Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition

Abstract

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses1. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer ‘persister’ cells2,3,4. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse5. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival6. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.

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Figure 1: Small-molecule screen in drug-tolerant persister cells.
Figure 2: Persister cells are vulnerable to GPX4 inhibition.
Figure 3: GPX4 inhibition causes ferroptosis in persister cells.
Figure 4: Persister cells have a disabled antioxidant program and depend on GPX4 in vivo.

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Acknowledgements

We acknowledge technical support from the UCSF ES Cell Targeting Core and UCSF Preclinical Therapeutics Core. This work was supported by grants from the National Cancer Institute (NCI) of the National Institutes of Health (NIH) (Cancer Target Discovery and Development Network grant U01CA168370 to M.T.M. and F.M., U01CA217882 to M.T.M., U01CA176152 to S.L.S., U01CA168397 to M.E.B., and R01CA212767 to M.T.M.), Susan G. Komen for the Cure Postdoctoral Fellowship KG1101214 to M.J.H., and the Howard Hughes Medical Institute (S.L.S.).

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M.T.M. and F.M. directed the project; M.J.H., V.S.V., S.L.S., F.M. and M.T.M. wrote the manuscript; M.J.H. performed chemical screens, RNA-seq analysis, and all cell culture experiments except V.S.V. and M.J.R. performed Kuramochi cell GPX4 inhibitor assays, D.B. assisted with ROS, glutathione and NADPH assays, and J.G. performed western blots; M.J.H. directed CRISPR editing of A375 cells and in vivo experiments that were performed at UCSF core facilities; V.S.V. and J.K.E. contributed reagents; A.M., H.D.D. and M.E.B. provided advice and project support.

Corresponding authors

Correspondence to Frank McCormick or Michael T. McManus.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks N. Chandel and P. Vandenabeele and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Additional data demonstrating persister cell ferroptosis sensitivity.

a, A375 melanoma persister cells are reversibly drug-resistant. Scale bars, 400 μm. b, Global antioxidant gene expression is downregulated in BT474 persister cells. P value calculated using a two-tailed Wilcoxon signed-rank test. c, d, MCF10A parental cells (c) and BT474 persister cells derived from carboplatin and paclitaxel (d) were treated with RSL3 or ML210 for 3 days. e, f, BT474 persister cells (e) or parental cells (f) were treated with erastin for 5 days. g, Western blot demonstrating GPX4 knockout in two distinct A375 clones (clone 1, KO1; clone 2, KO2). For gel source data, see Supplementary Fig. 1. h, BT474 parental cells co-treated with 2 μM lapatinib and RSL3 or ML210 for 3 days. Data are means and s.d. from 3 (c) or 2 (dh) biologically independent samples. c, NS, not significant (P > 0.05); two-tailed t-test. All data are representative of two separate experiments. Source data

Extended Data Figure 2 Additional data demonstrating that GPX4 inhibition causes ferroptosis in persister cells.

a, b, A375 persister cells were treated with RSL3 (a) or ML210 (b) and ferroptosis rescue compounds for 3 days. c, PC9 persister cells were treated with RSL3 or ML210 and ferroptosis rescue compounds for 3 days. d, Relative concentration of total labile iron in BT474 parental and persister cells. Data are means and s.d. from 2 biologically independent samples. All data are representative of two separate experiments. Source data

Extended Data Figure 3 Additional data demonstrating that persister cells have a disabled antioxidant program and depend on GPX4 in vivo.

a, Reduced glutathione (GSH) and reduced plus oxidized glutathione (GSH + GSSG) levels in BT474 cells. bd, Fractional viability of BT474 persister cells treated with RSL3 and antioxidant compounds (b), endogenous ROS-generating compound DMNQ (c), or the SOD1 inhibitor LCS-1 (d) for 3 days. e, ROS levels (DCF staining) in BT474 cells treated with ML210 for 1 h. f, BT474 persister cells were co-treated with ALDH inhibitor disulfiram and ferrostatin-1 for 3 days. g, Tumour volume measurements for the full time course of the experiment presented in Fig. 4d. Ferrostatin-1 was withdrawn on day 10. See Source Data for individual data points. h, Untreated A375 GPX4 wild-type or GPX4 knockout clone 1 tumour formation without ferrostatin-1 dosing. See Source Data for individual data points. i, ROS levels (DCF staining) in cells regrown without lapatinib for 15 days from BT474 persister cells and then treated with 1 μM RSL3 for 1 h. j, Persister cell GPX4 dependence model. Data are means and s.d. from three (a, e, i) or two (bd, f) biologically independent samples or four (g) or five (h) animals. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (P > 0.05); two-tailed t-tests. All data are representative of two separate experiments. Source data

Extended Data Table 1 Expression of epithelial–mesenchymal transition and stem markers
Extended Data Table 2 Expression of NRF2 target genes

Supplementary information

Supplementary Information

This file contains the raw western blot files for Extended Data Figure 1g. (PDF 179 kb)

Life Sciences Reporting Summary (PDF 83 kb)

Supplementary Table 1

This Supplementary Table contains RNAseq expression data (FPKM) and differential expression analysis for human RefSeq genes in BT474 parental and persister cells. (XLSX 1205 kb)

Supplementary Table 2

This Supplementary Table contains Ingenuity Pathway Analysis (IPA) “Functions” results derived from analysis of BT474 parental and persister cell RNAseq data. (XLSX 135 kb)

Supplementary Table 3

This Supplementary Table contains Ingenuity Pathway Analysis (IPA) "Canonical Pathways" results derived from analysis of BT474 parental and persister cell RNAseq data. (XLSX 58 kb)

Supplementary Table 4

This Supplementary Table contains Ingenuity Pathway Analysis (IPA) "Upstream Regulators" results derived from analysis of BT474 parental and persister cell RNAseq data. (XLSX 157 kb)

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Hangauer, M., Viswanathan, V., Ryan, M. et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 551, 247–250 (2017). https://doi.org/10.1038/nature24297

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