Cancer drug addiction is relayed by an ERK2-dependent phenotype switch

Abstract

Observations from cultured cells1,2,3, animal models4 and patients5,6,7 raise the possibility that the dependency of tumours on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we must first define the mechanism that underlies it. We performed an unbiased CRISPR–Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/threonine-protein kinase BRAF, in order to functionally mine their genome for ‘addiction genes’. Here we describe a signalling pathway comprising ERK2 kinase and JUNB and FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. This occurred in both cultured cells and mice and was irrespective of the acquired drug resistance mechanism. In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of the epithelial–mesenchymal transition. In melanoma cells, this reprogramming caused the shutdown of microphthalmia-associated transcription factor (MITF), a lineage survival oncoprotein; restoring this protein reversed phenotype switching and prevented the lethality associated with drug addiction. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with the phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target, B-cell lymphoma 2 (BCL-2), and by inducing DNA damage in cancer cells. Our results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

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Figure 1: Genome-wide CRISPR–Cas9 knockout screen to break cancer drug addiction identifies several signalling pathway components.
Figure 2: Conserved drug addiction pathway in multiple cell lines with distinct therapy resistance mechanisms.
Figure 3: ERK2 and JUNB act in an MITF-dependent genetic phenotype switch that controls drug addiction.
Figure 4: Conservation of drug addiction mechanism in human lung cancer cells, and drug withdrawal synergizing with dacarbazine in tumour cell elimination.

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Acknowledgements

We are grateful to K. Suda and T. Mitsudomi for HCC827 and HCC827CLR cells, J. Villanueva for 451Lu cells, I. de Krijger for technical advice on FISH, A. Broeks and the NKI Biobank for help with patient samples, R. Beijersbergen for providing the GeCKO library and A. Huitema for providing DTIC. We thank all members of the Peeper and Blank labs for advice. This work was financially supported by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013) /ERC synergy grant agreement number 319661 COMBATCANCER (X.K., D.S.P.), and a Fellowship (T.K.), a Queen Wilhelmina Award (D.S.P.) and a research grant (nr. 10304; X.K., D.S.P.), all by the Dutch Cancer Society/Koningin Wilhelmina Fonds.

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X.K., T.K. and D.S.P. designed all experiments. All in vitro experiments were carried out by X.K., with the exception of a reverse transcription PCR analysis done by K.K. Xenograft experiments were done by A.S. T.K. performed all bioinformatic analyses. J.-Y.S. analysed mouse tumours. J.B., E.A.R., M.H.G.F. and H.W.M.N. analysed human melanoma samples in collaboration with C.U.B. X.K. and D.S.P. wrote the manuscript. All authors revised and approved the manuscript. The project was supervised by D.S.P.

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Correspondence to Daniel S. Peeper.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks R. Marais, D. Solit 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 Drug addiction phenotype in acquired-drug-resistant melanoma cells.

Acquired BRAFi-resistant 451LuBR cells were cultured in the presence or absence of 1 μM BRAFi; acquired BRAFi + MEKi-resistant A375BMR, A101DBMR and Mel888BMR cells were cultured in the presence or absence of 0.5 μM BRAFi + 0.05 μM MEKi. Photographs were taken after seven days. The images are representative of three independent biological experiments.

Extended Data Figure 2 Generation of a diverse melanoma cell line panel with distinct drug resistance mechanisms.

a, 451Lu and 451LuBR cells were treated with increasing concentrations of dabrafenib (0, 0.01, 0.1, 1 and 10 μM) for six hours. Total cell lysates were subjected to immunoblotting with indicated antibodies. ERK1/2 served as a loading control. b, A375 and A375BMR cells were treated with increasing concentrations of the BRAFi dabrafenib + MEKi trametinib (0 + 0 μM, 0.01 + 0.001 μM, 0.1 + 0.01 μM and 1 + 0.1 μM) for six hours. Total cell lysates were subjected to immunoblotting with indicated antibodies. ERK1/2 served as a loading control. c, Quantification of BRAF amplification by quantitative PCR on genomic DNA of A375 and A375BMR cells. CRAF was included as a negative control. Cycle threshold values were normalized to LINE. P value calculated by unpaired two-sided Student’s t-test. Data in graphs are mean ± s.d. from three technical replicates; confidence interval 95%. d, Mel888 and Mel888BMR cells were treated with vehicle or 0.5 μM BRAFi + 0.05 μM MEKi for six hours. Total cell lysates were subjected to immunoblotting with indicated antibodies. ERK1/2 served as a loading control. For gel source images, see Supplementary Fig. 1. Data in ad are representative of three independent biological experiments.

Extended Data Figure 3 Disruption of MEK1 prevents mortality in drug-addicted 451LuBR cells after drug withdrawal.

a, 451LuBR, A375BMR, A101DBMR and Mel888BMR cells were infected with lentivirus that expressed sgRNAs targeting genes MAP2K1 (encoding MEK1), MAP2K2 (encoding MEK2) or a scrambled sequence (sgCtrl). Cells were seeded in the presence or absence of MAPK pathway inhibitors, and fixed, stained and photographed after 14 days (BRAFi or BRAFi + MEKi group) or 21 days (no drug group). b, Cell lysates from a were subjected to immunoblotting using the indicated antibodies. HSP90 served as a loading control. For gel source images, see Supplementary Fig. 1. Data in a and b are representative of three independent biological experiments.

Extended Data Figure 4 ERK rebound contributes to drug withdrawal lethality irrespective of drug resistance mechanism.

a, Four parental drug-sensitive cell lines were cultured without MAPK pathway inhibitors, and their drug-resistant counterparts were cultured in the presence or absence of MAPK pathway inhibitors, for the indicated number of days. Total cell lysates were subjected to immunoblotting with indicated antibodies. HSP90 served as a loading control. b, 451LuBR cells were cultured in the presence of 1 μM BRAFi, a high concentration (5 μM) of ERKi, a low concentration (0.05 μM) of ERKi, or vehicle control (no drug). A375BMR, A101DBMR and Mel888BMR cells were cultured with 0.5 μM BRAFi + 0.05 μM MEKi, 5 μM ERKi, 0.05 μM ERKi or vehicle control (no drug). Cells were fixed, stained and photographed after 14 days. For gel source images, see Supplementary Fig. 1. Data in a and b are representative of two independent biological experiments.

Extended Data Figure 5 Drug addiction is relayed by an MITF-dependent phenotype switch.

a, Drug-addicted A375BMR cells were cultured in the absence of 0.5 μM BRAFi + 0.05 μM MEKi for 0, 6, 24 and 48 h, and 451LuBR cells were cultured in the absence of 1 μM BRAFi for 0, 1, 3 and 5 days. Total RNA was isolated and subjected to sequencing analysis. Unsupervised clustering of the 200 most variably expressed genes is represented for the first (either 0 h or 0 days) and last time points (either 48 h or 5 days) in a heat map. b, Sequence data in a (all time points) were subjected to GSEA to determine the correlation of the indicated gene sets with the drug addiction effect in A375BMR and 451LuBR cells. For P value calculation, see Methods. c, 451LuBR and A101DBMR cells were infected with lentivirus that expressed sgRNAs targeting gene encoding cJUN (sgJUN) or a scrambled sequence (sgCtrl). Cells were seeded in the presence or absence of MAPK pathway inhibitors, and fixed, stained and photographed after 14 days (BRAFi or BRAFi + MEKi group) or 21 days (no drug group). d, Total cell lysates of the samples in c were subjected to immunoblotting analysis with indicated antibodies. HSP90 served as a loading control. e, 451LuBR and A101DBMR cells were cultured in the presence or absence of the MAPK pathway inhibitors for 24 h and then subjected to a transwell migration assay. The cells that migrated through the membrane were fixed, stained and photographed after 8 (A101DBMR) or 24 (451LuBR) hours. f, Quantification of the migration capacity (e) from three representative images from each sample; P value calculated by unpaired two-sided Student’s t-test. Data in graphs are mean ± s.d. For gel source images, see Supplementary Fig. 1. Data in ce are representative of two independent biological experiments.

Extended Data Figure 6 Phenotype switch is not observed in non drug-addicted cells upon drug withdrawal.

a, D10BMR cells were cultured with 1 μM BRAFi + 0.1 μM MEKi, 0.5 μM BRAFi + 0.05 μM MEKi or vehicle control (no drug); SK-Mel-28BR cells were cultured with either 3 μM BRAFi dabrafenib, 1 μM dabrafenib or vehicle control (no drug); Mel888PLXR and A375PLXR cells were cultured with 3 μM BRAFi PLX4720, 1 μM PLX4720 or vehicle control (no drug); Cells were fixed, stained and photographed after ten days. b, On the third day, total cell lysates of the samples in a were subjected to immunoblotting analysis with indicated antibodies. HSP90 served as a loading control. For gel source images, see Supplementary Fig. 1. Data in a and b are representative of two independent biological experiments.

Extended Data Figure 7 ERK2 and JUNB act in an MITF-dependent genetic phenotype switching program controlling drug addiction.

a, Mel888BMR cells were infected with lentivirus expressing sgRNAs targeting genes encoding JUNB (sgJUNB), ERK2 (sgERK2) or a scrambled sequence (sgCtrl). Cells were cultured in the presence or absence of 0.5 μM BRAFi + 0.05 μM MEKi for five days. Total RNA was isolated and subjected to sequencing analysis. Unsupervised clustering of the 200 most variably expressed genes is represented in a heat map. b, Genes involved in phenotype switching were selected from the sequence data in a and are represented in a heat map. c, Sequence data in a were subjected to GSEA to determine the correlation of the indicated gene sets with the extent of rescue of the drug addiction phenotype by sgJUNB and sgERK2 in Mel888BMR cells. dw, drug withdrawal. For P value calculation, see Methods.

Extended Data Figure 8 Pharmacological ERK inhibition blocks phenotype switch in drug-addicted cells upon drug withdrawal.

a, Mel888BMR, A101DBMR and A375BMR cells were cultured with 0.5 μM BRAFi + 0.05 μM MEKi, vehicle or 0.05 μM ERKi, 451LuBR cells were cultured with 1 μM BRAFi, vehicle or 0.05 μM ERKi for three days. Total cell lysates were subjected to immunoblotting with indicated antibodies. HSP90 served as a loading control. b, Photographs taken of cells in a, on the third day. c, Mel888 MEK1T55delinsRT and A375 MEK1T55delinsRT cells were cultured in the presence of 0.3 μM BRAFi, low concentration (0.05 μM) of ERKi or vehicle control (no drug). Cells were fixed, stained and photographed after ten days. d, On the fifth day, total cell lysates from cells in c were subjected to immunoblotting with indicated antibodies. HSP90 served as a loading control. e, HCC827CLR cells were cultured in the presence of 1 μM EGFR TKI CL-387785, a high concentration (5 μM) of ERKi, a low concentration (0.5 μM) of ERKi, or with vehicle control (no drug). Cells were fixed, stained and photographed after 14 days. For gel source images, see Supplementary Fig. 1. Data in ae are representative of three independent biological experiments.

Extended Data Figure 9 ERK2 and ERK1 have distinct functions in drug addiction.

a, 451LuBR control cells (sgCtrl), ERK2 knockout cells (sgERK2), ERK1 (v5-tagged)-overexpressing sgERK2 cells (sgERK2, ERK1 OE) and ERK1 knockout cells (sgERK1) were cultured in the presence or absence of 1 μM BRAFi for up to six days. Total cell lysates were subjected to immunoblotting analysis with indicated antibodies. HSP90 served as a loading control. b, Cells described in a were cultured in the presence or absence of BRAFi, and fixed, stained and photographed after 14 days (BRAFi group) or 21 days (no drug group). For gel source images, see Supplementary Fig. 1. Data in a and b are representative of two independent biological experiments.

Extended Data Figure 10 Dynamic phenotype of drug resistance and drug addiction in melanoma.

a, Schematic flow chart indicating drug withdrawal resistant cells were generated from drug-addicted cells with long-term drug withdrawal. b, Parental (A375, 451Lu and A101D) cells, acquired-drug-resistant (A375BMR, 451LuBR and A101DBMR) cells and spontaneously developed drug withdrawal-resistant (A375WR, 451LuWR and A101DWR) cells were cultured with MAPK pathway inhibitors or vehicle. Cells were fixed, stained and photographed after 14 days. c, Total cell lysates from cells in b were subjected to immunoblotting with pERK and ERK antibodies after six hours treatment. For gel source images, see Supplementary Fig. 1. Data in b and c are representative of three independent biological experiments.

Supplementary information

Reporting Summary (PDF 67 kb)

Supplementary Figures

This file contains the uncropped blots from Figures 1-4, Extended Data Figures 2-6 and 8-10. (PDF 28527 kb)

Supplementary Tables

This file contains 3 tables. Table 1 contains full sequencing data of screen clones, table 2 contains primer and oligo sequences for PCR and sgRNA plasmid construction. Table 3 contains xenograft tumor volume measurements. (PDF 295 kb)

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Kong, X., Kuilman, T., Shahrabi, A. et al. Cancer drug addiction is relayed by an ERK2-dependent phenotype switch. Nature 550, 270–274 (2017). https://doi.org/10.1038/nature24037

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