Drug-induced hepatotoxicity is a leading cause of both the withdrawal of approved drugs from the market and the attrition of new chemical entities during the drug development process; however, the mechanisms underlying drug-induced hepatotoxicity are not fully understood. We have used efavirenz, an antiretroviral drug that is hepatotoxic in certain patients, as a model compound to investigate cellular signaling mechanisms that may play a causal role in drug-induced hepatocyte death. Previously, using primary human hepatocytes, we demonstrated that efavirenz and the major oxidative metabolite of efavirenz, denoted as 8-hydroxyefavirenz (8- OHefavirenz), stimulate cell death in a manner that is dependent upon activation of the stress kinase c-Jun N- terminal kinase and upregulation of the proapoptotic protein BimEL (Bcl-2 interacting mediator of cell death extra long). Subsequently, we have reported that efavirenz can also activate inositol requiring enzyme 1α (IRE1α), a key regulator of cell stress that lies upstream of JNK and BimEL. The goal of this proposal is to determine the mechanism by which efavirenz and 8-OHefavirenz activate BimEL and IRE1α, while also gaining a mechanistic understanding of how genetic variation in IRE1α might impact efavirenz and 8-OHefavirenz-induced cell death. Importantly, we will leverage the insights we have gained through using efavirenz as a model compound and employ prototypic hepatotoxic drugs beyond efavirenz, namely carbamazepine, diclofenac and isoniazid, in order to establish BimEL and IRE1α as central regulators of drug-induced hepatotoxicity across a range of drug classes. The aims are as follows: (1) to test the hypothesis that BimEL acts as an executioner of cell death in response to efavirenz and other prototypic hepatotoxic drugs: BimEL null mice will be used to determine whether the absence of BimEL prevents hepatotoxicity stimulated by the hepatotoxic drugs being investigated here; CRISPR/Cas9 systems will be used to determine the role of effector proteins, Bax and Bak, that are downstream of BimEL in modulating hepatocyte death; CRISPR/Cas9 and reporter gene assays will be used to define the mechanism by which efavirenz, 8-OHefavirenz and other hepatotoxic drugs regulate the transcription of BimEL; efavirenz analogs will be employed in order to elucidate the structure-activity relationship of BimEL activation by efavirenz; (2) to test the hypothesis that IRE1α is a central upstream regulator of drug-induced hepatotoxicity that is stimulated by several classes of drugs: we will determine whether efavirenz, 8-OHefavirenz, and other hepatotoxic drugs stimulate formation of the IRE1α/TRAF2/ASK1/JNK complex that results in IRE1α-dependent activation of JNK; we will test the impact of naturally occurring genetic variants of IRE1α on activity and cell death. It is expected that these studies will define BimEL and IRE1α activation as important molecular mechanisms by which a range of drugs induce-hepatotox...