Ferroptosis as a modulator of checkpoint inhibitor immunotherapy
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Experimental Pathology
First Advisor
Bosenberg, Marcus
Abstract
Melanoma is the deadliest form of skin cancer, and its incidence continues to rise. While advances in immunotherapy, in the form of anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors, have led to extended cancer free outcomes for many patients with advanced disease, most patients do not have durable responses. Therefore, improvements in treatment of melanoma patients will depend on the identification of methods to enhance existing immunotherapy regimens. In this thesis, we investigated the role of ferroptosis, a regulated cell death modality driven by lipid peroxides, in antitumor immune responses against melanoma. To begin, we analyzed YR1.7 melanoma tumors experiencing successful and unsuccessful immune responses by single cell RNA sequencing (scRNA-seq) and discovered a prominent ferroptosis signature in successfully clearing tumors. Using multiple ferroptosis inhibitors, we confirmed that ferroptosis is a necessary component of immunotherapy-induced tumor clearance. In contrast to ferroptosis inhibitors, ferroptosis inducers enhanced responses to immunotherapy. They cause cancer cells to secrete proinflammatory cytokines like interferons, which increase tumor antigen presentation, and IL-6, which sensitizes cells to ferroptosis in vitro and correlates with improved survival in human melanoma patients. scRNA-seq analysis of immunotherapy-resistant human melanoma tumors revealed gene expression changes associated with ferroptosis resistance, and we validated this finding in immunotherapy-resistant murine melanomas. Given the therapeutic potential of ferroptosis inducers, we next sought to identify existing classes of FDA-approved medications that might target this pathway. First, we interrogated the ability of the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen to induce cell death in vitro and promote antitumor immunity in vivo. We discovered that ibuprofen increases levels of arachidonic acid and induces ferroptosis in both settings, and arachidonic acid on its own replicates these effects. Increased metabolism of arachidonic acid by lipoxygenases (LOXs) enzymatically generates lipid peroxides that propagate ferroptosis. At the same time, arachidonic acid decreases GPX4 and SLC7A11, impairing an essential pathway for ferroptosis suppression. We also interrogated the ability of statins to induce cell death and ferroptosis in YR1.7 melanoma and MC38 colon cancer cell lines. We demonstrated that lipophilic statins induce cell death with multiple features of ferroptosis, and transcriptomics and metabolomics also revealed system level changes to ferroptosis-related pathways. Mechanistically, mevalonate is necessary for isoprenylation of the sec-tRNA synthetase, which is required for synthesis of the ferroptosis suppressor GPX4. Like the other ferroptosis inducers that we tested, statins promoted antitumor immune responses and synergized with PD-1 blockade in vivo. Extending these findings to human melanoma patients, we showed that statins induced cell death in patient-derived melanoma cell lines and decreased expression of cholesterol synthesis genes is associated with improved overall survival. Despite the widespread attention that ferroptosis inducers have received as novel candidates for the treatment of melanoma and other cancers, how the metabolic changes associated with specific oncogenic mutations affect ferroptosis sensitivity remains largely unexplored. To address this, we tested the ferroptosis sensitivity of 12 murine and human melanoma cell lines and discovered that BRAFV600-mutant melanomas are relatively resistant to ferroptosis. Compared to their wild type (WT) counterparts, BRAF-mutant melanomas upregulate several lipid metabolism pathways. Increased lipid droplet biosynthesis enables BRAF-mutant cells to sequester intracellular lipids in their neutral form, protecting cells from ferroptotic cell death. Manipulating lipid droplet abundance with diacylglycerol O-acyltransferase (DGAT) inhibitors, oleic acid, or atglistatin modifies melanoma sensitivity to ferroptosis. Collectively, these results offer crucial insight into the role of ferroptosis in antitumor immunity and highlight the promise of ferroptosis-targeting agents as immunotherapy adjuvants. Cyclooxygenase activity, cholesterol metabolism, and lipid droplet biosynthesis as important regulators of ferroptosis sensitivity and potential therapeutic targets in melanoma.
Recommended Citation
Talty, Ronan, "Ferroptosis as a modulator of checkpoint inhibitor immunotherapy" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1074.
https://elischolar.library.yale.edu/gsas_dissertations/1074
