Identification Of An Integrated Stress Response-Inducing Agent (Nh125) That Modulates Dr5-Dependent Trail Synergy And Atf3-Driven Cell Death In Gliomaspheres

Purpose: The high incidence of glioblastoma recurrence necessitates additional therapeutic strategies to improve outcomes. One potential strategy targets treatment resistant cells via the integrated stress response (ISR) pathway as a means of sensitizing these cells to TNF-related apoptosis-inducing ligand (TRAIL). Methods: We generated a novel cell-based death receptor assay and used this assay to screen compounds from three commercially available libraries (total of 223 compounds) at 2 or 10 μM (Z prime = 0.64). A single hit was identified that was further characterized by immunoblotting and immunofluorescence. A compound dilution series was applied to a panel of cell lines including patient-derived glioma stem cells (GSC). IC 50 values were calculated by fitting normalized viability data to a modified Hill Equation. TRAIL synergy was assessed using the combination index method following treatment with compound and TRAIL. Knockout of C/EBP Homologous Protein (CHOP) was used to determine the role of integrated stress in TRAIL synergy. GSC were also treated with temozolomide followed by assessment of cell viability. RNA-seq analysis was performed on compound treated GSC, U251, and Normal Human Astrocytes. Short hairpin RNAs were used to establish the role of ATF3 in compound-treated GSC. Results: Using a novel cell-based death receptor we identified 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) as a potent inducer of DR5 expression. NH125 signals through the EIF2α-ATF4-CHOP axis, culminating in DR5 expression at low micromolar doses. CHOP is required for NH125-mediated TRAIL synergy. Compared to other cancer cell lines, GSC demonstrate an increased sensitivity to NH125 treatment, and synergize with TRAIL at lower doses than U251. NH125 produced a significant decrease in GSC viability when compared to temozolomide treatment (p u003c 0.05). This enhanced sensitivity of GSC is partially due to increased expression of ATF3 following NH125 treatment. Conclusions: NH125 generates an integrated stress response leading to CHOP-mediated TRAIL synergy. Patient-derived glioma stem cells demonstrate increased sensitivity to NH125 treatment that is partially driven by increased ATF3 expression. Engaging the ISR to enhance the efficacy of TRAIL represents a promising strategy to target the treatment refractory GSC that have been implicated in glioblastoma recurrence. Citation Format: Saad Sheikh, Xiaobing Tian, Deeksha Saxena, Xiangsheng Xu, Gary Kao, Jay F. Dorsey. Identification of an integrated stress response-inducing agent (NH125) that modulates DR5-dependent TRAIL synergy and ATF3-driven cell death in gliomaspheres [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A063.