Granulosa cell tumors (GCT) constitute only ~5% of ovarian neoplasms yet have significant consequences, as up to 80% of women with recurrent GCT will die of the disease. This study investigated the effectiveness of procaspase-activating compound 1 (PAC-1), an activator of procaspase-3, in treating adult GCT (AGCT) in combination with selected apoptosis-inducing agents. Sensitivity of the AGCT cell line KGN to these drugs, alone or in combination with PAC-1, was tested using a viability assay. Our results show a wide range in cytotoxic activity among the agents tested. Synergy with PAC-1 was most pronounced, both empirically and by mathematical modelling, when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This combination showed rapid kinetics of apoptosis induction as determined by caspase-3 activity, and strongly synergistic killing of both KGN as well as patient samples of primary and recurrent AGCT. We have demonstrated that the novel combination of two pro-apoptotic agents, TRAIL and PAC-1, significantly amplified the induction of apoptosis in AGCT cells, warranting further investigation of this combination as a potential therapy for AGCT.

Interleukin-2 (IL-2) has been used clinically for the treatment of some malignancies, but the toxicities associated with systemic IL-2 therapy are a major challenge. Here we have determined whether transcriptional targeting of IL-2 to breast cancer (BrCa) using an engineered human mammaglobin promoter/enhancer (MPE2) is a feasible option for reducing IL-2-associated toxicities while still achieving a meaningful antitumor effect. We have constructed nonreplicating adenovirus vectors encoding either a reporter gene (luciferase) or human IL-2 (hIL-2) complementary DNA under control of the MPE2 sequence, the murine cytomegalovirus immediate early (MCMV) promoter or the human telomerase reverse transcriptase (hTERT) promoter. Luciferase and hIL-2 complementary DNAs under the control of the MPE2 sequence in adenovirus vectors were expressed at high levels in BrCa cells and at lower levels in normal cells of human and murine origin. Cancer specificity of the hTERT promoter was found to be similar to that of the MPE2 promoter in cells of human origin, but reduced specificity in murine cells. The MPE2 regulatory sequence demonstrated excellent tissue specificity in a mouse tumor model. Whereas the MCMV promoter-controlled IL-2 vector generated high liver toxicity in mice, the MPE2-controlled IL-2 vector generated little or no liver toxicity. Both IL-2 vectors exerted significant tumor growth delay; however, attempts to further enhance antitumor activity of the IL-2 vectors by combining with the proapoptotic drug procaspase activating compound 1 (PAC1) were unsuccessful.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

Ovarian cancer is commonly diagnosed in its late stages, and new treatment modalities are needed to improve patient outcomes and survival. We have recently established the synergistic effects of combination tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and procaspase activating compound (PAC‐1) therapies in granulosa cell tumors (GCT) of the ovary, a rare form of ovarian cancer, using a mathematical model of the effects of both drugs in a GCT cell line. Here, to understand the mechanisms of combined TRAIL and PAC‐1 therapy, study the viability of this treatment strategy, and accelerate preclinical translation, we leveraged our mathematical model in combination with population pharmacokinetics (PKs) models of both TRAIL and PAC‐1 to expand a realistic heterogeneous cohort of virtual patients and optimize treatment schedules. Using this approach, we investigated treatment responses in this virtual cohort and determined optimal therapeutic schedules based on patient‐specific PK characteristics. Our results showed that schedules with high initial doses of PAC‐1 were required for therapeutic efficacy. Further analysis of individualized regimens revealed two distinct groups of virtual patients within our cohort: one with high PAC‐1 elimination and one with normal PAC‐1 elimination. In the high elimination group, high weekly doses of both PAC‐1 and TRAIL were necessary for therapeutic efficacy; however, virtual patients in this group were predicted to have a worse prognosis when compared to those in the normal elimination group. Thus, PAC‐1 PK characteristics, particularly clearance, can be used to identify patients most likely to respond to combined PAC‐1 and TRAIL therapy. This work underlines the importance of quantitative approaches in preclinical oncology.

Evasion of apoptosis is a hallmark of cancer, and direct induction of apoptosis without dependence on signaling upstream of Caspase-3 (CASP3) is an attractive target for cancer therapy. CASP3 sits at the hub of apoptotic pathways and it is a primary target for inhibition by anti-apoptotic proteins like the X-linked inhibitor of apoptosis (XIAP). Granulosa cell tumour (GCT) is a rare form of ovarian cancer, highly lethal in the event of recurrence, and has no standard chemotherapy because it is resistant to most common drugs. A recently discovered small-molecule drug, procaspase activating compound-1 (PAC1), has been shown to effectively cleave procaspase-3 into its active form by removal of an inhibitory zinc ion, facilitating autocleavage of the zymogen and direct induction of apoptosis. Initial in vitro experiments in our lab have shown PAC1 capable of significantly reducing viability of GCT cells (represented by the KGN cell line), and combining PAC1 with a drug inhibiting XIAP further increases the killing effect. In addition, combining the small-molecule XIAP inhibitor with carboplatin, a standard chemotherapy agent, displays significant drug interaction while killing GCT cells, in vitro. Results: GCT cell line KGN was treated with various concentrations of PAC1, for 24 and 48 hour time points, and assessed for cell viability using a metabolic assay. Results showed significant reduction of cell viability (p<0.05) in both time and dose-dependent manners. The dose-response curve for these assays indicate an EC50 of ~10 µM PAC1. High-content screening of PAC1-treated GCT cells produced quantified imagery that suggests treatment with PAC1 is, in fact, inducing activation of CASP3-mediated apoptosis and an assay inhibiting CASP3 displayed reduction in PAC1-induced killing. Combining 10 µM PAC1 with selected concentrations of embelin, a monovalent XIAP-inhibitor, showed strong additive effect in initial experiments, indicating it is an area worthy of further research. Combination of embelin with carboplatin also displayed significant drug interaction as well as single drug effect (p<0.05). This presentation suggests that PAC1 reduces viability of GCT cells in a time and dose-dependent manner, in vitro, and the mechanism of that loss of viability is caspase-mediated apoptosis. Furthermore, combining PAC1 with drugs that inhibit XIAP enhances the killing effect. Combining small molecule drugs that promote apoptosis display strong interaction with carboplatin, a standard chemotherapeutic agent, and may have the potential to allow carboplatin dose reduction. Further research to determine activity in animal models and/or primary human GCT explants is warranted and planned. Citation Format: Powel Crosley, Kate Agopsowicz, Michael Weinfeld, Mary Hitt. Combining small molecule drugs and standard chemotherapy for treatment of granulosa cell tumour cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1410.

INTRODUCTION: Procaspase activating compound-1 (PAC1) is a small-molecule drug shown in vitro to sequester inhibitory zinc ions from caspase-3. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand that binds membrane-bound death receptors, triggering the extrinsic apoptotic pathway. Both agents display low toxicity in humans. Vaccinia virus (VACV) is a double-stranded DNA virus that has shown therapeutic efficacy in clinical trials and has an established safety profile in humans due to its use as the smallpox vaccine. First-line standard of care for ovarian cancer is combination taxane and carboplatin, which has significant potential toxicity and 70% of women who receive it suffer relapse. Clinical trials involving TRAIL, both alone and combined with other drugs, have shown that while it is well-tolerated it is ineffective partly due to insufficient dosing at the tumour site. In an effort to uncover a safer, more effective therapeutic for ovarian cancer we report here on the successful construction of a recombinant oncolytic vaccinia virus expressing TRAIL (VACVTRAIL). Secretion of TRAIL by VACV-infected cancer cells will result in localized administration of TRAIL at higher dosages and minimize potential side-effects. We posit that treatment with PAC1 and VACVTRAIL represents a potentially safe, effective treatment for ovarian cancers. RESULTS: Testing in cell line models of granulosa cell tumour (GCT) have shown that recombinant human (rh)TRAIL is effective in combination with PAC1. Dose-response assays established that combination of PAC1 (20 μM) with rhTRAIL (10 ng/mL) dramatically reduced viability of cancer cells while being substantially less toxic to normal cells. Replication of those assays on patient-derived primary and recurrent GCT cells confirmed PAC1 combined with rhTRAIL was dramatically more cytotoxic than treatment with rhTRAIL or PAC1 alone. To optimize delivery of TRAIL to tumour cells, we constructed a recombinant VACVTRAIL virus that secretes TRAIL in the range of 70–80 ng/mL. Dose-response curves showed VACVTRAIL to be strongly cytotoxic with an ED50 of 0.1 plaque forming unit (PFU) per cell. Comparing toxicity of VACVTRAIL to a non-TRAIL-expressing VACV established that secretion of TRAIL is the basis for VACVTRAIL superiority in killing GCT cells, and supernatant collected from infected cells is more effective at reducing cell viability when combined with PAC1 than is rhTRAIL combined with PAC1. CONCLUSION: We have successfully constructed a TRAIL-expressing oncolytic VACV which produces effective levels of active TRAIL from infected cells. Results in vitro suggest combining PAC1 with oncolytic VACVTRAIL will allow localized delivery of TRAIL resulting in a safe, synergistic, self-amplifying therapy. Citation Format: Powel Crosley, Kate Agopsowicz, Kyle Potts, Ryan Noyce, Marjut Pihlajoki, Markku Heikinheimo, Anniina Färkkilä, David Evans, Mary Hitt. TRAIL-EXPRESSING ONCOLYTIC VACCINIA VIRUS COMBINED WITH SMALL-MOLECULE DRUG PAC1 IS A POTENTIALLY EFFECTIVE TREATMENT ALTERNATIVE FOR OVARIAN CANCERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-089.

Ovarian cancer is commonly diagnosed in its late stages, and new treatment modalities are needed to improve patient outcomes and survival. We have recently established the synergistic effects of combination tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and procaspase activating compound (PAC-1) therapies in granulosa cell tumours (GCT) of the ovary, a rare form of ovarian cancer, using a mathematical model of the effects of both drugs in a GCT cell line. Here, to understand the mechanisms of combined TRAIL and PAC-1 therapy, study the viability of this treatment strategy, and accelerate preclinical translation, we leveraged our mathematical model in combination with population pharmacokinetics (PopPK) models of both TRAIL and PAC-1 to expand a realistic heterogeneous cohort of virtual patients and optimize treatment schedules. Using this approach, we investigated treatment responses in this virtual cohort and determined optimal therapeutic schedules based on patient-specific pharmacokinetic characteristics. Our results showed that schedules with high initial doses of PAC-1 were required for therapeutic efficacy. Further analysis of individualized regimens revealed two distinct groups of virtual patients within our cohort: one with high PAC-1 elimination, and one with normal PAC-1 elimination. In the high elimination group, high weekly doses of both PAC-1 and TRAIL were necessary for therapeutic efficacy, however virtual patients in this group were predicted to have a worse prognosis when compared to those in the normal elimination group. Thus, PAC-1 pharmacokinetic characteristics, particularly clearance, can be used to identify patients most likely to respond to combined PAC-1 and TRAIL therapy. This work underlines the importance of quantitative approaches in preclinical oncology.

BACKGROUND: Granulosa cell tumour (GCT) constitutes ~5% of ovarian neoplasms. Surgery remains the primary treatment modality due to a generally poor response to chemotherapy in GCT patients. Procaspase activating compound-1 (PAC1) is a small-molecule drug shown in vitro to sequester inhibitory zinc ions from the Caspase-3 (CASP3) zymogen allowing CASP3 to automature and execute apoptosis. PAC1 has demonstrated safety and efficacy in vivo against several cancers and is currently in Phase I trials for advanced malignancies. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptosis ligand that can bind membrane-bound death receptors. This binding triggers the extrinsic apoptotic pathway resulting in activation of CASP3 to execute its proteolytic role and programmed cell death. TRAIL has been found to induce apoptosis selectively in cancer cells in vitro and in vivo, and has been well tolerated in human patients. We hypothesise that combining PAC1 activation of CASP3 with induction of apoptotic signaling by exogenous TRAIL will significantly heighten biologic effect, reduction of disease, and that these effects will be obtained at doses lower than those required by either agent alone. Here we report on in vitro experiments in support of this hypothesis, suggesting that combining PAC1 with TRAIL may be effective therapy for treatment of GCT. METHODS: The GCT cell line KGN was treated in vitro with a 6-log range of PAC1 concentration for 48 hours to establish a dose-response curve (using a real-time cell analyzer, RTCA). In parallel, KGN cells were treated with a 6-log range of TRAIL concentration to establish its dose-response curve. Calculated EC50 values were then used for both PAC1 (20 μM) and TRAIL (10 ng/mL) to evaluate the biologic response of simultaneous treatment with PAC1 and TRAIL, and TRAIL delayed 24 hours after PAC1 treatment (using resazurin viability assay and RTCA). Separately, cells from fresh primary and recurrent tumour samples were cultured in vitro for 5 days, then treated with PAC1 (20 μM), TRAIL (10 ng/mL), or the combination, and finally assayed for viability and caspase 3/7 activity 48 and 72 hours later. RESULTS: Dose-response assays indicate treatment with PAC1 strongly reduces viability of KGN cells compared to untreated control (p<0.05) in a dose-dependent manner and treatment with PAC1 alone significantly reduced viability compared to untreated control (p<0.05). Similar assays with TRAIL only reduced viability of KGN cells at the highest concentration tested (1 µg/mL). The assays also suggest a ~24 hour delay in PAC1 reduction of GCT viability while TRAIL appears to display a time-limited response. Combination treatment was assessed using calculated EC50 concentrations for both PAC1 (20 µM) and TRAIL (10 ng/mL). Assays for both KGN and patient-derived primary GCT cells tested each drug alone, both drugs applied concurrently and TRAIL applied 24 hours after PAC1. Combination of PAC1 with TRAIL was dramatically more cytotoxic than TRAIL or PAC1 treatment alone (p<0.05) CONCLUSION: Combining CASP3 activator PAC1 with apoptosis-inducing agents may be an effective strategy for treatment of GCT and warrants preclinical assessment. Citation Format: Powel Crosley; Kate Agopsowicz; Marjut Pihlajoki; Markku Heikinheimo; Anniina Färkkilä; Mary Hitt. PAC–1 COMBINATION WITH TRAIL ENHANCES APOPTOSIS IN CELL–LINE AND PRIMARY CULTURED ADULT GRANULOSA CELL TUMOUR CELLS [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 NTOC-086.