The impact of sex and cell death on checkpoint inhibitor hepatotoxicity

Project Summary Immune checkpoints have evolved to control self-reactivity, providing a break on the immune system. Cancer cells evade the immune system by overexpressing these checkpoints. Immune Checkpoint Inhibitors (ICI) block these evasion tactics and enhance anti-tumor responses, promoting cytotoxic T cell (CTL) survival, restoring tumor surveillance, and clearing malignant cells. These include monoclonal antibodies against two major immune checkpoints (although antibodies against other targets are in development): 1) The cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) and 2) Programmed death-1 (PD-1) and PD-ligand 1 (PD-L1). Although immunotherapy is extremely effective, leading to significant increase in life expectancy in many tumors such as malignant melanoma, ICIs have serious immune related adverse effects. One such side effect is hepatotoxicity, referred to as Immune-mediated Liver Injury from Checkpoint Inhibitors (ILICI). Severe ILICI (ALT/AST >5-20x upper limit of normal), occurs in up to 30% of patients on dual ICIs. When this occurs, immunotherapy must be discontinued, and patients require immunosuppression with high dose steroids, which can be detrimental for their cancer. Furthermore, patients with severe immune-related side effects such as ILICI are excluded from most future immunotherapy clinical trials. Female sex and the use of combination therapy to block both CTLA4 and PD-1/PD-L1 pathways are independent risk factor for ILICI. Given the wide use of immunotherapy for cancer and the high prevalence of hepatotoxicity, there is a critical need to define mechanisms of injury and the role of sex in ILICI to aid in developing innovative, targeted treatments as alternatives to systemic steroids. We have previously developed a novel mouse model of ILICI using a combination of two ICIs (PD-L1 inhibitor and CTLA4 inhibitor) in CTLA4+/- mice and shown apoptosis in hepatocytes coincides with the activation of the NLRP3 inflammasome in macrophages and pyroptosis in the liver. Using image mass cytometry (IMC), we found interactions between NLRP3hi macrophages, CD8+ CTLs, and apoptotic hepatocytes. We next confirmed these findings in human liver biopsies from patients enrolled in the Drug-Induced Liver Injury Network (DILIN). Additionally, when we stratified mice by sex, we noted an increase in liver inflammation and injury in female vs male mice with ILICI. In this proposal, we aim to further our research by investigating the impact of sex and estrogen signaling on immune activation and liver injury, exploring the interconnection of NLRP3 inflammasome and pyroptosis with hepatocyte apoptosis in ILICI, and evaluating the susceptibility of male and female hepatocytes treated with ICIs to TNF-mediated apoptosis in our novel ILICI cancer mouse model. Our ultimate goal is to find new and innovative liver-specific and targeted therapies to uncouple the unintended hepatotoxicity of ICIs from the desired anti-tumor response, allowing for continuation of life-saving cancer immunotherapy.