Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism

Abstract

Ferroptosis is a specialized iron-dependent cell death that is associated with lethal lipid peroxidation. Modulation of ferroptosis may have therapeutic potential since it has been implicated in various human diseases as well as potential antitumor activities. However, much remains unknown about the underlying mechanisms and genetic determinants of ferroptosis. Given the critical role of kinases in most biological processes and the availability of various kinase inhibitors, we sought to systemically identify kinases essential for ferroptosis. We performed a forward genetic-based kinome screen against ferroptosis in MDA-MB-231 cells triggered by cystine deprivation. This screen identified 34 essential kinases involved in TNFα and NF-kB signaling. Unexpectedly, the DNA damage response serine/threonine kinase ATM (mutated in Ataxia-Telangiectasia) was found to be essential for ferroptosis. The pharmacological or genetic inhibition of ATM consistently rescued multiple cancer cells from ferroptosis triggered by cystine deprivation or erastin. Instead of the canonical DNA damage pathways, ATM inhibition rescued ferroptosis by increasing the expression of iron regulators involved in iron storage (ferritin heavy and light chain, FTH1 and FTL) and export (ferroportin, FPN1). The coordinated changes of these iron regulators during ATM inhibition resulted in a lowering of labile iron and prevented the iron-dependent ferroptosis. Furthermore, we found that ATM inhibition enhanced the nuclear translocation of metal-regulatory transcription factor 1 (MTF1), responsible for regulating expression of Ferritin/FPN1 and ferroptosis protection. Genetic depletion of MTF-1 abolished the regulation of iron-regulatory elements by ATM and resensitized the cells to ferroptosis. Together, we have identified an unexpected ATM-MTF1-Ferritin/FPN1 regulatory axis as novel determinants of ferroptosis through regulating labile iron levels.

Fig. 1

Kinome screening identified 34 kinase essential for cystine-deprived death. a Scatter plot displayed the effects of siRNA-mediated silencing of individual kinase on the viability of MDA-MB-231 under cystine-deprived (Y-axis) and full media (X-axis) upon the siRNA-mediated silencing of individual kinase in MADA-MB-231 breast cancer cells. Y-axis and X-axis are the relative cell viabilities after knockdown each kinase normalized against nontargeting control siRNA in cystine deprived and normal medium, respectively. Each purple dot represents individual kinase-targeting pooled siRNA; each red dot represents significant “hits” from the screen based on the log-transformed normalized viability and calculated by ANOVA (threshold: p < 0.0001). b List of 34 “hit” essential kinases is shown with their normalized viability (against nontarget siRNA) in the control full media or cystine-deprived media (ANOVA p < 0.0001). The kinases were ranked by the normalized viability under cystine deprivation. c STRING analysis of all the hit kinases based on the protein–protein interaction networks, highlighting the essential role of ATM and ATR as highly connecting nodes among the kinases essential for the cystine-deprived death

Fig. 2

Validation of ATM as a critical mediator of ferroptosis. a Depletion of ATM and ATR confers resistance to cystine-deprived death. MDA-MB-231 cells were transfected with siNC, siATM, or siATR for 48 h, then incubated with normal full (200 μM cystine) or cystine-deprived (5 μM cystine) medium for 68 h. Cell viability was determined by CellTiter-Glo. Lower panel, the knockdown efficiency of ATM and ATR were determined by Western blots. b Depletion of ATM, ATR, or both ATM/ATR rescued MDA-MB-231 cells from erastin-induced death. MDA-MB-231 cells were transfected with siNC, siATM, siATR, or combined siATM/ATR for 72 h are then incubated with DMSO or erastin (10 μM) for 16 h (upper panel) or indicated time points up to 71 h (lower panel). Cell viability or death was determined by CellTiter-Glo (upper panel) or CellTox Green (lower panel), respectively. c Depletion of ATM by two individual siRNAs (siATM-27 and siATM-28) recued MDA-MB-231 cells from erastin-induced death. d, e ATM inhibitor Ku-55933 (d) and Ku-60019 (e) rescued cells from erastin-induced death. MDA-MB-231 cells were treated with Ku-55933 for 72 h, and then treated with DMSO or erastin for 24 h before viability determination by CellTiter-Glo. (The data are shown from one representative experiment with three biological replicates, and the data were reproduced from at least two independent experiments; Student’s t-test; *p < 0.05; **p < 0.01) f ATM inhibitor Ku-55933 reduced the S1981 phosphorylation of nuclear ATM. The amount of ATM protein or ATM S1981 phosphorylation in the nuclear or cytosol were analyzed by Western blots in the presence of erastin or Ku-55933. g Overexpression of wild-type (WT) but kinase dead (KD) ATM sensitized cells to ferroptosis. Vector, flag-WT-ATM, or flag-KD-ATM was transiently expressed into 293T cells. 24 h after transfection, cells were seeded into 96 well and pretreated with Ku-55933 (10 μM) for additional 24 h. The cell death was monitored by CellTox-Green 18 h after erastin (10 μM) treatment. The data was normalized to time 0 (immediately after addition of erastin). The data presented are mean ± S.D. from four biological replicates (Student’s t-test; *p < 0.05). In parallel, transfected cells lysates were collected for the protein expression of flag-WT-ATM or flag-KD-ATM by Western blots with indicated antibodies (right panel)

Fig. 3

The induction of iron regulatory genes by ATM inhibition is essential for protection against ferroptosis a Depletion of ATM upregulates the mRNA expression of FTH1, FTL, and FPN1. MDA-MB-231 cells were transfected with siNC or siATM for 48 h and the mRNA levels of indicated genes were determined by qRT-PCR. b ATM inhibitor Ku-55933 upregulated the mRNA expression of FTH1, FTL, and FPN1. MDA-MB-231 cells were treated with Ku-55933 for 48 h, and the mRNA levels of indicated genes were determined by qRT-PCR. c, d Inhibition of ATM by siRNA (c) or Ku-55933 (d) increased the level of FTH1 protein. Cell lysates from siATM (69 h) or Ku-55933 (48 h) were analyzed by Western blots with indicated antibodies. e, f FPN1 and FTH1 are essential for ferroptosis protection by ATM inhibition. Cells were transfected with indicated siRNA for 78 h, and incubated with DMSO or erastin (10 μM) for 18 h before determining the viability by CellTiter-Glo. (The data are shown from one representative experiment with three biological replicates, and the data were reproduced from at least two independent experiments; Student’s t-test; *p < 0.05; **p < 0.01) g FTH1 is essential for ferroptosis protection by ATM inhibition for longer time course. MDA-MB-231 cells were transfected with indicated siRNA for 72 h, and incubated with DMSO or erastin for 70 h. The cell death was monitored from time 0 to 70 h by CellTox-Green. All the data were normalized to DMSO control in each group (The data are shown from three biological replicates.)

Fig. 4

ATM inhibition reduces the intracellular labile iron pool. a Erastin treatment increases intracellular labile iron pool (LIP). MDA-MB-231 cells were treated with indicated concentrations of erastin for 6 h, and incubated with Calcien-AM. The relative LIP was calculated based on the differences in the fluorescence intensity before and after DFO chelation. (The data was acquired from six biological replicates performed in parallel; Student’s t-test; *p < 0.05, **p < 0.01) b ATM inhibition by Ku-55933 decreases intracellular labile iron pool. MDA-MB-231 cells were treated with Ku-55933 (5 μM) for 24 h, and incubated with Calcien-AM to measure the relative LIP. (The data was acquired from eight biological replicates performed in parallel; Student’s t-test; **p < 0.01) c Depletion of ATM by siRNA decreases intracellular labile iron pool. MDA-MB-231 cells were transfected with indicated siRNA for 72 h, and incubated with Calcien-AM to measure the relative LIP. (The data was acquired from four biological replicates performed in parallel; Student’s t-test; *p < 0.05) d Iron supplementation sensitizes ATM-depleted cells against erastin. MDA-MB-231 cells were transfected with siNC or siATM for 72 h, and then incubated with DMSO, erastin (10 μM), with or without ferric citrate (50 μM) for indicated time point. The amount of cell death was evaluated by CellTox-Green at each time point. The data presented are mean ± S.D. from four biological replicates, and the data were reproduced from two independent experiments. P-values were calculated by Student’s t-test, **p < 0.01

Fig. 5

MTF1 is responsible for induction of iron regulators and ferroptosis resistance of ATM inhibition. a, b MTF1 but not NRF2, p65, or IRP1/2 is responsible for the induction of FTH1, FTL, and FPN1 mRNA associated with ATM silencing. MDA-MB-231 cells were transfected with indicated siRNAs for 48 h, and the mRNA expression of indicated genes were analyzed by qRT-PCR. The data was acquired from three biological replicates. P-values were calculated by Student’s t-test, *p < 0.05, **p < 0.01, ns stands for non-significant. The level of each mRNA was normalized to β-actin mRNA. The P-value was calculated by comparing the expression of each group to the siNC control. c Depletion of MTF1 sensitized ATM-knockdown cells to ferroptosis. MDA-MB-231 cells transfected with indicated siRNAs for 72 h, treated with DMSO or erastin for 30 h and the viability was determined by CellTiter-Glo. The data was acquired from six biological replicates. P-values were calculated by Student’s t-test, *p < 0.05, **p < 0.01. d The subcellular localization of MTF1 upon TPEN, ZnCl₂ or Ku-55933 treatment. MDA-MB-231 cells treated with DMSO, TPEN (5 μM), ZnCl₂ (100 μM)_, or Ku-55933 (5 μM) were fixed and stained with DAPI (nuclear), phalloidin (F-actin), and MTF1 antibody. Representative images are shown to visualize the subcellular locations of MTF1 upon different treatments. e, f Ku-55933 increased nuclear translocation. The cytosol and nuclear fractions of MDA-MB-231 were collected after treatment of Ku-55933 (5 μM) for 24 h. The successful cytosolic and nuclear fractions were assessed GADPH and Lamin A/C, respectively. The relative amount of MTF1 in each fraction was quantified by Western blots (e) and ImageJ (f). The data presented are mean ± S.D. from three biological replicates. P-values were calculated by Student’s t-test, *p < 0.05, **p < 0.01

Fig. 6

Working model of ATM regulates ferroptosis. Cystine deprivation or erastin treatment reduces intracellular cystine, cysteine, and GSH (reduced glutathione) level while increases ROS (reactive oxygen species) and oxidized glutathione (GSSG). These changes make the cells susceptible to iron-mediated cell death, ferroptosis. ATM-inactivation increases the nuclear translocation of MTF1 protein, which induces the expression of ferritin (FTH1 and FTL) and ferroportin (FPN1) to sustain low-labile iron condition and confer resistance to ferroptosis