Abstract 4500: Inhibition of PERK by small molecule inhibitors enhances the response to ionizing radiation in vitro and animal tumor models

Endoplasmic reticulum (ER) is a well organized membranous network, responsible for synthesis and folding of secretory and membrane proteins, lipid and sterol biosynthesis and intracellular calcium storage. Perturbations in ER lumen caused by depletion of Ca²⁺ levels, hypoxia, nutrient deprivation, affect the ER homeostasis, a condition known as an ER stress. In order to cope with this stress, cells have developed an orchestrated biochemical response, termed Unfolded Protein Response (UPR). Hypoxia, an ER stress inducer, is an important and unique characteristic of the solid tumor microenvironment, promotes resistance to radiotherapy and contributes to poor patient prognosis. Cancer cells overcome these stress conditions through activation the PERK-eIF2α arm of the UPR, leading to inhibition of global protein synthesis and cell survival. We hypothesize that the use of small molecule PERK inhibitors will decrease the survival of hypoxic cells and eventually sensitize them to irradiation. In this study we used highly radioresistant melanoma and head and neck squamous cell carcinoma cell lines B16F10-ova and SQ20B, under nomroxia and hypoxia, respectively. We used two specific PERK inhibitors (PERKi) and tested for their efficacy both in vitro and in vivo. Induction of ER stress in both cell lines, either by treatment with the pharmacologic agents thapsigargin (TG) and tunicamycin (TN), or by exposure to hypoxic conditions (0.2%), was evaluated by activation of PERK and its downstream targets. Pre-treatment of stressed B16F10-ova and SQ20B cells for 2h with PERKi (1μM), caused complete inhibition of PERK-eIF2a-ATF4-CHOP arm of the UPR. Moreover, both cell lines pre-treated with PERKi presented a significant decrease in survival fraction under ER-stress induced by TG (0.5μM) as well as under hypoxia (0.2%) along with increasing doses of irradiation (IR). For the in vivo experiments, nude and C57BL/6 mice were treated with either 100mg/kg of PERKi by oral gavage, twice a day for 3weeks, or IR or a combination of PERKi and IR. Mice treated with PERKi showed a small but not significant delay of tumor growth in both backgrounds of mice. However, combined treatment with IR (12 and 15Gy) caused a significant reduction of tumor volume as well as a delayed tumor growth which was significantly more pronounced in the immunocompetent mice. Data from flow cytometry analysis of one of the the PERKi+IR treated tumors from C57BL/6 mice showed that there is a significant infiltration of CD3+ cells as well as significant higher number of CD3+CD8+ cells compared to the untreated mice or mice treated with either PERKi or IR alone. Collectively, our results show that PERKi effectively reduces the survival of hypoxic B16F10-ova and SQ20B cells and sensitize them to IR in vitro and in tumors. Further experiments will clarify whether PERKi have a dual role on reducing the hypoxic fraction and direct recruitment of the cytotoxic T-cells.

Citation Format: Ioannis Verginadis, Joel Encarnación, Souvik Dey, Constantinos Koumenis. Inhibition of PERK by small molecule inhibitors enhances the response to ionizing radiation in vitro and animal tumor models [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 4500. doi:10.1158/1538-7445.AM2017-4500