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Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers 1 . The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions 2 – 5 . However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR–Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome–ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis. The cellular organelles peroxisomes contribute to the sensitivity of cells to ferroptosis by synthesizing polyunsaturated ether phospholipids, and changes in the abundances of these lipids are associated with altered sensitivity to ferroptosis during cell-state transitions.

Genomic instability is one of the hallmarks of cancer, which underlies cancer development and progression. This DNA repair vulnerability presents cancer researchers with a therapeutic opportunity. Targeted therapies designed to overwhelm DNA repair pathways in cancer have proven largely effective as anti-cancer treatments. However, there are currently three major issues with their widespread use in cancer. Firstly, for cancers such as glioblastomas (GBMs), there have been no targeted therapies approved for decades. Secondly, for most targeted therapies there is an incomplete understanding of their precise mechanism of action, which limits their clinical utility. Thirdly, cancers develop resistance to most targeted therapies, preventing durable drug responses in patients. For my thesis, I aimed to overcome these drawbacks associated with targeted therapies. In Chapter 2 of my thesis, I present my research outlining the development and characterization of a novel DNA repair inhibitor for use in GBMs. This inhibitor can be explored in combination with radiation therapy, the current standard of care treatment for GBMs, to allow greater tumor cell killing while sparing the surrounding normal tissue. In Chapter 3 of my thesis, I present my research towards understanding the mechanism of action of poly (ADP-ribose) glycohydrolase inhibitors (PARGi), which are currently in clinical development for the treatment of DNA repair-deficient cancers. These results will inform clinical trials with PARGi moving forward. In Chapter 4 of my thesis, I outline our current understanding of resistance mechanisms to poly (ADP-ribose) polymerase inhibitors (PARPi), which have been approved for use in breast, ovarian, prostate, and pancreatic cancer. I present mechanistic insight into a specific mechanism of PARPi resistance, and propose recommendations for better stratifying patients in PARPi clinical trials. In all, these three chapters are focused on improving targeted therapies for use in DNA repair-deficient cancers with the aim of positively affecting cancer patient response and survival.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including , as hits. We found that loss of leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the microenvironment. Blocking the type-I interferon receptor in mice grafted with tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 tumors. Together, these results indicate a critical role for and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

PARG inhibitors are currently under clinical development for the treatment of DNA repair-deficient cancers, however, their precise mechanism of action is still unclear. Here we report that PARG inhibition causes increased nuclear PARylated PARP1 that limits PARP1 chromatin binding in response to DNA damage. This PARylated PARP1 accumulates as aggregates at sites distinct from the site of DNA damage, leading to the mis-localization of PARP1. Additionally, these aggregates are formed through PAR chains as abrogating PARP1 catalytic activity prevents their formation. Finally, these PARP1 nuclear aggregates persist long-term and are associated with cleaved cytoplasmic PARP1, a cell death hallmark, which ultimately leads to a non-apoptotic form of cell death. Overall, our data uncovers a novel mechanism of PARG inhibitor cytotoxicity, which will inform ongoing clinical studies.