death. Strong in vitro monotherapy activity was demonstrated in a set of 11 ovarian cancer cell lines with IC50s between 3 and 90nM. The in vivo efficacy of anetumab ravtansine dosed at 2.5 mg/kg Q3Dx3 i.v. anetumab ravtansine was evaluated in 9 human ovarian cancer models with varying mesothelin expression levels. Strong in vivo efficacy at this moderate dose with T/C <0.2 was seen in OVCAR-3 cell line and the PDX model OvCa6668. Efficacy was generally higher in tumors with strong mesothelin expression. No activity was seen in mesothelin-negative ovarian cancer models. Combination with the PI3K inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell line in vitro, showing increased apoptosis in the combination treatment. In vivo, anetumab ravtansine + copanlisib in OVCAR-3 tumors consistently resulted in greater efficacy than with either treatment alone. Both single treatments and the combination were well tolerated. Combination of anetumab ravtansine with bevacizumab, the only antibody therapy approved for ovarian cancer to date, also showed increased in vivo efficacy in the combination while being well tolerated. Next combination with PEGylated doxorubicin (PLD) was investigated. The combination of anetumab ravtansine plus PLD in OVCAR-8 cells showed additive effects and in vivo in OVCAR-8 tumors, the combination consistently showed better therapeutic efficacy than either therapy alone. Overall, these data support the development of anetumab ravtansine in ovarian cancer and suggest combinations with copanlisib, bevacizumab or PLD. Citation Format: Christoph A. Schatz, Maria Quanz, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Beatrix Stelte-Ludwig, Sven Golfier, Charlotte Kopitz, Cem Elbi, Karl Ziegelbauer, Dominik Mumberg. Anetumab ravtansine has single-agent activity in mesothelin-expressing human ovarian cancer models and potentiates the activity of chemotherapeutics and targeted agents [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 1780.

Momelotinib decreased cancer stem cell associated tumor burden and prolonged disease-free remission period in a mouse model of human ovarian cancer.

Despite a good initial response to front-line chemotherapy, majority of the ovarian cancer patients relapse with consecutive phases of recurrences; and nearly 60% die within 5 years due to the development of a chemoresistant disease. This study investigated whether inhibition of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway by momelotinib is sufficient in suppressing tumor burden and prolonging the disease-free survival period in a mouse model of ovarian cancer. We demonstrate that paclitaxel treatment enhanced JAK2/STAT3 activation which resulted in the enrichment of cancer stem cell (CSC)-like phenotype in the surviving ovarian cancer cells in vitro and in in vivo mouse xenografts. Combined treatment with paclitaxel and momelotinib inhibited paclitaxel-induced JAK2/STAT3 activation and CSC-like development in mice xenografts, and consequently reduced the tumor burden significantly greater than that achieved by paclitaxel-treatment alone. However, robust recurrent tumor growth with enhanced JAK2/STAT3 activation and CSC-like phenotype was observed in all mice groups after termination of treatments, but was delayed significantly in the paclitaxel and momelotinib treated group compared to other treatment groups. Daily oral gavage of momelotinib after termination of paclitaxel treatment showed sustained inhibition of tumor growth and a prolonged disease-free survival period in 50% of the mice. The other 50% of mice that developed tumors with ongoing momelotinib treatment also showed significantly increased survival benefit and a smaller tumor burden. These preliminary findings may have a profound clinical impact in developing an effective momelotinib-based ‘maintenance-therapy’ in ovarian cancer patients' post-chemotherapy treatment.

Abstract B119: Relationship of NaPi2b expression and efficacy of XMT-1536, a NaPi2b targeting antibody-drug conjugate (ADC), in an unselected panel of human primary ovarian mouse xenograft models

Abstract NaPi2b (SLC34A2) is expressed in many human ovarian and lung cancers. Previous human clinical trials with a NaPi2b targeting MMAE ADC have shown objective tumor responses, but have not shown a strong relationship between NaPi2b expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprising a novel humanized antibody conjugated with 10-15 auristatin F-HPA (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is capable of controlled bystander-effect killing, resulting in efficacy in tumors with heterogeneous antigen expression, and is metabolized intratumorally to an active nonpermeable metabolite to enable greater systemic tolerability. Previously presented preclinical studies using XMT-1536 have demonstrated efficacy in vivo in the NaPi2b-expressing OVCAR3 ovarian cancer line model. Here, we describe the evaluation of XMT-1536 in a panel of patient-derived xenograft models of human ovarian cancer, unselected for NaPi2b expression. The efficacy data from this study were compared to characteristics of each model, including NaPi2b expression, to predict a model for stratification of patients in XMT-1536 clinical trials. Primary ovarian cancer models were derived from serous ovarian or fallopian tube cancers and implanted in immunocompromised mice. Once tumors reached a stratified mean volume of 125-250 mm3, mice were treated with 3 mg/kg XMT-1536 weekly for three weeks in groups of n=3. Untreated animals in groups of n=2-4 were included as a control. The planned study endpoint was a tumor volume of 1 cm3 or 45 days. In a case of complete response, mice were followed for a longer time course to evaluate for tumor regrowth. An immunohistochemistry (IHC) assay to detect NaPi2b was established. Tumor blocks from untreated study animals were evaluated to determine an efficacy/staining pattern relationship. The established protocol was also applied to a series of human primary ovarian tumors. As of July 2017, treatment response data are available for 19 primary ovarian cancer mouse xenograft models, and NaPi2b IHC results are available for 15 models. XMT-1536 induced at least 50% reduction in tumor volume relative to baseline in 10/19 (53%) of all tested models, with tumor volume reduction greater than 50% in 8/15 (53%) of models with available IHC data. There was an association between NaPi2b IHC H-score and tumor volume change after XMT-1536 treatment (Spearman rank coefficient 0.72). Among tumors with H-score ≥70, 8/10 (80%) models achieved 50% or greater reduction in tumor volume after XMT-1536 treatment vs 0/5 (0%) models with H-score <70. Applying the same IHC assay to primary human ovarian tumors, 12/20 (60%) tested tumors had an H-score ≥70. This study demonstrates broad activity of XMT-1536 in a panel of primary ovarian cancer mouse xenograft models without molecular selection for NaPi2b expression. NaPi2b expression measured by IHC assay is associated with the probability of tumor model response to XMT-1536 treatment and may be useful for patient stratification/selection in the clinic. XMT-1536 is scheduled to enter phase 1 evaluation in patients with ovarian cancer and other NaPi2b-expressing tumors in early 2018. The trial will include mandatory tissue collection to evaluate the relationship of NaPi2b expression with activity of XMT-1536. Citation Format: Rebecca Mosher, Laura Poling, LiuLiang Qin, Natalya Bodyak, Donald Bergstrom. Relationship of NaPi2b expression and efficacy of XMT-1536, a NaPi2b targeting antibody-drug conjugate (ADC), in an unselected panel of human primary ovarian mouse xenograft 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 B119.

MYC Targeted Long Noncoding RNA DANCR Promotes Cancer in Part by Reducing p21 Levels.

The MYC oncogene broadly promotes transcription mediated by all nuclear RNA polymerases, thereby acting as a positive modifier of global gene expression. Here, we report that MYC stimulates the transcription of DANCR, a long noncoding RNA (lncRNA) that is widely overexpressed in human cancer. We identified DANCR through its overexpression in a transgenic model of MYC-induced lymphoma, but found that it was broadly upregulated in many human cancer cell lines and cancers, including most notably in prostate and ovarian cancers. Mechanistic investigations indicated that DANCR limited the expression of cell-cycle inhibitor p21 (CDKN1A) and that the inhibitory effects of DANCR loss on cell proliferation could be partially rescued by p21 silencing. In a xenograft model of human ovarian cancer, a nanoparticle-mediated siRNA strategy to target DANCR in vivo was sufficient to strongly inhibit tumor growth. Our observations expand knowledge of how MYC drives cancer cell proliferation by identifying DANCR as a critical lncRNA widely overexpressed in human cancers.Significance: These findings expand knowledge of how MYC drives cancer cell proliferation by identifying an oncogenic long noncoding RNA that is widely overexpressed in human cancers. Cancer Res; 78(1); 64-74. ©2017 AACR.

Targeting ovarian cancer and endothelium with an allosteric PTP4A3 phosphatase inhibitor.

Overexpression of protein tyrosine phosphatase PTP4A oncoproteins is common in many human cancers and is associated with poor patient prognosis and survival. We observed elevated levels of PTP4A3 phosphatase in 79% of human ovarian tumor samples, with significant overexpression in tumor endothelium and pericytes. Furthermore, PTP4A phosphatases appear to regulate several key malignant processes, such as invasion, migration, and angiogenesis, suggesting a pivotal regulatory role in cancer and endothelial signaling pathways. While phosphatases are attractive therapeutic targets, they have been poorly investigated because of a lack of potent and selective chemical probes. In this study, we disclose that a potent, selective, reversible, and noncompetitive PTP4A inhibitor, JMS-053, markedly enhanced microvascular barrier function after exposure of endothelial cells to vascular endothelial growth factor or lipopolysaccharide. JMS-053 also blocked the concomitant increase in RhoA activation and loss of Rac1. In human ovarian cancer cells, JMS-053 impeded migration, disrupted spheroid growth, and decreased RhoA activity. Importantly, JMS-053 displayed anticancer activity in a murine xenograft model of drug resistant human ovarian cancer. These data demonstrate that PTP4A phosphatases can be targeted in both endothelial and ovarian cancer cells, and confirm that RhoA signaling cascades are regulated by the PTP4A family.

NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells.

Understanding the mechanisms supporting tumor-initiating cells (TIC) is vital to combat advanced-stage recurrent cancers. Here, we show that in advanced ovarian cancers NFκB signaling via the RelB transcription factor supports TIC populations by directly regulating the cancer stem-like associated enzyme aldehyde dehydrogenase (ALDH). Loss of RelB significantly inhibited spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in subcutaneous and intrabursal mouse xenograft models of human ovarian cancer. RelB also affected expression of the ALDH gene ALDH1A2 Interestingly, classical NFκB signaling through the RelA transcription factor was equally important for tumorigenesis in the intrabursal model, but had no effect on ALDH. In this case, classical signaling via RelA was essential for proliferating cells, whereas the alternative signaling pathway was not. Our results show how NFκB sustains diverse cancer phenotypes via distinct classical and alternative signaling pathways, with implications for improved understanding of disease recurrence and therapeutic response. Cancer Res; 77(24); 6927-40. ©2017 AACR.

Evaluation Fucoidan Extracts From Undaria pinnatifida and Fucus vesiculosus in Combination With Anticancer Drugs in Human Cancer Orthotopic Mouse Models.

Objective: To determine the activity of fucoidan from Undaria pinnatifida (UPF) and Fucus vesiculosus (FVF) when given in combination of chemotherapy drugs using selected human breast or ovarian cancer orthotopic mouse models. Methods: Mice were inoculated with 1 × 106 cells of TOV-112d, MCF-7, or ZR-75 subcutaneously or SKOV3-GFP-Luc intraperitoneally on day 0. MCF-7 and ZR-75 mice were administered with estradiol valerate 2 mg/kg in 0.2 mL castor oil subcutaneously two days prior to cell inoculation. Mice were randomized to one of six arms (N = 10/arm) paclitaxel, UPF/paclitaxel, FVF/paclitaxel, tamoxifen, UPF/tamoxifen, or FVF/tamoxifen. Tumors were measured three times per week for 28 days. Results: Improved activity was observed with UPF or FVF in combination with tamoxifen in both the MCF-7 and ZR-75D breast cancer mouse models. Decreased activity of paclitaxel was observed when given in combination with UPF or FVF in both breast cancer mouse models. The combination of FVF/tamoxifen in the TOV-112d ovarian cancer mouse model had improved activity but no there was difference observed with the UPF/tamoxifen in either ovarian cancer mouse model. No difference was observed with combination of UPF or FVF with paclitaxel in human ovarian cancer SKOV3 or TOV-112d orthotopic mouse models. Conclusion: This study did confirm that UPF/FVF in combination with tamoxifen did not decrease tamoxifen activity in both breast and ovarian cancer, with some potential to improve activity compared to tamoxifen alone in breast cancers. Previous in vitro studies had suggested UPF and FVF had overall synergistic activity with paclitaxel; however, in the current in vivo human cancer mouse model studies there was no change in paclitaxel activity when given in combination with UPF or FVF in either of the two human ovarian cancer models. Furthermore, this study demonstrated that UPF or FVF given in combination with paclitaxel had a potential antagonistic effect in breast cancer models. Additional studies are warranted to delineate mechanisms contributing to variation in the in vivo activity when given in combination with paclitaxel. As a first step, a clinical pharmacokinetic study evaluating impact of FVF/UPF given in combination with chemotherapy in patients with solid tumors is underway.

Hyaluronic acid-green tea catechin micellar nanocomplexes: Fail-safe cisplatin nanomedicine for the treatment of ovarian cancer without off-target toxicity

The green tea catechin, (–)-epigallocatechin-3-O-gallate (EGCG), has gained significant attention as a potent adjuvant to enhance the antitumor efficacy of cisplatin while mitigating its harmful side effects. Herein we report the development of a fail-safe cisplatin nanomedicine constructed with hyaluronic acid-EGCG conjugate for ovarian cancer therapy. A simple mixing of this conjugate and cisplatin induces spontaneous self-assembly of micellar nanocomplexes having a spherical core-shell structure. The surface-exposed hyaluronic acid enables efficient delivery of cisplatin into CD44-overexpressing cancer cells via receptor-mediated endocytosis whereas the internally packed EGCG moieties offer an environment favorable for the encapsulation of cisplatin. In addition, the antioxidant effect of EGCG moieties ensures fail-safe protection against off-target organ toxicity originating from cisplatin-evoked oxidative stress. Pharmacokinetic and biodistribution studies reveal the prolonged blood circulation and preferential tumor accumulation of intravenously administered nanocomplexes. Moreover, the nanocomplexes exhibit superior antitumor efficacy over free cisplatin while displaying no toxicity in both a subcutaneous xenograft model and peritoneal metastatic model of human ovarian cancer. Our findings demonstrate proof of concept for the feasibility of green tea catechin-based micellar nanocomplexes as a safe and effective cisplatin nanomedicine for ovarian cancer treatment.

1662P - Synergistic antitumor effects of OT-101 (trabedersen), a transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) and chemotherapy in preclinical tumor models

Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combination strategies. Methods: OT-101 was administered as single agent (1-64 mg/kg, qdx3/wk or qdx21) and in combination with Gemcitabine (GEM, 15 mg/kg, qdx2/wk), Dacarbazine (DTIC, 1-10 mg/kg, qdx4/wk) or Paclitaxel (PTX, 10 mg/kg, qdx5) to nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) SC glioblastoma (U87) or (iv) SC ovarian (SKOV-3) tumors. Mice were monitored for adverse effects, body weight loss, tumor size and survival outcome. Lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-BrdUrd and CD31 antibodies to determine tumor cell proliferation and vascularization, respectively. Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p < 0.0001) versus untreated control in the PAC model. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. DTIC alone), glioblastoma (U87, p = 0.001 vs. PTX) and ovarian (SKOV-3, p < 0.05 vs. PTX) cancer models when combined with either DTIC (C8161) or PTX (U87 and SKOV-3). No synergy was observed with GEM (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at HED of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day. Conclusions: The preclinical data laid the groundwork for establishing combination therapies in the clinic. Of interest is the preferential synergy between OT-101 and PTX or DTIC, but not with GEM. Legal entity responsible for the study: Autotelic Inc Funding: None Disclosure: All authors have declared no conflicts of interest.

Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation.

Cell-free circulating linear DNA is being explored for noninvasive diagnosis and management of tumors and fetuses, the so-called liquid biopsy. Previously, we observed the presence of small extrachromosomal circular DNA (eccDNA), called microDNA, in the nuclei of mammalian tissues and cell lines. Now, we demonstrate that cell-free microDNA derived from uniquely mapping regions of the genome is detectable in plasma and serum from both mice and humans and that they are significantly longer (30%-60% >250 bases) than cell-free circulating linear DNA (∼150 bases). Tumor-derived human microDNA is detected in the mouse circulation in a mouse xenograft model of human ovarian cancer. Comparing the microDNA from paired tumor and normal lung tissue specimens reveals that the tumors contain longer microDNA. Consistent with human cancers releasing microDNA into the circulation, serum and plasma samples (12 lung and 11 ovarian cancer) collected prior to surgery are enriched for longer cell-free microDNA compared with samples from the same patient obtained several weeks after surgical resection of the tumor. Thus, circular DNA in the circulation is a previously unexplored pool of nucleic acids that could complement miRNAs and linear DNA for diagnosis and for intercellular communication.Implications: eccDNA derived from chromosomal genomic sequence, first discovered in the nuclei of cells, are detected in the circulation, are longer than linear cell-free DNA, and are released from normal tissue and tumors into the circulation. Mol Cancer Res; 15(9); 1197-205. ©2017 AACR.

Abstract 4900: Cysteamine suppresses tumor metastasis by inhibiting activity of matrix metalloproteases without inducing toxicity in mouse models of human ovarian cancer

Abstract Previously, we demonstrated that cysteamine, a small molecule inhibitor of matrixmetalloproteases (MMPs), inhibited tumor invasion in vitro and metastasis in vivo in mouse models of human pancreatic and ovarian cancer. We have also shown that subcutaneous cysteamine administration improved the survival of mice with orthotopically transplanted pancreatic tumors. Herein, we examined the effect of cysteamine on total MMP activity, MMP isoforms and activity by substrate gel zymography in tumors obtained from orthotopic mouse model of human ovarian cancer. We also investigated any gastro-intestinal and general toxicity in vital organs of mice treated with the highest dose of cysteamine (250 mg/kg). We developed orthotopic tumors by injecting two human ovarian cancer cell lines in the ovary of female athymic nude mice. After 4 days of tumor implant, the mice were treated with different doses of cysteamine and followed for 5 weeks. Total MMP activity by fluorogenic substrate, MMP-2, 9 and 14 by ELISA and MMP activity by substrate gel zymography were measured in tumor lysates and compared with untreated tumors. We performed H&amp;E staining in vital organs such as heart, lung, liver, kidney, spleen and brain for evaluating general toxicity of cysteamine. In addition, we examined stomach and duodenum from cysteamine treated and vehicle treated mice if they developed any ulcerations. In two orthotopic murine models of human ovarian cancer, consistent with our previous observations, tumor metastasis was significantly decreased compared to controls in cysteamine treated mice. Similarly, total MMP activity was also significantly decreased in primary tumors treated with cysteamine in a dose dependent manner (P≤0.05). Zymographic MMP activity was also decreased significantly in cysteamine treated tumors compared to control corroborating with total MMP activity (P≤0.05). Interestingly, ELISA did not show any changes in MMP expression in cysteamine treated and untreated tumors. Furthermore, we did not observe any symptoms of general toxicity in animals treated at the highest dose of cysteamine and major vital organs and gastro-intestinal organs from treated animals showed no evidence of histological changes as evident by H&amp;E staining. In conclusion, our results suggest that cysteamine is a potent inhibitor of MMP activity without affecting expression. Cysteamine does not cause any gastro-intestinal or vital organ toxicity and may be useful therapeutic agent for ovarian cancer either alone or in combination therapy with known anti-cancer agents. Citation Format: Akiko Suzuki, Rukmini Bhardwaj, Pamela Leland, Bharat H. Joshi, Raj K. Puri. Cysteamine suppresses tumor metastasis by inhibiting activity of matrix metalloproteases without inducing toxicity in mouse models of human 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 4900. doi:10.1158/1538-7445.AM2017-4900

Abstract 5650: Enhancement of CAR-T cell activity via PD-1 knockdown by self-deliverable RNAi

Abstract Background: A growing body of evidence indicates that the blockade of PD-1 and other immunosuppressive receptors can increase the efficacy of adoptively transferred cells in immunotherapy for cancer. The expression of PD-1 can be suppressed by ex vivo treatment of transferred lymphocytes by self-deliverable RNAi (sd-rxRNA®) compounds. sd-rxRNAs are based on proprietary chemical modifications of siRNA compounds making them stable to nuclease degradation and capable of penetration into target cells without the use of transfection reagents or physical methods. Aim: To determine whether the reduction of PD-1 by ex vivo treatment of mesothelin-targeting CAR-T cells with sd-rxRNA will improve the efficacy of meso-CAR-T cells in a mouse model of ovarian cancer. Methods: Second-generation human CAR-T cells expressing P4 antibody fragments targeting mesothelin (meso-CAR-T) were treated ex vivo with PD-78, a self-deliverable siRNA silencing the PD-1 gene. The meso-CAR-T cells were then injected into tumors in a xenograft mouse model of human ovarian cancer (A1847 cell line expressing mesothelin and PD-L1) in NSG mice. The tumor volumes were monitored over the course of one month. After the animals were sacrificed, human CD45-containing cells extracted from the tumor were analyzed for PD-1 expression by flow cytometry. Results: We observed a statistically significant reduction in tumor growth in the group of mice injected with meso-CAR-T cells pretreated ex vivo with PD-78. The post-mortem tumor analysis by flow cytometry showed that the expression of PD-1 in meso-CAR-T cells was significantly suppressed for the full duration of the experiment (one month). Conclusion: Our results suggest that the silencing of immunosuppressive genes by ex vivo treatment with sd-rxRNA may improve efficacy of CAR-T cells in the treatment of solid tumors. A similar approach may be applied to other types of adoptive cell transfer. Citation Format: Alexey Wolfson, Alexey Eliseev, Taisia Shmushkovich, Monica Betancur-Boissel, Monica Betancur-Boissel, Nathalie Scholler. Enhancement of CAR-T cell activity via PD-1 knockdown by self-deliverable RNAi [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 5650. doi:10.1158/1538-7445.AM2017-5650