Identification and characterization of a potent activator of p53-independent cellular senescence via a small-molecule screen for modifiers of the integrated stress response.

The Integrated Stress Response (ISR) is a signaling program that enables cellular adaptation to stressful conditions like hypoxia and nutrient deprivation in the tumor microenvironment. An important effector of the ISR is activating transcription factor 4 (ATF4), a transcription factor that regulates genes involved in redox homeostasis and amino acid metabolism and transport. Because both inhibition and overactivation of the ISR can induce tumor cell death, modulators of ATF4 expression could prove to be clinically useful. In this study, chemical libraries were screened for modulators of ATF4 expression. We identified one compound, E235 (N-(1-benzyl-piperidin-4-yl)-2-(4-fluoro-phenyl)-benzo[d]imidazo[2,1-b]thiazole-7-carboxamide), that activated the ISR and dose-dependently increased levels of ATF4 in transformed cells. A dose-dependent decrease in viability was observed in several mouse and human tumor cell lines, and knockdown of ATF4 significantly increased the antiproliferative effects of E235. Interestingly, low μM doses of E235 induced senescence in many cell types, including HT1080 human fibrosarcoma and B16F10 mouse melanoma cells. E235-mediated induction of senescence was not dependent on p21 or p53; however, p21 conferred protection against the growth inhibitory effects of E235. Treatment with E235 resulted in an increase in cells arrested at the G2/M phase with a concurrent decrease in S-phase cells. E235 also activated DNA damage response signaling, resulting in increased levels of Ser15-phosphorylated p53, γ-H2AX, and phosphorylated checkpoint kinase 2 (Chk2), although E235 does not appear to cause physical DNA damage. Induction of γ-H2AX was abrogated in ATF4 knockdown cells. Together, these results suggest that modulation of the ISR pathway with the small molecule E235 could be a promising antitumor strategy.