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Pharmacological activation of REV-ERBs is lethal in cancer and oncogene-induced senescence

Nature volume 553, pages 351–355 (2018)Cite this article

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Abstract

The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response1,2. Perturbations of these processes are hallmarks of cancer3 and chronic circadian rhythm disruption predisposes individuals to tumour development1,4. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. REV-ERBs, the nuclear hormone receptors REV-ERBα (also known as NR1D1) and REV-ERBβ (also known as NR1D2), are essential components of the circadian clock5,6. Here we show that two agonists of REV-ERBs—SR9009 and SR9011—are specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and have no effect on the viability of normal cells or tissues. The anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as HRAS, BRAF, PIK3CA and others) and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 has a critical role in evoking an apoptotic response in malignant cells. Notably, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective antitumour strategy, identifying a class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are inhibitors of autophagy and de novo lipogenesis, with selective activity towards malignant and benign neoplasms.

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Figure 1: SR9009 is selectively lethal in cancer cell lines driven by different oncogenic signalling.
Figure 2: SR9009 agonist of REV-ERBs inhibits autophagy.
Figure 3: SR9009 and SR9011 treatment evokes an apoptotic response and induces inhibition of autophagy in OIS cells.
Figure 4: SR9009 impairs viability of NRAS-driven naevi and glioblastoma growth and extends survival.

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Acknowledgements

We thank K. V. Ly, L. Fijany, Y. Soda, M. Soda and M. Schmitt for technical assistance; F. d’Adda di Fagagna, S. Minucci, A. Viale, G. Gargiulo and J. Karlseder for discussion and feedback; the Narita, Gage, Burris, Amati and Shaw laboratories, and F. F. Lang for reagents; and the Salk Institute’s Waitt Advanced Biophotonics Center and Gene Targeting and Transfer, and M. Shokhirev and the Razavi Newman Integrative Genomics and Bioinformatics Core. G.S. is supported by the AIRC/Marie Curie International Fellowships in Cancer Research (12298), Istituto Superiore di Sanità, TRAIN ‘Training through Research Application Italian iNitiative’. M.V.P. is supported by the NIH (NIAMS grants R01-AR067273 and R01-AR069653) and a Pew Charitable Trust grant. X.W. is supported by a CIHR postdoctoral fellowship (MFE-123724). I.M.V is an American Cancer Society Professor of Molecular Biology. A.R. is supported by the NCI T32 grant, Salk Women in Science, Salk Excellerators Award and the Stavros Niarchos Foundation New Frontiers Salk Research Specialist Award. M.J.K. is supported by F30 DK112604. A.S. is supported by the NCI Cancer Center Support Grant P30 (CA014195 MASS core) and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals. This work was supported in part by a Worldwide Cancer Research grant and an American Federation of Aging Research mid-career grant M14322 to S.P. Additional support came from a Cancer Center Core Grant (P30 CA014195-38), the H. N. and Frances C. Berger Foundation, the Glenn Center for Aging Research and the Leona M. and Harry B. Helmsley Charitable Trust (grant #2012-PG-MED002).

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Authors and Affiliations

  1. Regulatory Biology Laboratory, Salk Institute for Biological Studies, La Jolla, 92037, California, USA

    Gabriele Sulli & Satchidananda Panda

  2. Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, 92037, California, USA

    Amy Rommel & Inder M. Verma

  3. Department of Developmental and Cell Biology, University of California, Irvine, Irvine, 92697, California, USA

    Xiaojie Wang & Maksim V. Plikus

  4. Clayton Foundation Laboratories of Peptide Biology, Salk Institute for Biological Studies, La Jolla, 92037, California, USA

    Matthew J. Kolar & Alan Saghatelian

  5. Department of Genomic Medicine, The University of Texas MD, Anderson Cancer Center, Houston, 77030, Texas, USA

    Francesca Puca

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Contributions

X.W. and M.V.P. performed experiments related to naevi. A.R. participated in xenograft experiments. F.P. performed experiments in Extended Data Figs 8g–l, 10h–j. M.J.K. performed lipidomic assays. G.S. designed the study, performed experiments, analysed data and wrote the manuscript. A.S, I.M.V. and M.V.P. supervised experiments and edited the manuscript. S.P. supervised, designed the study and reviewed the data and manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Gabriele Sulli or Satchidananda Panda.

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Reviewer Information Nature thanks S. Kay, L. Zender 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 SR9011, an additional agonist of REV-ERBs, selectively kills cancer cell lines.

a, Viability assay shows that SR9011 is cytotoxic specifically in cancer cells (72 h). One-way ANOVA. n indicates biological replicates: astrocytes, n = 7 (mock), n = 7 (2.5 μM), n = 9 (5 μM), n = 13 (10 μM) and n = 13 (20 μM), ***P = 0.0004; astrocytomas, n = 21 (mock), n = 15 (2.5 μM), n = 7 (5 μM), n = 8, (10 μM), n = 7 (20 μM), ****P < 0.0001; BTICs, n = 10 (mock), n = 8 (2.5 μM), n = 9 (5 μM), n = 13 (10 μM), n = 10 (20 μM), ****P < 0.0001. b–d, Proliferation assay showing that SR9011 treatment does not affect normal BJ cells, but is deleterious for transformed BJ-ELR cells and the cancer cell lines MCF-7 and HCT116 (20 μM, 7 days). Depletion of REV-ERBs by shRNA impairs apoptosis induction by the SR9011 agonist of REV-ERBs; n = 3 biologically independent experiments. e, Human acute T-cell leukaemia cells are affected by the SR9011 agonist of REV-ERBs (72 h, one-tailed Mann–Whitney test, ****P < 0.0001; n = 24 (mock) and 12 (SR9011) biological replicates). f, Immunoblot analysis of cleaved caspase 3 shows that agonists of REV-ERBs trigger apoptosis in the A375 melanoma cell line (representative of n = 2 biologically independent experiments). g–j, Immunostaining for cleaved caspase 3 and TUNEL assay confirm apoptosis induction by SR9011 in the cancer cell lines MCF-7 and A375. h, j, quantification of g and i, respectively. n indicates biologically independent samples; MCF-7, n = 5 (mock) or 11 (SR9011); A375, n = 8 (mock) or 16 (SR9011). One-tailed Mann–Whitney test, MCF-7 cleaved caspase 3, *P = 0.0117; TUNEL assay, *P = 0.0231; A375 cleaved caspase 3, ****P < 0.0001; TUNEL assay, ****P < 0.0001. Scale bars, 50 μm. k, Electron microscopy confirms induction of apoptosis, as indicated by extensive presence of swollen mitochondria (representative of n = 3 biologically independent samples in two experiments). Arrows, normal mitochondria; asterisks, swollen mitochondria. Nu, nucleus. Scale bar, 1 μm. l, Downregulation of NR1D1 and NR1D2 is confirmed by qRT–PCR (A375). n = 3 biologically independent samples. One-tailed Mann–Whitney test, *P = 0.05. All panels representative of three biologically independent experiments unless otherwise specified. All data are mean ± s.e.m. a.u., arbitrary units. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Figure 2 Induction of apoptosis by agonists of REV-ERBs is independent of p53 and oxidative stress.

a–j, Treatment with agonists of REV-ERBs triggers apoptosis independently of p53 status, as shown by proliferation assay (7 days, 20 μM; a, c, f, h) and TUNEL assay (3 days, 20 μM; b, i, j) in cancer cell lines affected by various types of p53 alteration. n indicates biologically independent samples; T47D, n = 8 (mock), n = 6 (SR9009) and n = 10 (SR9011), one-way ANOVA, ****P < 0.0001; PANC-1, n = 4 (mock), n = 6 (SR9009) and n = 7 (SR9011), one-way ANOVA; TUNEL assay, *P = 0.0108; cleaved caspase 3, ****P < 0.0001; SKMEL28, n = 4 (mock), n = 5 (SR9009, SR9011), one-way ANOVA; TUNEL assay, ****P < 0.0001; cleaved caspase 3, **P = 0.0035. d, e, Apoptosis is induced in both wild-type and p53-null HCT116 cells (TUNEL assay, 4 days, 20 μM, mean ± s.e.m.). One-tailed Mann–Whitney test. n indicates biologically independent samples. HCT116 wild type, n = 5 (mock, SR9009), **P = 0.004; HCT116 p53 knockout, n = 8 (mock) or 6 (SR9009), ***P = 0.0003. f, Immunoblot analysis of cleaved caspase 3 shows that agonists of REV-ERBs trigger apoptosis in the RIGH cell line (one experiment). k, Co-treatment with the reducing agent NAC (10 mM) does not rescue the viability of A375 cells (20 μM, 7 days). n indicates biological replicates, n = 5 (mock, −NAC) or 6 (all other dot plots). One-way ANOVA, ****P < 0.0001. l, Results obtained under hypoxic conditions (20 μM, 6 days) were similar to those obtained in co-treatments with NAC. n indicates biological replicates, n = 3 (mock −NAC, mock hypoxia and SR9009 hypoxia) or 6 (SR9009 normoxia, SR9011 normoxia and SR9011 hypoxia). One-way ANOVA, ****P < 0.0001). m, n, Hypoxia or co-treatment with NAC does not alter the ability of agonists of REV-ERBs to induce apoptosis in A375 cells. One-way ANOVA. n indicates biologically independent samples: normoxia, n = 3 (mock), n = 5 (SR9009) or n = 11 (SR9011); TUNEL assay, *P = 0.0432; cleaved caspase 3, ***P = 0.0004; hypoxia, n = 6 (mock), n = 13 (SR9009) or n = 14 (SR9011); TUNEL assay, ***P = 0.0005; cleaved caspase 3, *P = 0.0028; NAC, n = 3 (mock), n = 4 (SR9009) or n = 3 (SR9011); TUNEL assay, *P = 0.0104; cleaved caspase 3, **P = 0.0042. Scale bars, 50 μm. All panels representative of three biologically independent experiments with similar results unless otherwise specified. All data are mean ± s.e.m. Norm, normoxia; Hypo, hypoxia. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Figure 3 Attenuation of oxidative stress does not affect the cytotoxic activity of agonists of REV-ERBs.

a, b, Treatment with agonists of REV-ERBs (20 μM) induces apoptosis on co-treatment with NAC and under hypoxic conditions, as shown by proliferation assays of HCT-116 cells. n indicates biological replicates. n = 3 (mock ± NAC), n = 6 (SR9009/SR9011 ± NAC), n = 9 (SR9009 + NAC), n = 11 (SR9011 + NAC), n = 3 (mock normoxia), n = 6 (SR9009/SR9011 normoxia and hypoxia), n = 5 (mock hypoxia) 6 days, one-way ANOVA, ****P < 0.0001. c, d, Apoptosis induction of HCT116 cells remained unchanged under hypoxia or on co-treatment with NAC; 20 μM, 6 days, one-way ANOVA. n indicates biologically independent samples: normoxia, n = 5 (mock), n = 6 (SR9009), n = 8 (SR9011); TUNEL assay, ***P = 0.0003; cleaved caspase 3, **P = 0.0021; hypoxia, n = 3 (mock), n = 5 (SR9009), n = 4 (SR9011); TUNEL assay, **P = 0.0015; cleaved caspase 3, **P = 0.0046; NAC, n = 4 (mock), n = 5 (SR9009), n = 5 (SR9011); TUNEL assay, ****P < 0.0001; cleaved caspase 3, ****P < 0.0001. e, In MCF-7 cells, apoptosis triggered by agonists of REV-ERBs is independent of oxidative state, as shown by proliferation assay (20 μM). n indicates biological replicates: n = 6 (mock, normoxia and hypoxia), n = 4 (09-011 normoxia), n = 7 (09 hypoxia), n = 5 (011 hypoxia); one-way ANOVA, ****P < 0.0001; f, g TUNEL assay and immunofluorescence analysis of cleaved caspase 3 confirm previous results (Extended Data Fig 3c, d). n indicates biologically independent samples. n = 3 (mock normoxia), n = 5 (mock hypoxia), n = 11 (09 normoxia), n = 8 (09 hypoxia), n = 10 (011 normoxia and hypoxia). One-way ANOVA. Normoxia, TUNEL assay, **P = 0.0049; cleaved caspase 3,**P = 0.0054; hypoxia, TUNEL assay, ****P < 0.0001; cleaved caspase 3, ****P < 0.0001. All panels representative of three biologically independent experiments with similar results. All data are mean ± s.e.m. Norm, normoxia; Hypo, hypoxia.

Source data

Extended Data Figure 4 Agonists of REV-ERBs inhibit de novo lipogenesis.

a, b, Agonists of REV-ERBs downregulate FASN and SCD1 mRNA, as assayed by qRT–PCR. A172 cell line 48 h, 20 μM; FASN and SCD1, n = 3 biologically independent samples, ****P < 0.0001. FASN and SCD1 protein levels (b) are reduced on treatment with SR9009 and SR9011. c–i, Agonists of REV-ERBs reduce free fatty acid concentrations, as quantified by liquid chromatography–mass spectrometry. c, Relative levels of free fatty acids that are the primary products of FASN (palmitic acid, C16:0; stearic acid, C18:0) and SCD1 (palmitoleic acid, C16:1; oleic acid, C18:1) are lower in samples treated with SR9009 than in control samples. d, The unsaturation index (changes in oleate:stearate ratio) is decreased in the SR9009-treated sample, compared to mock-treated samples, owing to the large decrease in monounsaturated oleate observed in the SR9009-treated sample. e, f, SR9009 treatment reduces polyunsaturated fatty acids levels compared to mock-treated samples, in agreement with previous results (Extended Data Fig. 3c, d). g–i, Decreases in free fatty acid levels can affect the concentrations of phospholipids that contain these fatty acids. Treatment with agonists of REV-ERBs leads to reductions in palmitic acid-containing phosphatidylcholine, arachidonic acid- and oleic acid-containing phosphatidylinositols, mono- and di-unsaturated phosphatidylglycerol (g) and phosphatidylethanolamines (h, i); A172 cell line 48 h, 20 μM, *P = 0.05. j, Simplified scheme illustrating the metabolic products of FASN and SCD1. k, Supplementation of oleic acid partially ameliorates the cytotoxity of agonists of REV-ERBs (A172, 20 μM, 4 days). l, Supplementation of palmitic acid does not impair the cytotoxicity of agonists of REV-ERBs (20 μM, A172, 4 days). All data are mean ± s.e.m. P value is calculated with one-way ANOVA in panel a, and with one-tailed Mann–Whitney test in the remaining panels. n = 3 biologically independent samples (d–i). For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Figure 5 REV-ERB agonist SR9011 inhibit autophagy.

a, b, Treatment with SR90011 reduces the number of autophagosomes both in MCF7 and T47D cell lines. n indicates biologically independent samples, MCF7 20 μM 24 h, n = 9 (mock), n = 4 (SR9011), **P = 0.0056; T47D 48 h 20 μM, n = 5 (mock), n = 4 (SR9011), **P = 0.0079. c, d, SR9011 induces accumulation of p62, as shown by immunofluorescence both in MCF7 and T47D cell lines. n indicates biologically independent samples. MCF7 p62 48 h, n = 3 (mock), n = 4 (SR9011), *P = 0.0286; T47D 48 h, n = 5 (mock and SR9011), **P = 0.004. e, Accumulation of p62 is confirmed by immunoblot (48h, 20 μM A375). f, g, Inhibition of autophagy precedes apoptosis induction, as shown by immunofluorescence of p62, cleaved caspase 3 and TUNEL assay. n indicates biologically independent samples: n = 4 (mock, 48 h), n = 5 (SR9011 p62), n = 3 (SR9011, 48 h), n = 6 (mock, SR9011 72 h, p62), n = 10 (mock, 72 h), n = 8 (SR9011, 72 h) A375 20 μM; cleaved caspase 3, 48 h, *P = 0.0286; cleaved caspase 3, 72 h, ****P < 0.0001; TUNEL assay, 48 h, *P = 0.0286; TUNEL assay, 72 h, ****P < 0.0001; p62, 48 h, **P = 0.0079; p62, 72 h, **P = 0.0011. All panels representative of three biologically independent experiments with similar results. All data are mean ± s.e.m. P value is calculated with one-tailed Mann–Whitney test in all panels. For gel source data, see Supplementary Fig. 1.

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Extended Data Figure 6 Agonists of REV-ERBs (SR9009 and SR9011) block autophagy.

a, Agonists of REV-ERBs block autophagy, which results in reduced autophagic flux. b, Quantification of LC3 puncta. n indicates biologically independent samples. n = 6 (mock, chloroquinine (CQ) ± SR9011), n = 11 (SR9009), n = 5 (SR9011), n = 7 (CQ + SR9009). One-way ANOVA: mock versus SR9009 and SR9011, **P = 0.0049; CQ versus CQ + SR9009 or CQ + SR9011, ****P < 0.0001. c, On treatment with SR9009 and SR9011, autophagy blockage can be observed by electron microscopy, even under starvation conditions. Arrows, representative autophagosomes; Nu, nucleus. Scale bars, 1 μM. n = 3 biologically independent samples of two independent experiments with similar results (mock ± SR9009 and SR9011) or one experiment (mock, SR9009 and SR9011 ± starvation). d, Agonists of REV-ERBs induce lysosome accumulation, as shown by immunofluorescence assay for the lysosome marker LAMP1. n indicates biologically independent samples: n = 11 (mock), n = 6 (SR9009), n = 12 (SR9011). T47D, 72 h 20 μM, one-way ANOVA, ****P < 0.0001. e, Lysosome accumulation is confirmed by LysoTracker Red (MCF-7, 72 h 20 μM). Scale bars, 50 μm. BF, bright field. f, Marked lysosomal turnover defects are revealed with electron microscopy. n = 3 biologically independent samples of two independent experiments with similar results. Arrows, lysosomes; Nu, nucleus. Scale bars, 1 μM. g, h, Starvation synergizes with treatment with the SR9009 agonist of REV-ERBs (MCF-7 48 h, 20 μM; A375, 3 days, 20 μM). i, j, Starvation has no effect on expression of REV-ERBs, as shown by qRT–PCR; two-tailed Mann–Whitney test. ns, not significant; FM, fresh medium; ST, starvation. k, l, Overexpression of ULK3, ULK2 and LKB1 impairs the induction of apoptosis by SR9011 (MCF-7, A375 6 days, 20 μM). m, WST-1 viability assay shows abrogation of apoptosis in cells that overexpress ULK2. n indicates biological replicates: n = 12 (empty vector (E.V.) mock, ULK2 mock, ULK2 SR9011), n = 27 (E.V. SR9011) A375, 6 days, 20 μM. One-tailed Mann–Whitney test, E.V. mock versus E.V. 011, ****P < 0.0001; ULK2 mock versus ULK2 011 *P = 0.0225). n, qRT–PCR shows overexpression of ULK3 (one-tailed Student’s t-test, **P = 0.0031). o, p, Immunofluorescence assay confirms overexpression of LKB1 and ULK2. n = 3 biologically independent samples (i, j, n). Scale bars, 50 μm. All panels representative of three biologically independent experiments with similar results, unless otherwise specified. All data are mean ± s.e.m.

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Extended Data Figure 7 Core autophagy genes are novel REV-ERBs targets.

a, Analyses of available ChIP–seq data5 indicate that REV-ERBs peaks are present in Ulk3, Ulk1, Becn1 and Atg7 (P < 0.00001 calculated by MACS using Poisson distribution, false discovery rate ≤ 0.05). b–e, Analysis of REV-ERBs-binding motif performed using HOMER indicates the presence of REV-ERBs-binding sites in Ulk3, Ulk1, Becn1 and Atg7 genes (mouse genome). f–i, Treatment with agonists of REV-ERBs leads to downregulation of autophagy central regulators (MCF-7 72 h 20 μM; one-way ANOVA, ****P < 0.0001). j, Autophagy genes are upregulated on expression of REV-ERBs shRNA. A375 cell line, n = 6 biologically independent samples. One-tailed Mann–Whitney test; ULK3, **P = 0.0011; ATG7 and BECN1, **P = 0.0011; ULK1, **P = 0.0043. k, The repression of autophagy genes caused by SR9009 and SR9011 is abrogated in A375 cells expressing REV-ERBs shRNA; control shRNA ± SR9009 or SR9011, one-way ANOVA; ULK1, *P = 0.0162; ATG7, **P = 0.0036. f–i, k, n = 3 biologically independent samples. All data are mean ± s.e.m. shREVs, shNR1D1 and shNR1D2; shCTRL, non-silencing shRNA.

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Extended Data Figure 8 REV-ERBs regulate autophagy core genes and block autophagy in slowly proliferating cancer stem cells.

a, b, Immunoblot analyses show a reduction of ULK1, ATG7, ULK3 and BECN1 protein levels on treatment with agonists of REV-ERBs (MCF-7 72 h 20 μM). c–e, REV-ERBs shRNA increases protein levels of autophagy regulators (A375). f, The reduction in ATG7 protein levels on treatment with SR9009 and SR9011 is abrogated in cells expressing REV-ERBs shRNA. g, WST-1 viability assays show that treatment with SR9009 and SR9011 is cytotoxic specifically in patient-derived glioblastoma stem cells; mean ± s.e.m., 5 days, one-way ANOVA. n indicates biological replicates: GSC 272, n = 4 (mock, SR9009), n = 6 (SR9011), ***P = 0.0002; GSC 6.27, n = 5 (mock), n = 10 (SR9009), **P = 0.003; GSC 8.11, n = 8 (mock), n = 5 (SR9009), n = 7 (SR9011), ****P < 0.0001; GSC 7.11, n = 9 (mock, SR9009), n = 7 (SR9011), ****P < 0.0001. h–k, Immunoblot analyses show accumulation of p62 in patient-derived glioblastoma stem cells (one independent experiment). l, MTS assays show that GSC 6.27, 7.11 and 272 are characterized by a slow proliferation rate; n = 4 biologically independent samples, four experiments, mean ± s.d. All panels representative of three biologically independent experiments with similar results, unless otherwise specified. For gel source data, see Supplementary Fig. 1.

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Extended Data Figure 9 Agonists of REV-ERBs do not affect viability of normal proliferating and quiescent OIS cells.

a, Immunofluorescence assay for RAS confirms RAS overexpression in OIS cells. b, SA-β-galactosidase assay shows induction of senescence; n = 3 biologically independent samples, one-tailed Student's t-test, ****P < 0.0001. c, Induction of cell cycle inhibitors CDKN2B and CDKN2A is assayed by qRT–PCR. n = 5 biologically independent samples. One-tailed Mann–Whitney test, CDKN2B, **P = 0.004; CDKN2A, **P = 0.0079. d, e, Agonists of REV-ERBs do not induce apoptosis in proliferating and quiescent normal diploid BJ fibroblasts (d–g), as shown by proliferation assay (d, 7 days, 20 μM) and immunofluorescence for cleaved caspase 3 (e, f, 7 days, 20 μM). One-way ANOVA. n indicates biologically independent samples: n = 7 (mock), n = 5 (SR9009, SR9011). Cell viability is also not affected in an additional normal diploid WI38 cell line (g, 10 days, 20 μM). h, i, SR9009 and SR9011 inhibit autophagy in OIS cells, as shown by the accumulation of lysosomes (LysoTracker Red) and the absence of LC3 puncta (3 days, 20 μM). Scale bars, 50 μm. Data in a–i are representative of three independent experiments with similar results, unless otherwise specified. All data are mean ± s.e.m.

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Extended Data Figure 10 SR9009 impairs tumour growth and improves survival of glioblastoma patient-derived xenografts.

a, Protein levels of autophagy genes in NRAS naevi are reduced upon treatment with SR9009, as assayed by immunoblot (n = 4 mice, one experiment). b, TUNEL assays show that apoptosis induction is absent in normal skin on treatment with SR9009 (n = 4 mice, one experiment, 12 days, SR9009 20 μM). Scale bar, 10 μm; BF, bright field. c, TUNEL assays show that apoptosis induction is absent in normal brain tissues on treatment with SR9009 (6 days, 200 mg kg−1 twice a day, n = 5 mice, one experiment). d, Treatment with SR9009 (100 mg kg−1 twice a day) is tolerated better than temozolomide administration (82.5 mg kg−1 once a day for 5 days), as shown by measurement of percentage body weight change. One-tailed Mann–Whitney test; day 6, 8, 10, *P = 0.0411, mean ± s.e.m. n = 5 (SR9009) or 6 (temozolomide) mice. e, The glioblastoma cell line A172 is sensitive to treatment with SR9009 and SR9011 (20 μM 6 days, three biologically independent experiments with similar results). f, Previous analyses30 of The Cancer Genome Atlas data show genetic alterations that affect NR1D1 and NR1D2 are absent. Gene expression analysis shows that no cases are present with downregulation of REV-ERBs, and only a small fraction with upregulation. n = 574 biologically independent samples. NR1D1: upregulation, 1.56%; homozygous deletion (Hom Del), 0.17%; unaltered, 98.27%. NR1D2: upregulation, 4.54%; unaltered, 95.46%. g, In vivo treatment with SR9009 results in the decrease of ATG7 protein levels (6 days, 200 mg kg−1 twice a day, n = 5 mice, one experiment). h, SR9009 treatment impairs in vivo growth of glioblastoma patient-derived xenografts (6 days, 200 mg kg−1, n = 5 mice). i, Quantification of tumour size by in vivo luciferase assays (mean ± s.e.m., n = 10 mice, one-tailed Mann–Whitney test, **P = 0.0057). j, SR9009 improves survival in mice that bear glioblastoma patient-derived xenografts. SR9009 200 mg kg−1 twice a day, n = 11 (vehicle), n = 11 (SR9009), n = 11 (temozolomide (82.5 mg kg−1 once a day for 5 days)) mice; two-tailed log-rank analyses. For gel source data, see Supplementary Fig. 1.

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Sulli, G., Rommel, A., Wang, X. et al. Pharmacological activation of REV-ERBs is lethal in cancer and oncogene-induced senescence. Nature 553, 351–355 (2018). https://doi.org/10.1038/nature25170

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