New small molecule inhibitors of UPR activation demonstrate that PERK, but not IRE1 alpha signaling is essential for promoting adaptation and survival to hypoxia

The unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1? and PERK.We utilized shRNAs and small-molecule inhibitors of IRE1? (4?8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1? activity. PERK activity was monitored by immunoblotting eIF2? phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors.Using knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1? dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4?8c and the PERK inhibitor are selective and potent inhibitors of IRE1? and PERK activation, respectively. However, despite potent inhibition of IRE1? activation, 4?8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents.Our results demonstrate that IRE1? but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts.