Abstract 214: Styrylbenzoic acid derivative DC10 potentiates radiotherapy by inhibiting glutathione synthesis, leading to increased oxidative stress and cancer cell death
Authors: Sarowar Shahin , Cirillo Davide , Jativa Pablo , Nilsen Mette H. , Otragane Sarah-Muheha A. , Heggdal Jan I. , Selheim Frode , Ceña Valentín , Bjørsvik Hans-René , Enger Per Øyvind 
Published: June 2022

Abstract
Metastatic tumors with moderate radiosensitivity account for most cancer-related deaths, highlighting the limitations of current radiotherapy regimens. The xCT-inhibitor sulfasalazine (SAS) sensitizes cancer cells by blocking xCT-mediated cystine uptake, and thereby glutathione (GSH) synthesis protecting against radiation-induced oxidative stress. However, SAS has limited clinical potential as a radiosensitizer due to side effects and low bio-availability. Using SAS as a starting point, we previously developed synthetic xCT-inhibitors through scaffold hopping & structure optimization aided by structure-activity relationship analysis (SAR). Notably, the compound DC10 exhibited potent inhibition of GSH synthesis. In this study, we validated DC10 as a radiosensitizer in the xCT-expressing cancer cell lines A172, A375 & MCF7 in vitro & in mice harboring melanoma xenografts. After DC10 treatment, we measured 14C-cystine uptake in the cancer cell lines using liquid scintillation counting, and intracellular levels of GSH & reactive oxygen species (ROS) using luminescence assays. We performed immunoblotting for γH2AX & ATM to assess DNA damage after treatment with DC10 and radiotherapy. We then assessed the effect of adding DC10 to radiation upon cancer cell colony formation. Blood samples from mice treated with DC10 underwent biochemical analysis to assess toxicity. Finally, mice with A375 melanomas in the flank, received DC10 and radiotherapy in combination, as monotherapies or no treatment. Notably, DC10 reduced cystine uptake and GSH synthesis & increased ROS levels in a dose-dependent manner. Furthermore, DC10 interacted synergistically with radiation to increase DNA damage & reduce colony formation. Mice receiving DC10 were clinically unaffected, whereas blood samples analyses to assess bone marrow suppression, liver or kidney toxicity revealed no significant differences between mice & untreated controls. Importantly, DC10 potentiated the anti-tumor efficacy of radiation in mice with melanoma xenografts. We conclude that DC10 acts as a radiosensitizer, is well tolerated, & mediates its effect by inhibiting cystine uptake, leading to GSH depletion and increased oxidative stress. Thus, our findings demonstrate the feasibility of using synthetic xCT-inhibitors to overcome radioresistance. The expression of xCT in multiple tumor types further implies it represents a target generic to cancer rather than confined to tumor subtypes. Hence, this therapeutic concept could be applicable to a wide range of radioresistant malignancies.
Citation Format: Shahin Sarowar, Davide Cirillo, Pablo Jativa, Mette H. Nilsen, Sarah-Muheha A. Otragane, Jan I. Heggdal, Frode Selheim, Valentín Ceña, Hans-René Bjørsvik, Per Øyvind Enger. Styrylbenzoic acid derivative DC10 potentiates radiotherapy by inhibiting glutathione synthesis, leading to increased oxidative stress and cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 214.