(D) DO34 lowers 2-AG content of HT1080 cells

(D) DO34 lowers 2-AG content of HT1080 cells. ferroptosis regulated by ABHD12. Ferroptosis is usually (S)-Mapracorat a form of cell death defined by runaway peroxidation of membrane phospholipids.1 This lipid peroxidation is dependent on iron, and ferroptotic cell death can be inhibited by iron chelators.1 Ferroptosis is implicated in several types of pathological cell death, often related to cell and/or organ damage and associated hJumpy degenerative disorders.2 Ferroptosis has more recently garnered interest as a way to promote malignancy cell death independent of the apoptotic pathways that are often defective in transformed cells.3 For instance, drug-resistant, or persister, malignancy cells4 and those that have undergone an epithelial-to-mesenchymal transition5 exhibit high susceptibility to ferroptosis. Ferroptosis can be induced by at least two known pharmacological mechanisms: blockade of the cellular import of cystine by inhibitors of the cystine/glutamate transporter SCL7A11 (or system xc?), such as erastin, or by direct inhibition of the phospholipid hydroperoxide glutathione peroxidase 4 (GPX4).6, 7 The lipid peroxidation step of ferroptosis occurs on phospholipids bearing polyunsaturated fatty acids (PUFAs), such as linoleic (C18:2), arachidonic (C20:4), and adrenic (C22:4) acids,8 and it is either catalyzed by lipoxygenases9 or iron-mediated autoxidation.10 Accordingly, prevention of PUFA incorporation into phospholipids by disruption of enzymes like ACSL411 or LPCAT312 attenuates ferroptosis. Whether inhibitors of SLC7A11 or GPX4 can induce ferroptosis in malignancy cells with sufficient selectivity to avoid general cell or organ toxicity remains unclear, and it would therefore be of interest to identify other proteins that modulate the ferroptotic potential of malignancy cells. Two recent studies used genetic screens of ferroptosis-resistance malignancy cells to identify the coenzyme Q oxidoreductase FSP1 (or AIFM2) as a suppressor of ferroptosis in cells that lack dependency on GPX4.13, 14 Here, we considered an alternative, chemical screen to discover potentiators of ferroptosis. Realizing that this hydrolysis of the potency of the compound for inhibiting ABHD12 (IC50 1 M)26 (Physique 1F and Physique S1B) and was blocked by co-treatment with the ferroptosis inhibitor ferrostatin-1 (Physique S2). DO264 did not potentiate other forms of cell death in HT1080 cells, such as apoptosis induced by staurosporine or necrosis induced by H2O2 (Physique S3), and was not independently cytotoxic in HT1080 or SU-DHL-5 cells at concentrations below 10 M (Physique S2). These data, (S)-Mapracorat taken together, show that ABHD12 inhibiton by DO264 specifically potentiates ferroptotic cell death. To provide further evidence that this ferroptosis-potentiating effects of DO264 occurred through inhibition of ABHD12, we generated ABHD12-knockout (ABHD12-KO) HT1080 cells using CRISPR-Cas9 genome editing. We performed these studies on a population level rather than by selecting clones to avoid potential clonality effects around the basal ferroptotic sensitivity of cells. We confirmed substantial loss of ABHD12 in gene-edited HT1080 cells using a tailored activity-based probe JJH35026 (Physique 2A). The ABHD12-KO cells displayed heightened sensitivity to GPX4-dependent ferroptosis compared to cells treated with control sgRNAs (Physique 2B). The impact of genetic disruption of ABHD12 was lower in magnitude than the effect of DO264 (Physique 2B), which could indicate a residual amount of ABHD12 expression in the ABHD12-KO cell populace or that part of the ferroptotic-enhancing effects of DO264 occur through (S)-Mapracorat an additional target. Regardless, these data demonstrate that this pharmacological or genetic impairment of (S)-Mapracorat ABHD12 enhances the ferroptotic sensitivity of malignancy cells. Open in a separate window Physique 2. Genetic disruption of ABHD12 enhances ferroptosis. (A) Loss of ABHD12 in (S)-Mapracorat CRISPR-Cas9-edited HT1080 cells (ABHD12-KO cells), as measured using the ABHD12-directed activity-based probe JJH350 (2 M, 45 min, 37 C), following explained protocols26. Populations of HT1080 cells were.