Abstract P5-05-07: Foxo3a activation contributes to the synergy of imipridone and radiation in triple-negative breast cancer
Authors: Wang Shang-Jui , Schiff Devora , Wu Hao , Jhawar Sachin , Haffty Bruce G 
Published: March 2020

Abstract
Purpose/Objective: Imipridone (IMIP) is a small molecule DRD2 antagonist that has shown efficacy in preclinical and early phase clinical studies in advanced cancers. Antitumor mechanisms of IMIP appears to be cell-type dependent, and may include Foxo3a-dependent TRAIL upregulation, ATF4/CHOP integrated stress response pathway, and upregulation of death receptors (DR4/DR5). Our previous work has demonstrated therapeutic synergy of combination treatment with IMIP and radiation (RT) in preclinical triple-negative breast cancer (TNBC) models. In this study, we investigated the potential mechanism of synergy in combination treatment with IMIP and RT in TNBC. Methods: In vitro experiments were performed in six TNBC cell lines (MB-468, BT-549, BT-20, MB-231, MB-436, and HCC1937). Cells were treated with IMIP alone, RT alone, or combination of IMIP and RT. RT (0-8 Gy) was delivered via a Gamma-cell 40 exactor and IMIP was given at doses of 0-2 µM. To evaluate the AKT-Foxo3a axis function, we examined the phosphorylation status of AKT and Foxo3a as a surrogate for their functional activities. Cells were treated with IMIP, followed by a single dose of RT (0, 4 or 8 Gy), and cells were harvested at 72 hours and 96 hours post-RT. Whole cell lysates were collected and Western blotting was performed with phosphorylation-specific antibodies of AKT and Foxo3a. Downstream Foxo3a target gene expression was evaluated via RT-qPCR, including TRAIL, PUMA, and various targets. To determine the dependency of Foxo3a, knockdown experiment by transfection of Foxo3a siRNA was performed. Results: Of the six TNBC cell lines tested, MB-468 and BT-20 cells were found to exhibit synergistic cell death from combination treatment with IMIP (1 µM) and RT (4 or 8 Gy). Western blotting of whole cell lysates of both MB-468 and BT-20 revealed a mild decrease in the inhibitory Foxo3a phosphorylation at S253 (Foxo3a-P S253) with RT alone at 96 hours post-RT, no effect with IMIP alone, but a marked reduction of Foxo3a-P S253 with combination of IMIP and RT, without significant changes in the total Foxo3a protein levels. No effect was observed with AKT phosphorylation at S473. In addition, cleaved caspase 3 levels were increased at 96-hours post-RT with combination treatment. RT-qPCR of extracted mRNA revealed Foxo3a target upregulation of apoptotic PUMA gene in both MB-468 and BT-20 cells, as well as death ligand TRAIL in BT-20 cells. Finally, transient depletion of Foxo3a level via siRNA demonstrated a partial rescue of treatment-induced cell death in MB-468 cells, but not in BT-20 cells. Conclusion: We demonstrated therapeutic synergy of IMIP and RT in select TNBC cell lines (MB-468 and BT-20). Synergistic decrease in the inhibitory S253 Foxo3a phosphorylation with combination treatment, independent of upstream AKT activity, correlated with increased cell death, increased cleaved caspase-3 levels, and activation of Foxo3a targets PUMA and TRAIL. Foxo3a knockdown partially rescued MB-468 cells, but not BT-20 cells, from combination treatment, suggesting that other cellular pathways may also contribute to the observed synergy of IMIP and RT in select TNBC cells.
Citation Format: Shang-Jui Wang, Devora Schiff, Hao Wu, Sachin Jhawar, Bruce G Haffty. Foxo3a activation contributes to the synergy of imipridone and radiation in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-07.