Summary Cancer immunotherapy restores and/or enhances effector function of CD8+ T cells in the tumor microenvironment1,2. CD8+ T cells activated by cancer immunotherapy execute tumor clearance mainly by inducing cell death through perforin-granzyme- and Fas/Fas ligand-pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent lipid peroxide accumulation5,6. Although it was mechanistically illuminated in vitro7,8, emerging evidence has shown that ferroptosis may be implicated in a variety of pathological scenarios9,10. However, the involvement of ferroptosis in T cell immunity and cancer immunotherapy is unknown. Here, we find that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumor cells, and in turn, increased ferroptosis contributes to the anti-tumor efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system xc-, restrains tumor cell cystine uptake, and as a consequence, promotes tumor cell lipid peroxidation and ferroptosis. In preclinical models, depletion of cyst(e)ine by cyst(e)inase in combination with checkpoint blockade synergistically enhances T cell-mediated anti-tumor immunity and induces tumor cell ferroptosis. Expression of system xc- is negatively associated with CD8+ T cell signature, IFNγ expression, and cancer patient outcome. Transcriptome analyses before and during nivolumab therapy reveal that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumor ferroptosis is a novel anti-tumor mechanism. Targeting tumor ferroptosis pathway constitutes a therapeutic approach in combination with checkpoint blockade.

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC– subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

A challenge in oncology is to rationally and effectively integrate immunotherapy with traditional modalities including radiotherapy. Here, we demonstrate that radiotherapy induces tumor cell ferroptosis. Ferroptosis agonists augment and ferroptosis antagonists limit radiotherapy efficacy in tumor models. Immunotherapy sensitizes tumors to radiotherapy by promoting tumor cell ferroptosis. Mechanistically, IFNγ derived from immunotherapy-activated CD8+ T cells and radiotherapy-activated ATM independently, yet synergistically repress SLC7A11, a unit of the glutamate-cystine antiporter xc−, resulting in reduced cystine uptake, enhanced tumor lipid oxidation and ferroptosis, and improved tumor control. Thus, ferroptosis is an unappreciated mechanism and focus for the development of effective combinatorial cancer therapy.

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than non-malignant cells due to genetic alterations and abnormal growth and as a result, maintenance of the anti-oxidant glutathione (GSH) is essential for their survival and proliferation1–3. Under elevated ROS conditions endogenous l-Cysteine (l-Cys) production is insufficient for GSH synthesis, necessitating l-Cys uptake, predominantly in its disulfide form l-Cystine (CSSC) via the xCT(−) transporter. Here we show that administration of an engineered, pharmacologically optimized, human Cyst(e)inase enzyme mediates sustained depletion of the extracellular l-Cys and CSSC pool in mice and non-human primates, selectively causes cell cycle arrest and death (PI and Annexin-V staining) in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS, yet results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice that develop disease resembling human chronic lymphocytic leukemia. The observation that enzyme-mediated depletion of the serum l-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods suggests that Cyst(e)inase represents a safe and effective therapeutic modality for inactivating anti-oxidant cellular responses in a wide range of malignancies4,5.

Lactonases from Bacillus species hydrolyze the N-acylhomoserine lactone (AHL) signaling molecules used in quorum-sensing pathways of many Gram-negative bacteria, including Pseudomonas aeruginosa and Erwinia carotovora, both significant pathogens. Because of sequence similarity, these AHL lactonases have been assigned to the metallo-beta-lactamase superfamily of proteins, which includes metalloenzymes of diverse activity, mechanism, and metal content. However, a recent study claims that AHL lactonase from Bacillus sp. 240B1 is not a metalloprotein [Wang, L. H., et al. (2004) J. Biol. Chem. 279, 13645]. Here, the gene for an AHL lactonase from Bacillus thuringiensis is cloned, and the protein is expressed, purified, and found to bind 2 equiv of zinc. The metal-bound form of AHL lactonase catalyzes the hydrolysis of N-hexanoyl-(S)-homoserine lactone but not the (R) enantiomer. Removal of both zinc ions results in loss of activity, and reconstitution with zinc restores activity, indicating the importance of metal ions for catalytic activity. Metal content, sequence alignments, and X-ray absorption spectroscopy of the zinc-containing lactonase all support a proposed dinuclear zinc binding site similar to that found in glyoxalase II.