Ferroptosis regulation by the NGLY1/NFE2L1 pathway

Abstract

SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

Keywords: NRF1; cancer; ferroptosis.

Fig. 1.

NFE2L2 and NFE2L1 negatively regulate ferroptosis. (A and B) Correlation between KEAP1 mutation status and sensitivity to ferroptosis inducers erastin or ML162. Data are derived from the DepMap portal. Each data point represents a single cell line. Higher AUC values indicate lower sensitivity to compound treatment. WT, wild type. Mut, mutant. (C) Protein levels in A549 Control^N and NFE2L2^KO1/2,N cell lines treated ± the proteasome inhibitor bortezomib (Btz). (D) Cell death in A549 Control^N and NFE2L2^KO1/2,N cell lines. Era2, erastin2. (E) Protein levels in A549 Control1^N and NFE2L2^KO1,N cells stably transduced with empty vector (EV) lentivirus or lentivirus directing the expression of NFE2L2. (F) Cell death in A549 Control1^N and NFE2L2^KO1,N cells described in E. Cpt, camptothecin. (G) Protein levels in A549 Control1/2^N and NFE2L1^KO1/2,N cell lines treated as indicated. (H) Cell death in A549 Control1/2^N and NFE2L1^KO1/2,N cell lines. (I) Protein levels in A549 Control1^N and NFE2L1^KO1,N cells transduced with EV lentivirus or lentivirus directing the expression of NFE2L1. (J) Cell death in A549 Control1^N and NFE2L1^KO1,N cells described in I. (K) Difference (delta) in normalized area under the curve (nAUC) lethal fraction values for A549 NFE2L1^KO1/2,N versus Control1^N cells after treatment with 261 different bioactive small molecules (5 µM). Higher ΔnAUC values indicate greater death sensitivity in NFE2L1^KO1/2,N versus Control1^N cell lines. (L) Effect of ferroptosis inducers on NFE2L1 protein levels. A549 cells were treated with vehicle (DMSO), erastin2 (4 µM), or ML162 (4 µM) ± Btz (200 nM) for 4 h. (C, E, G, and I) Treatment was ±Btz for 4 h. (D, L, H, and J) Fer-1 was used at 1 µM. Data represent mean ± SD of three (D and H) or four (F and J) independent experiments. Data in K represent the average ΔnAUC of three biological replicates. ***P ≤ 0.001.

Fig. 2.

NGLY1-mediated protein sequence editing of NFE2L1 regulates ferroptosis. (A) Schematic of NFE2L1 processing and activation, adapted from ref. . MAF, bZIP transcription factors that heterodimerize with NFE2L1. ARE, antioxidant response element. (B) Protein levels in A549 Control^N and NGLY1^KO1/2,N cell lines. (C) Cell death in A549 Control^N and NGLY1^KO1/2,N cell lines. (D) Protein levels in A549 Control1^N and NFE2L1^KO1,N cells stably transduced with EV lentivirus control or lentivirus directing the expression of NGLY1. (E) Cell death in cell lines described in D. (F) Protein levels in A549 Control1^N cells stably transduced with EV lentivirus control or lentivirus directing the expression of wild-type NGLY1 or NGLY1^L408X. (G) Cell death in cell lines described in F. (H) Protein levels in A549 Control1^N or NFE2L1^KO1,N cells stably transduced with EV lentivirus control, or lentivirus directing the expression of wild-type NFE2L1 or the sequence-edited NFE2L1^8ND mutant. (I) Cell death in cell lines described in H. (B, D, F, and H) Treatment was ±Btz for 4 h. (C, G, E, and I) Fer-1 was used at 1 µM. Data represent mean ± SD from three (C) or four (E and I) independent experiments.

Fig. 3.

NFE2L1 regulates ferroptosis via GPX4. (A) A549 cell protein expression in the indicated conditions. Note: The NFE2L2 panel (A, Right) is the same as shown in Fig. 1C, as other results were obtained from the same lysates. (B) Reduced GSH levels determined by liquid chromatography coupled to mass spectrometry (LC-MS) in A549 cell lines treated with vehicle (DMSO) or erastin2 (4 µM) for 4 h. (C) Cell death in A549 cell lines examined ± 24-h BSO pretreatment and the indicated conditions. (D) Protein expression in Control1^N and NGLY1^KO1/2,N cells. (E) Protein expression in Control1^N (Cntrl1^N) and NFE2L1^KO1,N cells stably transduced with EV lentivirus control or lentivirus directing the expression of cGPX4. (F) Cell death in cell lines described in E. (G) Protein levels in A549 Control1^N and NFE2L1^KO1,N cell lines stably transduced with EV lentivirus control or lentivirus directing the expression of NFE2L1 or NFE2L1^8ND. (H) Protein levels in HT-1080 Control^N and GPX4^KO,N cell lines stably transduced with EV lentivirus control or lentivirus directing the expression of NFE2L1 or NFE2L1^8ND. (I) Protein levels in HT-1080 Control^N and GPX4^KO,N cell lines stably transduced with control EV lentivirus or lentivirus directing the expression of NFE2L2. (J) Cell death in stably transduced HT-1080 Control^N cells (from I) treated with erastin2 (125 nM), ML162 (250 nM), or bortezomib (100 nM). (K) Effect of NFE2L1 or NFE2L2 overexpression, as in H and I, respectively, on cell death in HT-1080 Control^N and GPX4^KO,N cells upon Fer-1 withdrawal. (A, D, E, and H) Treatment was ±Btz for 4 h. (F, J, and K) Fer-1 was used at 1 µM. B, J, and K depict individual data points from independent experiments. Data represent mean ± SD of two (C) or three (F) independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns, not significant.

Fig. 4.

Functional compensation between NFE2L1 and NFE2L2. (A, Left) Protein levels in A549 Control1/2^N and NFE2L1^KO1/2,N cells. (A, Right) Protein levels in A549 Control1/2^N and NFE2L2^KO1/2,N cells. (B) Protein levels in A549 Control1^N and NFE2L1^KO1,N cells stably transduced with EV lentivirus control or lentivirus directing the expression of NFE2L2. (C) Cell death in A549 Control1^N and NFE2L1^KO1,N cell lines described in B. (D) Protein levels in A549 Control1^N and NGLY1^LOF2,N cells stably transduced with EV lentivirus control or lentivirus directing the expression of NFE2L2. (E) Cell death in A549 Control1^N and NGLY1^LOF1,N cell lines described in D. (F) Protein levels in A549 Control1^N and NFE2L2^KO1,N cells stably transduced with EV lentivirus control or lentivirus directing the expression of wild-type NFE2L1 or mutant NFE2L1^8ND. (G) Cell death in A549 Control1^N and NFE2L2^KO1,N cell lines described in F. (A, B, and F) Treatment was ±Btz for 4 h. (C, E, and G) Fer-1 was used at 1 µM. Data represent mean ± SD of three (C and G) or two (E) independent experiments.