Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response. (25th April 2022) - Pitera, Aleksandra P
Abstract: Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl‐prolyl‐tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild‐type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF‐induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2‐to‐eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic. SYNOPSIS: Treatment with Halofuginone (HF), an inhibitor of the glutamyl‐prolyl‐tRNA synthetase, activates the integrated stress response (ISR) leading to both gene expression reprogramming and translational attenuation. However, the decrease of translation induced by HF is atypical as it occurs independently of GCN2 and eIF2α phosphorylation. ISR‐related gene expression changes induced by HF depend on GCN2 and eIF2α phosphorylation. HF‐induced translation attenuation is independent of GCN2 and eIF2α phosphorylation. HF induces translation elongation defect. Proline‐dependent cancer cell lines are more sensitive to HF‐induced cytotoxicity. Abstract : Pharmacological glutamyl‐prolyl‐tRNA synthetase inhibition invokes both atypical ISR‐independent translation attenuation and ISR‐mediated changes in gene expression.