SKOV3 Models Research Articles

Abstract 2051: Innovations in ADC technology platform with legumain-cleavable KSP-inhibitor payloads adaptable to various aspects of cancer biology

Abstract Despite recent advances in the antibody-drug conjugate (ADC) field it remains challenging to achieve a sufficient therapeutic window. To address key issues of current ADCs such as aggregate formation and payload-related toxicities, we have developed a platform technology with tailored solutions for ADCs and small molecule-drug conjugates (SMDCs). Key features of the ADC technology comprise of kinesin spindle protein inhibitors (KSPi) as a novel payload class, a unique linker selectively cleaved by the tumor-associated protease legumain, and a CellTrapper® modification of the KSPi payload to reduce membrane permeability and provide accumulation in tumor cells beneficial for efficacy and safety1,2. Our advanced ADC VIP943 is targeting CD123 for the treatment of AML and MDS and is currently in Phase 1 clinical trial (NCT06034275). Here we show the extension and applicability of the platform in combination with various antibodies. Two case studies demonstrate how the payload-linker optimization increases the selectivity for tumor over healthy cells and potentially overcomes transporter-mediated drug resistance. Tuning the cleavable linker of cetuximab-ADCs for a higher challenge of legumain-mediated payload release resulted in a 52-fold improved selectivity of cytotoxicity against tumor cells versus HaCaT skin cells. A trastuzumab-ADC using a KSPi payload modified with the CellTrapper, shows low nanomolar potency against JIMT-1 cells resistant to trastuzumab-deruxtecan and thus may overcome resistance. An increased potency was not observed with an ADC releasing a permeable KSPi without CellTrapper. To address heterogenous target expression in solid tumors, novel antibody-2-drug conjugates (A2DCs) were synthesized to combine the advantages of intracellular accumulation of non-permeable payloads with the release of a second, membrane permeable KSPi payload. In vitro studies comparing the A2DCs with their respective mono-drug ADCs on 4 different targets shows the highest cytotoxicity achieved with A2DCs independent of the efficacy driver in mono ADCs. Lastly, synthesis of homogenous KSPi ADCs with a drug to antibody ratio of ~8 is compatible with different antibodies without significant aggregate formation. Furthermore, an easy and efficient process for ring-opening of the thiosuccinimide ring after conjugation was developed and shown to successfully avoid ADC de-conjugation via retro-Michael reaction. Selected examples of each of these subclasses were coupled to trastuzumab; all conjugates were highly efficacious and resulted in tumor regressions in the SKOV-3 model similar to trastuzumab-deruxtecan. Conclusion: Our technology platform offers options for efficient and safe cancer treatments and allows for versatile adaptations to the various aspects of cancer biology. 1. Kirchhoff et al, Cancers (2020). 2. Lerchen et al, Bioconj Chem. (2020). Citation Format: Hans-Georg Lerchen, Anne-Sophie Rebstock, Mareike Wiedmann, Beatrix Stelte-Ludwig, Amy J. Johnson, Raquel Izumi, Ahmed Hamdy. Innovations in ADC technology platform with legumain-cleavable KSP-inhibitor payloads adaptable to various aspects of cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2051.

Abstract 6001: Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer

Abstract Introduction: Vascular endothelial growth factor (VEGF) is a key promoter of angiogenesis and disease progression in epithelial ovarian cancer (OC). Bevacizumab, a humanized anti-VEGF monoclonal antibody, is approved for treatment of patients with ovarian and other cancers. Despite the wide use of anti-VEGF therapy for cancer treatment, most patients develop resistant disease. Therefore, new combinations/strategies are needed to reduce adaptive resistance to anti-VEGF antibody (AVA) therapy. Our aim is to determine the potential role of epigenetic changes in AVA-adaptive resistance. We identified and characterized epigenetic changes in BARD1, a tumor suppressor, at the emergence of AVA resistance. Methods: To define epigenetic changes during AVA therapy induced resistance, we performed series of in vitro (methylation array, RNA sequencing) and in vivo (orthotopic OC (PDX-MDA-OVCA1, immunocompetent-IG10, and immunocompromised-SKOV3) and fibrosarcoma-HT1080, an inherently VEGF resistant model) experiments. The biological effects of DNA methylation agent (5`-Azacytidine; 5`-Aza) with B20 (murine anti-VEGF antibody) treatment were subsequently tested using in vivo models. Results: To determine whether 5`-Aza can enhance the efficacy of AVA therapy and overcome resistance to AVA in OC, we established AVA-resistant SKOV3 model. In AVA-resistant mice treated with B20 + 5`-Aza, there was a statistically significant reduction in tumor weight (p=0.03). Next, we performed a Human 450k methylation and mRNA arrays from xenografts of SKOV3 tumors. Based on both analyses, we demonstrated that BARD1 was one of the top candidate which was highly expressed (p=0.02) and less methylated (p<0.05) in the combination of B20 and 5`-Aza group compared to B20 alone. Our data also showed that BARD1 expression was restored in the combination therapy. The sequential treatment with 5`-Aza reverted the resistance towards the AVA therapy in SKOV3 model. Furthermore, the combination of B20 and 5`-Aza significantly decreased tumor weight in SKOV3 (p=0.005), IG10 (p<0.05), MDA-OVCA1 (p=0.01), and HT1080 (p<0.05). BARD1 expression levels were substantially increased in combination therapy compared to the B20 alone group (>4-fold, p=0.006). Tumor weight and BARD1 expression were inversely correlated. To determine the biological significance of BARD1, we knockdown BARD1 using Tet-inducible shRNA system. Our data showed a significant reduction in tumor weight in the BARD1 knockdown group in SKOV3 model (p=0.021). RNA-sequencing of BARD1 knockdown showed an enrichment of the angiogenesis pathway. Moreover, BARD1 expression levels were decreased in hypoxic conditions both in vitro and in vivo. Conclusion: Our findings indicate a previously unrecognized role for BARD1 in mediating resistance to AVA therapy. Use of epigenetic drugs to control the expression of tumor suppressor genes may enhance the efficacy of AVA therapy in OC. Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Sudhir Kumar, Elaine Stur, Lingegowda S. Mangala, Stephen Baylin, Gabriel Lopez-Berestein, Sunila Pradeep, Anil K. Sood. Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6001.

Abstract 2153: Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1

Abstract CCNE1 gene amplification is associated with overexpression of Cyclin E1 protein and is an important oncogenic driver. CCNE1 amplification is common in high grade serous ovarian carcinomas (HGSOC) and is associated with platinum resistance and poor patient outcomes. Importantly, Cyclin E1 overexpression can also occur in the absence of gene amplification in ovarian cancer. Overexpression of Cyclin E1 increases CDK2 activity and accelerates entry into S phase of cell cycle, resulting in replication stress and rendering cells more dependent on DNA repair. WEE1 participates in the DNA damage response by controlling essential checkpoints of the cell cycle, preventing cells from entering mitosis and allowing for DNA repair before cell cycle progression. Thus, we hypothesize that ovarian cancers overexpressing Cyclin E1, either via CCNE1 gene amplification or through other independent mechanisms, are more sensitive to WEE1 inhibition. Here, we tested this hypothesis in a range of preclinical cancer models using azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor currently in clinical development. Ovarian cancer cell lines overexpressing Cyclin E1 protein (Cyclin E1high) were more sensitive to azenosertib than ovarian cancer cell lines with lower levels of Cyclin E1 protein (Cyclin E1low) inducing significant cytotoxic effects (GRmax < -0.5). In Cyclin E1low ovarian cancer cell lines, overexpression of Cyclin E1 increased sensitivity to azenosertib. In contrast, silencing of CDK2 in Cyclin E1high cells reduced sensitivity to azenosertib. In vivo, higher levels of baseline expression of Cyclin E1 in tumor cells were associated with better response to azenosertib, with tumor growth inhibition (TGI) ranging from 51.5% in a Cyclin E1low SKOV3 model to 88% in a Cyclin E1high OVCAR3 model. Azenosertib synergized with chemotherapy agents in multiple ovarian cancer models. In vitro, we report that Cyclin E1high cancer cells were more sensitive to the combination of azenosertib and chemotherapeutics than Cyclin E1low cancer cells. In vivo, the synergy between azenosertib and paclitaxel was stronger in Cyclin E1high OVCAR3 model (46% reduction of initial tumor volume/104% TGI) than Cyclin E1low A2780 model (85% TGI). Taken together, these data suggest that Cyclin E1 overexpression via gene amplification or independent mechanisms sensitize ovarian cancer cells to azenosertib alone or in combination with chemotherapy. This observation supports the use of Cyclin E1 expression to enrich for azenosertib responders in ovarian cancer and possibly other tumor types. Two subsets of Cyclin E1-driven platinum resistant ovarian cancer patients, as defined by gene amplification or protein overexpression, are being enrolled in an azenosertib monotherapy trial. Citation Format: Jianhui Ma, Wen Liu, Heekyung Chung, Hooman Izadi, Petrus DeJong, Olivier Harismendy, Jia li, Fernando Doñate, Ahmed Samatar, Mark Lackner, Laure Escoubet, Kevin Bunker, Daehwan Kim, Nathan Jameson, Tugba Yildiran Ozmen, Kangjin Jeong, Dong Zhang, Wen-An Pan, Gordon Mills. Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2153.

8P NXP800 versus cisplatin in ARID1a-mutated ovarian clear cell carcinoma xenograft models

NXP800 (CCT361814) is a Heat Shock Factor (HSF1) pathway inhibitor currently being studied in a phase 1 clinical trial (NCT05226507). NXP800 demonstrated substantial antitumor activity in cancer cell lines of different histologies, including ovarian clear cell and endometrioid carcinoma (OCCC and EOC), gastric carcinoma, and haematological cancers. Loss of function of ARID1A was identified as a predictive marker for response to NXP800 in OCCC and EOC. Here we describe an in vivo study of NXP800 versus cisplatin as an active control in cisplatin-resistant and cisplatin-sensitive ARID1A-mutated OCCC xenografts, supporting the clinical development of NXP800 in this indication. In vivo studies were performed by generating subcutaneous tumors in Nude (nu/nu) mice using TOV-21G and SKOV3 cell lines. Loss of ARID1A was confirmed in western blot. Treatment with NXP800 resulted in substantial tumor growth inhibition (TGI) in both models. In the TOV-21G model, baseline tumor volumes for the vehicle, cisplatin and NXP800 groups were 201.71, 205.66 and 202.20 mm3, respectively; on Day 28, average tumor volumes were 399.84, 236.68 and 121.47 mm3, respectively, representing increases in tumor volume of 98% for the vehicle control group and 15% for the cisplatin group, and a decrease in tumor volume of 41% for the NXP800 group. In the SKOV3 model, baseline tumor volumes for the vehicle, cisplatin and NXP800 groups were 136.2, 138.5 and 134.7 mm3, respectively; on Day 28, average tumor volumes were 312.94, 253.71 and 53.33 mm3, respectively, representing increases in tumor volume of 130% for the vehicle control group and 80% for the cisplatin group, and a decrease in tumor volume of 60% for the NXP800 group. NXP800 exhibits notable therapeutic activity in xenografts of OCCC – a condition of high unmet medical need with limited treatment options – including sustained tumour growth inhibition and regression both in cisplatin resistant (SKOV3) and sensitive (TOV-21G) OCCC models. Planned phase 1b expansion cohorts will include ovarian clear cell carcinoma and ovarian endometrioid carcinoma patients with ARID1A mutation.

Synthesis and antiproliferative activities of steroidal lactam conjugates bearing a new nitrogen mustard

Alkylating agents are potent anticancer compounds that exert their anticancer properties through the inhibition of cell replication and transcription leading to cell death. Despite the numerous benefits, these agents also have serious drawbacks such as their high toxicity and low specificity towards cancer cells. As previously reported by our group, conjugation of alkylating agents with azasteroids can reduce their systemic toxicity and enhance their anticancer activity. In this work, novel steroidal alkylating agents bearing POPAM-OH were synthesized and their anticancer efficacy was evaluated in vitro and in vivo. All the novel hybrids demonstrated high antiproliferative effects against 5 different cancer cell lines in the low micromolar range. Treatment of SCID mice bearing SKOV-3 or PC-3 tumor xenografts with the most potent hybrid 19 led to significant reduction of tumor size (tumor inhibition TI = 95% in SKOV3 models and TI = 85.2% in PC3 models). Importantly, the acute toxicity of hybrid 19 (LD10 = 36 μΜ, LD50 = 62 μΜ) in CB17 SCID mice exhibited three-fold decrease compared to the acute toxicity of previously reported hybrids of POPAM-NH2. This is an important finding since systemic cytotoxicity is a critical limitation of alkylating agents. Collectively, the steroidal conjugates of POPAM-OH displayed significant anticancer efficacy and reduced toxicity in vitro and in vivo rendering them as good candidates for cancer therapy.

Alpha-fetoprotein mediated targeting of polymeric nanoparticles to treat solid tumors.

Background: Serious side effects caused by paclitaxel formulation, containing toxic solubilizer Cremophor®EL, and its nonspecific accumulation greatly limit clinical paclitaxel application. Aim: To designpaclitaxel-loaded copolymer of lactic and glycolic acidsnanoparticles decorated with alpha-fetoprotein third domain (rAFP3d-NP) to increase paclitaxel safety profile. Methods: rAFP3d-NP was obtained via carbodiimide technique. Results: The particles werecharacterized with high paclitaxel loading content of 5% and size of 280nm. rAFP3d-NP revealed biphasic profile with 67% release of paclitaxel during 220h. Increased area under the curveinf and mean residence timevalues after rAFP3d-NP administration confirmed prolonged blood circulation compared with paclitaxel. rAFP3d-NP demonstrated significant tumor growth inhibition at 4T1 and SKOV-3 models. Conclusion: rAFP3d-NP is a promising delivery system for paclitaxel and can be applied similarly for delivery of other hydrophobic drugs.

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%–75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with 125I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [125I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [125I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [125I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [125I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [125I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [125I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

Abstract 4575: Chimeric antigen receptor macrophages (CARMA) for adoptive cellular immunotherapy of solid tumors

Abstract Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated profound results in hematologic malignancies but clinical efficacy in the solid tumor setting has not been observed. Barriers to T cell entry and function may partially explain this observation. As solid tumors actively recruit myeloid cells, we hypothesized that macrophages have the potential to be a powerful cellular immunotherapeutic agent in this setting if properly activated and redirected. We here describe the development of CAR macrophages (CARMA), demonstrating the feasibility, mechanism, and efficacy of this platform. To examine the function of CARs in macrophages, first generation anti-CD19, anti-mesothelin, or anti-HER2 CARs with a CD3ζ intracellular domain were introduced into the THP1 macrophage model. In vitro function was assessed via quantitative phagocytosis and luciferase-based specific killing assays. CARMA selectively phagocytosed and cleared cognate antigen-bearing tumor cells. To demonstrate the requirement for CAR-mediated intracellular signaling for activity, a CD3ζ-null CAR construct was tested in vitro. The deletion of CD3ζ significantly reduced the phagocytic and killing capacity (p<0.01) of CARMA. We identified Ad5f35, a chimeric adenovirus, as a novel and highly efficient viral vector for the transduction of normal donor and cancer patient macrophages (>70% CAR expression). Ad5f35 transduction polarized human macrophages toward a durable immunostimulatory M1 phenotype and rendered CARMA resistant to subversion toward the immunosuppressive M2 phenotype, as defined by surface markers and metabolomics. CARMA enhanced the proliferative capacity of CD8+ T cells in phytohemagglutinin activation assays and secreted factors that activated by-stander macrophages. Primary human anti-HER2 CARMA demonstrated targeted phagocytosis and killing of HER2 expressing ovarian and breast cancer cell lines, and exhibited a six-fold higher luciferase-based killing capacity of SKOV3 cells compared to trastuzumab in vitro (p=0.002). Anti-HER2 CARMA was evaluated in vivo in an intraperitoneal (IP) SKOV3 ovarian cancer xenograft model. Mice that received IP CARMA had a decrease in tumor burden of approximately two orders of magnitude and had a 30-day survival benefit relative to untreated or control macrophage treated mice (p=0.018). In a systemically disseminated SKOV3 model, a single dose of IV CARMA led to a durable anti-tumor response (38-fold reduction relative to control on day 31 post-treatment; p=0.016) Lastly, we demonstrated that the blockade of the anti-phagocytic CD47/SIRPα axis enhanced the phagocytic capacity of CARMA. In summary, we here demonstrate that human macrophages engineered with a CAR exhibit targeted anti-tumor function in both in vitro and in vivo preclinical models. This novel cellular immunotherapeutic approach has a clear translational potential for the treatment of solid tumors. Citation Format: Michael Klichinsky, Marco Ruella, Olga Shestova, Saad S. Kenderian, Miriam Y. Kim, Roddy O'Connor, John Scholler, Carl June, Saar Gill. Chimeric antigen receptor macrophages (CARMA) for adoptive cellular immunotherapy of 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 4575. doi:10.1158/1538-7445.AM2017-4575

Abstract 3529: Immunotherapy targeting folate receptor induces autophagy in ovarian cancer

Abstract Cancer cells are highly dependent on folate metabolism mediated by the folate receptor (FRa). Gene encoding FRa (FOLR1) is overexpressed in endometrial and ovarian cancers. We aim to investigate the functional mechanisms of blocking FRa with MORAb-003 antibody, a humanized monoclonal antibody directly against FRa. We first analyzed clinical data from the Cancer Genome Atlas (TCGA) for correlation between copy numbers of FOLR1 gene and disease-free survival in patients with high-grade serous ovarian carcinoma (HGSC). Next, we determined the surface−expression of FRa on HGSC cells using flow cytometry (FACS). We then used xenograft mouse models to test tumor inhibition by MORAb-003 antibody-induced FRa blockade. We performed reverse-phase protein array (RPPA) analysis in FRa-positive and FRa-negative HGSC tumors to compare their responses to MORAb-003 treatment. To recapitulate the in vivo environment, we cultured FRa-positive and -negative epithelial ovarian cancer cells in three-dimensional (3D) condition and targeted the specific factors identified by RPPA using shRNAs knockdown. MORAb-003 monotherapy significantly reduced tumor weights and numbers of tumor nodules in both FRa-positive IGROV1 and SKOV3 models; combination of MORAb-003 and docetaxel significantly reduced both tumor weights and numbers of tumor nodules compared to docetaxel monotherapy. As expected, we did not observe these effects in the A2780 model, which has low FRa expression. Immunohistochemistry using PCNA and TUNEL staining in resulting tumors revealed that MORAb-003 reduced proliferation, but had no significant effect on apoptosis. Systematic analysis from RPPA identified a number of autophagy factors and cell-death initiators as being significantly upregulated by MORAb-003 treatment in FRa-positive tumors including Beclin-1 and PEA-15 (a factor involved in activation of autophagic cell death). cDNA microarrays further showed that MORAb-003 treatment up-regulated the expression of BECN1, ATG8, ATG3 and LC3B in FRa-positive tumors. Using FACS analysis with acridine orange (AO) staining, we observed significantly more AO positive population in 3D cultured SKOV3 cells treated with MORAb-003 alone (16.9%) or MORAb-003+docetaxel (21.2%) than in cells treated with control IgG (5.24%). Such differences were not observed in A2780 cells. We also observed significant increase of autophagy flux in SKOV3 cells but not in A2780 cells. We also found that blocking autophagy with hydroxychloroquine or bafilomycin A1, or knockdown Beclin-1 or PEA-15 with shRNAs reversed the growth inhibition induced by MORAB-003 in 3D cultured SKOV3 cells. We reported autophagy-associated cell death is a previously unrecognized mechanism for targeting of FRa in addition to the known mechanisms including antibody-dependent cellular cytotoxicity. Our studies also suggested that Beclin-1 and PEA-15 could be further explored as prognostic markers for FRa-targeted therapy. Citation Format: Yun-Fei Wen, Anil Sood. Immunotherapy targeting folate receptor induces autophagy in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3529.

PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples

BackgroundDespite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer. MethodsPG545’s anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models. ResultsPG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response. ConclusionOur results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.

Abstract 4027: Pathway analysis of chemoresistance in ovarian cancer cell lines.

Abstract Introduction: Ovarian Cancer is the most deadly female reproductive cancer and fifth most common cause of death from cancer among women. Limited characterization of the chemoresistant population demonstrates the need for improved therapy for ovarian cancer. Pathway analysis of microarray data of paired chemosensitive and chemoresistant ovarian cancer cell lines may help to elucidate important contributors to chemoresistance, and paired analyses can eliminate the significant heterogeneity among patient samples. Significant genes may serve as potential targets for novel drug therapies. Some genes likely contribute to resistance of multiple chemotherapies and represent common pathways of resistance. Methods: Microarray data of three pairs of chemosensitive and chemoresistant ovarian cancer cell lines were analyzed using GeneSpring Single Experiment Analysis of 517 pathways. These pairs include SKOV3ip1 and SKOV3TRip2 (taxane-resistant), HeyA8 and HeyA8MDR (multi-drug resistant), and A2780cp20 and A780cp55 (increasing cisplatin resistance). Significant genes within significant pathways were compared between types of drug resistance. Results: Between SKOV3ip1 and SKOV3TRip2, 8 pathways were significantly (p<0.001) modulated, dominated by the selenium pathway and folate/B12 metabolism. Between HeyA8 and HeyA8MDR, 9 pathways were significantly modified, led by DNA regulation and cell cycle progression. These differences between the SKOV3 model were interesting, given that both chemoresistant lines were developed in the same way. In A2780cp20 and A2780cp55, 7 pathways were significantly affected, highlighted by members of the Electron Transport Chain and controls on oxidative phosphorylation and autophagy. Validation of individual genes as important to chemoresistance will follow from this analysis. Conclusions: Multiple pathways are implicated in chemoresistance, even when selected by the same chemotherapeutic agent. Pathways contributing to metabolism, DNA repair, cell cycle control, and stress responses dominated. The high variability in pathway overexpression highlights the importance of patient-specific approaches to overcoming chemoresistance in ovarian cancer. Citation Format: William P. Jackson, Ashwini A. Katre, Zachary A. Dobbin, Adam D. Steg, Charles N. Landen. Pathway analysis of chemoresistance in ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4027. doi:10.1158/1538-7445.AM2013-4027

Topotecan and Doxorubicin Combination to Treat Recurrent Ovarian Cancer: The Influence of Drug Exposure Time and Delivery Systems to Achieve Optimum Therapeutic Activity

To provide proof-of-concept data to support use of Doxil-liposomal topotecan (Topophore C) combinations to treat ovarian cancer. ES-2, OVCAR-3, and SKOV-3 ovarian cancer cell lines were treated with doxorubicin-topotecan combinations by exposing the cells to drugs from 1 to 72 hours. Pharmacokinetic analysis was conducted following administration of liposomal formulations of these drugs alone and in combination. Efficacy assessments were completed in ES-2 and SKOV-3 ovarian cancer models. On the basis of drug doses capable of achieving 50% reduction in cell viability over 72 hours, doxorubicin-topotecan combinations were additive in SKOV-3 but highly synergistic in ES-2 and OVCAR-3 cells. Favorable drug-drug interactions increased with increased drug exposure time. Topophore C pharmacokinetic remained unaffected when co-administered with Doxil. In the ES-2 model, Doxil at maximum tolerated dose (MTD 7.5 mg/kg) in combination with free topotecan (MTD 15 mg/kg) did not enhance median survival time (MST) over that achieved with topotecan alone. In contrast, MST was increased to 52 days with combination of Topophore C (MTD 2.5 mg/kg) and Doxil (7.5 mg/kg) compared with untreated animals (MST 18 days) or those treated with Topophore C alone (MTD 5 mg/kg, MST 40 days). In the SKOV-3 model, combination treatments showed better therapeutic efficacy than the individual drugs. Topotecan-doxorubicin combinations produced additive or synergistic effects which were best achieved when the tumor cells were exposed to drugs over extended time. Doxil-Topophore C combinations are therapeutically superior as judged in two ovarian cancer models. Clin Cancer Res; 19(4); 865-77. ©2012 AACR.

An injectable depot system for sustained intraperitoneal chemotherapy of ovarian cancer results in favorable drug distribution at the whole body, peritoneal and intratumoral levels

The current study characterizes the impact of docetaxel (DTX) distribution on efficacy following sustained intraperitoneal (IP) chemotherapy in murine models of ovarian cancer. A polymer-lipid biodegradable depot (PoLigel) was used to deliver DTX in a sustained manner over 21-days following IP administration. Distribution and efficacy studies were carried out in SCID mice bearing SKOV3 IP solid tumors or C57BL/6 mice with ID8 IP ascites fluid. In addition, a subcutaneous (SC) SKOV3 model was used to determine whether systemic drug levels that result from IP administration of the PoLigel influence antitumor efficacy. Immunostained IP and SC SKOV3 tumor sections were used to study cell death, intratumoral drug distribution and tumor penetration. Sustained concentrations of DTX were observed in plasma, tissue, tumor and ascites over the entire study period. Drug accumulation was several fold greater in tumors and ascites when compared to plasma levels. Sustained chemotherapy resulted in significant reduction in tumor burden and ascites volume. IP tumors showed greater cell death compared to the SC tumors as seen by higher TUNEL and caspase-3 expression. At the intratumoral level, DTX distributed more towards the core of IP tumors compared to the SC tumors. Tumor penetration of drug from nearest blood vessel was 1.5 fold greater in the IP tumors than the SC tumors. Overall, favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure contribute to the high efficacy observed. These results encourage the clinical use of IP sustained chemotherapy for ovarian cancer.

Abstract A108: LY2606368, a second generation Chk1 inhibitor, inhibits growth of ovarian carcinoma xenografts either as monotherapy or in combination with standard-of-care agents.

Abstract Chk1 is a key component of the cellular response to DNA damage resulting from exposure to various genotoxic agents. When damaged DNA is present, Chk1 maintains genomic integrity by stalling cell cycle progression thereby providing time for the appropriate repair to occur. Inhibition of Chk1 kinase activity leads to a loss in the regulation of this damage response and results in abrogation of cell cycle arrest and ultimately to cell death as cells with damaged DNA proceed inappropriately into mitosis. Thus first generation small molecule inhibitors of Chk1 kinase were developed to act as companion compounds that would be used together with established standard -of-care (SoC) cancer treatments that target DNA to enhance the effectiveness of such treatments. As a second generation inhibitor, LY2606368 distinguishes itself from other molecules in this class in that it has been shown to inhibit tumor growth when used either as monotherapy or in combination with SoC agents. The single agent activity of LY2606368 is likely related to the ability of this molecule to potently inhibit Chk1 thereby effectively interfering with its function to support normal DNA replication via regulation of origin firing and/or aspects of chromosomal dynamics during mitosis which are thought to be dependent upon Chk1 kinase activity. The studies presented here have evaluated the potential of LY2606368 to inhibit ovarian tumor growth both as monotherapy and in combination with cisplatin or paclitaxel. Treatment of tumor-bearing animals on a BID X 3 schedule with LY2606368 alone resulted in profound growth inhibition such that greater than 80% inhibition was observed at doses of 12mg/kg in both subcutaneous and orthotopic models. Moreover, the studies in the orthotopic SKOV3 model demonstrated that treatment with LY2606368 not only robustly inhibited growth of the primary tumor but also significantly reduced the incidence of metastases and accumulation of ascites fluid. Subsequent studies in SKOV3 subcutaneous xenografts revealed that significant growth inhibition could be achieved with various combination strategies which include agents that are commonly used in the treatment of ovarian cancer. In particular, the extent of growth inhibition that could be achieved with cisplatin or paclitaxel alone was augmented by combining these agents with LY2606368. Overall these animal studies indicate the potential therapeutic benefit in ovarian cancer that could be achieved with LY2606368 either as monotherapy or in combination with SoC agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A108.

Growth control of human ovarian cancer cells by the dynein light chain and TGFβ receptor interacting protein km23-1

Abstract We have identified km23-1 as a TGFβ signaling intermediate that is also a light chain of the minus-end directed motor protein dynein (DLC). Our results suggest that km23-1 (also termed DYNLRB1/robl1/mLC7-1/Dnlc2a) can function as a “motor receptor” to recruit TGFβ signaling components to the dynein motor for endosomal trafficking prior to nuclear translocation. For example, we have shown that overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocks TGFβ-stimulated nuclear translocation of Smad2. Further, siRNA blockade of km23-1 resulted in a reduction both in nuclear levels of phosphorylated (ie, activated) Smad2 and of TGFβ/Smad2-dependent transcriptional activity (JBC 282: 19122, 2007). Similarly, we have shown that km23-2 is required for Smad3 TGFβ signaling (JCP 221: 707, 2009). However, many human ovarian cancer cells (HOCCs) have lost growth inhibitory responsiveness to TGFβ. In order to assess whether km23-1 could also regulate the growth of aggressive, TGFβ-resistant HOCCs, we developed a tet-off-inducible model system for induction of wild-type km23-1 expression in SKOV-3 HOCCs. Here we show that overexpression of km23-1 (by 10-20 fold) suppressed the growth of the cells in vitro and markedly impeded tumor formation in vivo. Fluorescence-activated cell sorting (FACS) analysis in the SKOV-3 model system demonstrated an increased fraction of cells in G2/M after induction of km23-1 expression (dox removal), compared with controls. Similar results were obtained for the TGFβ-resistant IGROV-1 HOCCs. Additional studies revealed that km23-1 overexpression in HOCCs increased the mitotic index, in association with activation of both MEK and ERK and an elevation of cyclin B1 protein expression levels. The elevated levels of phospho-MEK, phospho-ERK, and cyclin B1, as well as the distribution of the cells in mitosis, suggest that the cells are delayed in prometaphase/ metaphase. This mitotic delay, followed by cell death, appears to be the mechanism by which high levels of km23-1 can suppress ovary tumor growth in vivo. Since dynein is known to have critical functions at this point in mitosis by inactivating the spindle assembly checkpoint (SAC), studies are underway to investigate how km23-1 overexpression regulates the SAC to cause the reduced tumor formation in vivo. This work was supported by NIH grants CA100239, CA92889, and CA90765 to KMM. Our results indicate that km23-1 is a critical ovarian cancer target for the development of novel diagnostic and therapeutic agents. In this regard, US patents have been issued to KMM related to km23-1-based methods for diagnostic/prognostic screening, as well as for the identification of km23-1-based therapeutic agents for ovarian cancer (US 7,229,758 B2; 6-07 & US 7,741,043; 6-10).

Abstract 4479: PF-04691502, a potent and selective PI3K/mTOR dual inhibitor with antitumor activity

Abstract The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, loss of function of PTEN or activation of receptor tyrosine kinases. Inhibition of key signaling proteins in the pathway, such as PI3K, AKT and mTOR, therefore represents a high value targeting strategy for diverse cancers. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in in vitro biochemical, cell and xenograft models. In in vitro biochemical assays PF-04691502 inhibited recombinant PI3Kα, β, γ and δ isoforms with Ki's of 1.2-2.2 nM and recombinant mTOR with a Ki of 9.1 nM. PF-04691502 demonstrated a high degree of selectivity for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the Class III PI3K hVps34. PF-04691502 also inhibited transformation of avian fibroblasts mediated by PI3K γ, δ, mutant PI3Kα E545K or membrane-localized AKT with IC50's of ∼100nM. In cell assays PF-04691502 inhibited PI3K/mTOR signaling in SKOV3 ovarian cancer cells with PI3Kα mutations and in U87MG glioblastoma cells with PTEN alteration, as indicated by reduced levels of phosphorylation of AKT(T308), AKT(S473) and S6 ribosomal proteins. Functional studies for anti-proliferative effects suggest PF-04691502 has broad efficacy across tumor types. In SKOV3 and U87MG xenograft models PF-04691502 treatment resulted in dose-dependent tumor growth inhibition (TGI) with maximum TGI of ∼70% at the maximum tolerated dose of 10 mg/kg, by once daily oral dosing. Inhibition of AKT(S473) phosphorylation and S6RP(S235/236)/PRAS40(T246)/4EBP1(T37/46) phosphorylation were used as quantitative and qualitative pharmacodynamic (PD) endpoints, respectively; a clear pharmacokinetic (PK)/PD relationship was established in both models after multiple dose oral administration. In the U87MG xenograft model AKT(S473) phosphorylation was inhibited with an estimated EC50 of 5.7 nM (free plasma concentration) based on PK/PD modeling. The free plasma Area Under Curve was estimated to be 850 nM*hr for 70% TGI at 10mg/kg and was found to be similar in the SKOV3 model. The projected human efficacious dose of 10 mg once daily oral dosing provides Caverage steady state exposure of 22.4 nM (free plasma concentration) which is sufficient for 50-80% inhibition of pAKT S473, and corresponds to 74% TGI. Phase 1 clinical trials of PF-04691502 as a single agent are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4479.

In Vivo Activity of ARRY-380, a Potent, Small Molecule Inhibitor of ErbB2 in Combination with Trastuzumab, Docetaxel or Bevacizumab.

Abstract ARRY-380 is an orally active, potent small molecule tyrosine kinase inhibitor targeting ErbB2. The compound is a reversible, ATP-competitive inhibitor with nanomolar potency against ErbB2 in both in vitro and in cell-based assays. This compound has very good in vivo and in vitro PK/ADME properties and has shown excellent activity in numerous mouse tumor models including breast (BT-474, MDA-MB-453), ovarian (SKOV-3) and gastric (N87) carcinoma models. Here we demonstrate excellent single agent activity and combinability with trastuzumab, docetaxel or bevacizumab in breast and ovarian carcinoma models. For the BT-474 studies, female SCID beige mice were implanted with tumor fragments. For the SKOV-3 tumor studies, female nude mice were inoculated with cells subcutaneously in the flank. Animals received: doses of ARRY-380 ranging up to 200 mg/kg/d, PO; and/or trastuzumab at 20 mg/kg, IP, Q3D or QW; and/or docetaxel at 10 mg/kg, IV, Q3D; and/or bevacizumab at 10 mg/kg, IP, Q4D x3. Tumor size was measured at regular intervals and subsets of animals were monitored for up to 90 days to determine tumor-free survival. In the BT-474 model, ARRY-380 demonstrated significant dose-related tumor growth inhibition (TGI; 50% at 50 mg/kg/d and 96% at 100 mg/kg/d) with numerous partial regressions (>50% reduction from baseline size) at the higher dose level in 9/12 animals. One complete response was observed at the higher dose. Trastuzumab alone provided a 45% TGI with no regressions. ARRY-380 (50 mg/kg/d) in combination with trastuzumab showed a 98% TGI with complete regressions in 9/12 animals and two partial regressions. At dose of 100 mg/kg/d of ARRY-380 in combination with trastuzumab, there was 100% TGI and all animals had complete responses. Docetaxel as a single agent produced a 55% TGI with no regressions. In combination with ARRY-380 (50 mg/kg/d), there was an 81% TGI and five partial regressions. In the SKOV-3 model, ARRY-380 demonstrated significant dose-related tumor growth inhibition (TGI; 39% at 50 mg/kg, BID and 96% at 100 mg/kg, BID) with partial regressions (>50% reduction from baseline size) at the higher dose level in all animals. Bevacizumab alone provided a 55% TGI with no regressions. ARRY-380 (50 mg/kg, BID) in combination with bevacizumab showed 80% TGI with partial responses in 7/8 animals and one stable disease. From this work we have demonstrated superb single agent activity for ARRY-380 in the BT-474 human breast carcinoma xenograft model and the SKOV-3 human ovarian carcinoma model. In addition, ARRY-380 has shown additive activity and tolerability with trastuzumab, docetaxel and bevacizumab. ARRY-380 has entered Phase I clinical trials in patients with advanced cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5104.

Abstract C172: Efficacy of AMG 479, alone and in combination with cisplatin, in ovarian carcinoma xenograft models

Abstract AMG 479, a fully human monoclonal antibody raised to the type I insulin-like growth factor receptor (IGF1R), is currently being studied in the treatment of patients with ovarian carcinoma. We have previously shown that AMG 479 inhibits the growth of pancreatic and sarcoma tumor models by inhibiting IGF1R signaling through ligand blockade and receptor internalization and degradation. In the present study, we tested the ability of AMG 479 to inhibit the growth of four human ovarian carcinoma xenografts (OV-90, SK-OV3, TOV-21G, and OV-CAR3) in nude mice. Recent evidence has shown that multiple players in the IGF-axis are differentially regulated in human ovarian serous carcinomas, suggesting that the IGF1R pathway may play an important role in the growth of these tumors. The OV-90 model showed exquisite sensitivity to single-agent AMG 479, reaching significant (p<0.0001) tumor growth inhibition (TGI) of more than 90% with 100 or 300 g AMG 479/dose administered twice per week for the duration of the experiment. The OV-CAR3 model was also sensitive to single-agent AMG 479 and showed significant (p<0.0001) TGI of about 50% with 300 g AMG 479/dose, twice per week. The TOV-21G and SK-OV3 models showed resistance to AMG 479 with no significant TGI achieved at any dose. The OV-90 (sensitive) and TOV-21G (resistant) models were used to further characterize the AMG 479 response in a sensitive and a resistant model. AMG 479 effectively inhibited both IGF1R and Akt activity in the OV-90 model but not in the TOV-21G model in vitro. Furthermore, analysis of tumor xenografts collected at the end of the experiment revealed that TGI in the OV-90 model correlated with 50% receptor downregulation (total IGF1R), 90% inhibition of receptor activation (phospho IGF1R), and 50% inhibition of Akt activity, while no measurable inhibition in these analytes were detected in the TOV-21G xenografts. The potential benefit of combining AMG 479 with platinum-based therapy was assessed by combination with cisplatin in the moderately sensitive OVCAR-3 model. Treatment with AMG 479 (300 g/dose, twice per week) in combination with cisplatin (2.5 mg/kg, twice per week) resulted in significant (p=0.0001 vs. AMG 479, p=0.0005 vs. cisplatin) TGI compared with either therapy alone, suggesting that the two agents can be combined clinically for the treatment of ovarian carcinoma patients. Together, the data suggest that AMG 479 has therapeutic potential in treating ovarian carcinoma patients both as monotherapy and in combination with standard of care agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C172.

Biologic characteristics of intraperitoneal transplantation model of human ovarian carcinoma in severe combined immunodeficiency mice

To develop a human ovarian carcinoma SKOV3 model in severe combined immunodeficiency (SCID) mouse and to study its biologic characteristics. Human ovarian carcinoma SKOV3 cells were injected intraperitoneally into female SCID mouse to establish a transplantation model of human ovarian carcinoma. The biological characteristics, metastasis and morphology of transplanted tumors were studied. All tumors grew progressively with no sign of regression. The tumor cells spread around the peritoneal cavity and mainly on the diaphragm, mesentery, peritoneum and around the liver, which was confirmed by histopathology. The morphology, growth pattern and CA125 secretion of primary culture of transplanted cells remained as same as those of ovarian carcinoma cell line SKOV3. An intraperitoneal transplantation model of human ovarian carcinoma SKOV3 in SCID mice has been developed successfully, which can simulate the biological behavior of peritoneal metastasis of human ovarian carcinoma.