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.

The 85-kDa cytosolic PLA 2 (cPLA 2 ) mediates agonist-induced arachidonic acid release in many cell models, including mouse peritoneal macrophages. cPLA 2 is regulated by an increase in intracellular calcium, which binds to an aminoterminal C2 domain and induces its translocation to the nuclear envelope and endoplasmic reticulum. Phosphorylation of cPLA 2 on S505 by mitogenactivated protein kinases (MAPK) also contributes to activation. In macrophages, zymosan induces a transient increase in intracellular calcium and activation of MAPK, which together fully activate cPLA 2 and synergistically promote arachidonic acid release. There are alternative pathways for regulating cPLA 2 in macrophages because PMA and okadaic acid induce arachidonic acid release without increasing calcium. The baculovirus expression system is a useful model to study cPLA 2 activation. Sf9 cells expressing cPLA 2 release arachidonic acid to either A23187 or okadaic acid. cPLA 2 is phosphorylated on multiple sites in Sf9 cells, and phosphorylation of S727 is preferentially induced by okadaic acid. However, the phosphorylation sites are non-essential and only S505 phosphorylation partially contributes to cPLA 2 activation in this model. Although okadaic acid does not increase intracellular calcium in Sf9 cells, calcium binding by the C2 domain is necessary for arachidonic acid release. A23187 and okadaic acid activate cPLA 2 by different mechanisms, yet both induce translocation to the nuclear envelope in Sf9 cells. The results demonstrate that alternative regulatory pathways can lead to cPLA 2 activation and arachidonic acid release. J. Leukoc. Biol. 65: 330-336; 1999.

Prostate cancer (PCa) is the most commonly diagnosed malignancy among western men and accounts for the second leading cause of cancer-related deaths. PCa tends to grow slowly and recent studies suggest that it relies on lipid fuel more than on aerobic glycolysis. However, the biochemical mechanisms governing the relationships between lipid synthesis, lipid utilization, and cancer growth remain unknown. To address the role of lipid metabolism in PCa we have used Etomoxir and Orlistat, clinically safe drugs that block lipid oxidation and lipid synthesis/lipolysis, respectively. Etomoxir is an irreversible inhibitor of the carnitine palmitoyltransferase (CPT1) enzyme that decreases beta oxidation in the mitochondria. Combinatorial treatments using Etomoxir and Orlistat resulted in synergistic decreased viability in LNCaP, VCaP and patient-derived benign and PCa cells. These effects were associated with decreased androgen receptor (AR) expression, decreased mammalian target of Rapamycin (mTOR) signaling and increased caspase-3 activation. Knockdown of CPT1A enzyme in LNCaP cells resulted in decreased palmitate oxidation but increased sensitivity to Etomoxir, with inactivation of AKT kinase and activation of caspase-3. Systemic treatment with Etomoxir in nude nice resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease PCa tumor growth.

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The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membranebound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.The human neutrophil is a critically important cell involved in host defense reactions. Part of the inflammatory response mounted by the neutrophil involves generation of leukotriene B 4 (LTB 4 ), 5 a metabolite of arachidonic acid (20:4) generated through the 5-lipoxygenase pathway. Synthesis of leukotrienes in the neutrophil is a highly complex process under considerable regulation. The initiating event involves elevation of free calcium ion concentration in the cytosol, translocation of cytosolic phospholipase A 2 ␣ (cPLA 2 ␣) and 5-lipoxygenase to perinuclear membranes, liberation of 20:4 mediated by cPLA 2 ␣, association of 5-lipoxygenase with its activating protein, and initiation of 20:4 oxygenation (1). We have reported that inhibition of 20:4 reacylation with thimerosal leads to a greater than 50-fold increase in LTB 4 production in human neutrophils, revealing the importance of 20:4 reacylation in limiting the availability of free 20:4 as a part of the regulation of eicosanoid biosynthesis within this cell type (2). The nature and identity of the lysophospholipid acyltransferase (or acyltransferases) inhibited by thimerosal has not been elucidated, yet it has been known for a number of years that the reacylation of free 20:4 into phospholipids by the Lands pathway is quite active in the human neutrophil (3, 4).Recent investigations have identified several new lysophospholipid acyltransferases (5-10), including some members of the membrane-bound O-acyltransferase (MBOAT) family such as human mboa-7 (henceforth referred to as MBOAT7 in this work), human MBOAT5, and mouse MBOATs 1, 2, and 5. Human MBOAT5 and MBOAT7 have been shown to use 20:4-CoA as a substrate. However, there have not been any...

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.

The 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) mediates agonist-induced arachidonic acid release and eicosanoid production. Calcium and phosphorylation on Ser-505 by mitogen-activated protein kinases (MAPKs) regulate cPLA 2 . Arachidonic acid release and eicosanoid production induced by stimuli that do (A23187, zymosan) or do not (phorbol myristate acetate (PMA), okadaic acid) mobilize calcium were quantitatively suppressed in cPLA 2 -deficient mouse peritoneal macrophages. The contribution of MAPKs to cPLA 2 -mediated arachidonic acid release was investigated. Both extracellular signal-regulated kinases (ERKs) and p38 contributed to cPLA 2 phosphorylation on Ser-505. However, although ERK inhibition did not affect A23187-induced arachidonic acid release, it suppressed zymosan-, PMA-, and okadaic acid-induced arachidonic acid release under conditions where phosphorylation of cPLA 2 on Ser-505 was unaffected. This indicates an additional regulatory mechanism for the ERK pathway. A role for transcriptional regulation is suggested by data showing that cycloheximide and actinomycin D inhibited arachidonic acid release induced by zymosan, PMA and, okadaic acid but not by A23187. Our results show that MAPK pathways contribute to arachidonic acid release in macrophages through alternative mechanisms in addition to their ability to phosphorylate cPLA 2 on Ser-505 and suggest a role for new protein synthesis.

Arachidonic acid release is induced in macrophages with diverse agonists including calcium ionophores, phorbol myristate acetate (PMA), okadaic acid, and the phagocytic particle, zymosan, and correlates with activation of cytosolic phospholipase A 2 (cPLA 2 ). The role of calcium and phosphorylation of cPLA 2 in regulating arachidonic acid release was investigated. Zymosan induced a rapid and transient increase in [Ca 2؉ ] i . This in itself is not sufficient to induce arachidonic acid release since ATP and platelet activating factor (PAF), agonists that induce transient calcium mobilization in macrophages, induced little arachidonic acid release. Unlike zymosan, which is a strong activator of mitogen-activated protein kinase (MAPK), ATP and PAF were weak MAPK activators and induced only a partial and transient increase in cPLA 2 phosphorylation (gel shift). However, ATP or PAF together with colony stimulating factor-1 (CSF-1) synergistically stimulated arachidonic acid release. CSF-1 is a strong MAPK activator that induces a rapid and complete cPLA 2 gel shift but not calcium mobilization or arachidonic acid release. Arachidonic acid release was more rapid in response to CSF-1 plus ATP or PAF than zymosan and correlated with the time course of the cPLA 2 gel shift. Although low concentrations of ionomycin induced a lower magnitude of calcium mobilization than ATP, the response was more sustained resulting in arachidonic acid release. A23187 and ionomycin induced weak MAPK activation, and a partial and transient cPLA 2 gel shift. The MAPK kinase inhibitor, PD 98059 suppressed A23187-induced MAPK activation and cPLA 2 gel shift but had little effect on arachidonic acid release. These results indicate that in macrophages a transient increase in [Ca 2؉ ] i and sustained phosphorylation of cPLA 2 can act together to promote arachidonic acid release but neither alone is sufficient. A sustained increase in calcium is sufficient for inducing arachidonic acid release. However, PMA and okadaic acid induce arachidonic acid release without increasing [Ca 2؉ ] i , although resting levels of calcium are required, suggesting alternative mechanisms of regulation.