Abstract
Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global histone H3 lysine 36 trimethylation, an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Notably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.
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Code availability
All software and packages used are listed in the Reporting Summary and are publicly available. The code relevant to the ChIP-seq analysis is hosted on Zenodo (https://zenodo.org, https://doi.org/10.5281/zenodo.6865749).
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Acknowledgements
We are grateful to members of J. Gil’s laboratory for reagents, comments and other contributions to this project. We thank members of the Genomics LMS facility (L. Game, K. Rekopoulou and A. Ivan) and the Bioinformatics LMS facility (G. Dharmalingam, M. Karimi and H. Pallikonda) for help with RNA-seq and data processing. We thank O.C. Bing (BRC, A*STAR) for the histopathology scoring of liver sections. For the purpose of open access, the author has applied a Creative Commons Attribution license. Core support from MRC (MC_U120085810), a Development Gap Fund grant from LifeArc and Cancer Research UK (C15075/A28647) funded this research in J. Gil’s laboratory. P.M.-C. acknowledges funding from RTI2018-096068-B-I00, ERC-2016-AdG-741966, La Caixa HR17-00040, UPGRADE-H2020-825825, MWRF, Fundació La Marató-TV3, AFM, MDA and DPP-E. This work was supported by grants from the Deutsche Krebshilfe (to J.J.), the Dutch Cancer Society (to G.d.H.) and the Tekke Huizinga Fund (S.B. and G.d.H.). L.R. was supported by the Pasteur - Paris University International PhD Program and by the Fondation pour la Recherche Médicale. O.B was supported by Fondation ARC pour la Recherche sur le Cancer, INSERM-AGEMED and ANR S-ENCODE - 19-CE13-0017-01. O.B. is a Centre National de la Recherche Scientifique Research Director DR2. T.W. was funded by National Medical Research Council, Singapore through NMRC/OFLCG/003b/2018 and A*STAR through the Central Research Fund for Applied/Translational Research.
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Contributions
A.G. designed, performed and analyzed the cell culture experiments and wrote the first draft of the manuscript. A.J.I., V.W., M.A. and N.M. designed, performed and analyzed experiments. L.R. performed the ChIP-seq experiments. P.-F.R. analyzed the ChIP-seq and RNA-seq data. O.B. designed and analyzed the ChIP-seq experiments, secured funding and wrote the manuscript. S.C.B., J.J. and A.A. designed, performed and analyzed the UCB experiments. G.d.H. designed and analyzed the UCB experiments, secured funding and wrote the manuscript. L.O. and V.M. designed, performed and analyzed the muscle stem cell experiments. E.P. and P.M.-C. designed, analyzed and wrote the muscle stem cell experiments and secured funding. A.P. designed, performed and analyzed the liver regeneration experiments. T.W. designed, analyzed and wrote the liver regeneration experiments and secured funding. J.G. conceived and designed the project, secured funding and wrote the manuscript, with all authors providing feedback.
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Competing interests
J.G. has acted as a consultant for Unity Biotechnology, Geras Bio, Myricx Pharma and Merck KGaA. Pfizer and Unity Biotechnology have funded research in J.G.’s laboratory (unrelated to the work presented here). J.G. owns equity in Geras Bio. J.G. and A.G. are named inventors in an MRC patent and J.G. is a named inventor in other Imperial College patents, both related to senolytic therapies (the patents are not related to the work presented here). T.W. is scientific co-founder of, and holds stakes in, Cargene Therapeutics, which develops nucleic-acid therapeutics for liver diseases (unrelated to the work presented here). The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Cellular models of senescence induced by oncogene activation and telomere uncapping.
a, IMR90 ER:RAS as a model of OIS. Quantification of immunofluorescence staining for BrdU (left) and p16INK4a (right) of IMR90 ER:RAS cells 4 days after treatment with 4OHT or vehicle (DMSO) (n = 3). b, IMR90 tet-TRF2△B△M as a model of telomere uncapping-induced senescence. c, Left, quantification of immunofluorescence staining for 53BP1 of IMR90 tet-TRF2△B△M cells after treatment with doxycycline or vehicle (DMSO) (n = 3). Right, representative immunofluorescence images. Scale bar, 20 μm. d, Left, quantification of immunofluorescence staining for p21CIP1 (n = 3). Right, representative immunofluorescence images. Scale bar, 50 μm. e, Left, quantification of immunofluorescence staining for p16INK4a (day 7, n = 2). Right, representative immunofluorescence images. Scale bar, 100 μm. f, Left, quantification of immunofluorescence staining for BrdU (day 3, n = 3). Right, representative immunofluorescence images. Scale bar, 50 μm. All statistical significances were calculated using unpaired two-tailed t-tests. All error bars represent mean ± s.d; n represents independent experiments.
Extended Data Fig. 2 Treatment with 3DA alleviates oncogene-induced senescence.
a, Representative immunofluorescence images of p16INK4a (red) in IMR90 ER:RAS cells cells 4 days after treatment with 4OHT and 10 μM 3DA or vehicle (DMSO). Scale bar, 100 μm. b, Quantification (n = 3). The statistical significance was calculated using unpaired two-tailed t-test. c, p21CIP1 protein expression in IMR90 ER:RAS cells treated with DMSO or 4OHT to induce senescence or 4OHT and 10 μM 3DA. Normalized nuclear intensity values and mean values on day 3 (left), day 6 (middle) and day 9 (right panel) are shown (n = 200 cells per condition). The statistical significance was calculated using unpaired two-tailed t-tests. d, Timeline of the experiment (left) and crystal violet staining (right). IMR90 ER:RAS cells were treated with 4OHT continuously to induce senescence. On day 6, DMSO or 10 μM 3DA were added (change of media every 3 days). Cells were fixed on day 14. e, Timeline of the experiment (left) and quantification of immunofluorescence staining for p16INK4a (middle) and BrdU (right). IMR90 ER:RAS cells were treated with DMSO for 4 weeks, or 10 μM 3DA for 2 weeks followed by DMSO for 2 weeks, or 10 μM 3DA for 4 weeks. Media was changed every three days. Treatment was started when cells were at passage 14 and ended when cells were at passage 20. At the end of the experiment, p16INK4a protein expression and BrdU-positive cells were quantified. For p16INK4a, normalized nuclear single-cell intensity values and mean of 200 cells are shown. To assess proliferation, percentage of BrdU-positive cells was measured (n = 3). Statistical significances were calculated using one-way ANOVA. All error bars represent mean ± s.d; n represents independent experiments unless otherwise stated.
Extended Data Fig. 3 Genetic or chemical inhibition of AHCY alleviates oncogene-induced senescence.
a, Left, representative images of immunofluorescence staining for AHCY (red). Scale bar, 50 μm. Right, single-cell intensities for AHCY (n = 1,000 cells per condition). The statistical significance was calculated using unpaired two-tailed t-test. b, Left, representative images of immunofluorescence staining for γH2AX (red). Right, quantification of immunofluorescence staining for γH2AX (n = 4). c, Quantification of immunofluorescence staining for BrdU of IMR90 ER:RAS cells four days after treatment with 4OHT or vehicle (DMSO) and 2.5 μM DZNep, 10 μM D-eritadenine and 4 μM TGF-β RI kinase inhibitor II as positive control. d, Expression levels of AHCY (n = 4). e, Expression levels of INK4a (encoding for p16INK4a, n = 4). f, Principal component analysis (PCA) for the experiment described in Fig. 3f. g,h, GSEA signatures from the same experiment. All statistical significances were calculated using one-way ANOVA. All error bars represent mean ± s.d; n represents independent experiments unless otherwise stated.
Extended Data Fig. 4 Contribution of histone methyltransferases and H3K36 methylation to senescence induction.
a, Quantification of immunofluorescence staining for BrdU of IMR90 ER:RAS cells 4 days after treatment with 4OHT or vehicle (DMSO) and increasing concentrations of GSK126 (an inhibitor of the H3K27 methylase EZH2), BRD4770 (an inhibitor of the H3 K9 methylase EHMT2) or EPZ004777 (an inhibitor of the H3 K79 methylase DOT1L). T, 4 μM TGF-β RI kinase inhibitor II as positive control (n = 3). All error bars represent mean ± s.d; n represents independent experiments. b, Immunoblot of protein extracts of IMR90 ER:RAS cells after 4OHT induction and treatment with 10 μM 3DA or vehicle (DMSO). Immunoblot of Histone H3 is included as a sample processing control. Immunoblots are a representative experiment out of three. c, Single-cell nuclear intensity values for H3K36me3 in a representative experiment out of 5 (n = 1000 cells per condition). The threshold used to quantify the cells stained for H3K36me3 cells in Fig. 4b, is shown as a red dashed line. d, Left, representative immunofluorescence images of histone H3 staining (red) 4 days after 4OHT induction and treatment with 10 μM 3DA or vehicle (DMSO). Scale bar, 100 μm. Right, single-cell nuclear intensity values for histone H3 in a representative experiment out of 3 (n = 1,000 cells). e, Left, representative immunofluorescence images of H3K36me3 staining (red) 6 days after treatment with 4OHT and 10 μM 3DA or vehicle (DMSO). Scale bar, 100 μm. Right, quantification (n = 4 independent experiments). All statistical significances were calculated using one-way ANOVA. All error bars represent mean ± s.d.
Extended Data Fig. 5 Transcriptional profiling after chemical or genetic inhibition of AHCY.
a, Principal component analysis (PCA) for the RNASeq experiment described in Fig. 4c. b, Principal component analysis (PCA) including data from the experiments in Figs. 3f, 4c. c, GSEA of RNA-Seq data using signatures for oncogene-induced senescence and SASP. d, GSEA of RNA-Seq data using a signature for Hallmark E2F targets. e, GSEA of H3K36me3 ChIP-Seq data using a signature for SASP. f-g, Representative genome browser snapshots showing H3K36me3 normalized signal at IL12RB2 (module 2, f) and CENPF (module 4, g) gene loci for DMSO (orange), DMSO + 4OHT (green) and 3DA + 4OHT (violet) conditions. Data are expressed as normalized counts per million reads (CPM) in 200 bp non-overlapping windows.
Extended Data Fig. 6 H3K36 methylation is needed for establishing oncogene-induced senescence.
a, Expression levels of NSD2 (n = 4). b, Expression levels of NSD3 (n = 3). c, Expression levels of SMYD2 (n = 4). d, Single-cell nuclear intensity values for H3K36me3 staining 7 days after treatment with 4OHT or vehicle (DMSO) of IMR90 ER:RAS cells infected with different pGIPZ shRNAs against NSD2, NSD3, SMYD2, AHCY or the parental pGIPZ vector (n = 1,000 cells per condition for a representative experiment out of 3). The threshold used to quantify the cells stained for H3K36me3 cells in Fig. 5b, is shown as a red dashed line. All statistical significances were calculated using one-way ANOVA. All error bars represent mean ± s.d.
Extended Data Fig. 7 3-deaazadenosine inhibits reprogramming-induced senescence.
a, Senescence induced in IMR90 cells upon expression of reprogramming factors (OSKM). b, crystal violet-stained, IMR90 cells transduced with either and empty vector or OSKM (a vector expressing reprogramming factors OCT4, SOX2, KLF4, cMYC) were treated with 1 μM 3DA or vehicle (DMSO). Images are a representative experiment out of three.
Extended Data Fig. 8 3DA rejuvenates geriatric satellite cells.
a-f, Analysis of the experiment described in Fig. 6a. a, Expression levels for mouse Cdkn1a mRNA (encoding for p21) in young (2–3 months, n = 7) versus geriatric satellite cells (28–31 months, n = 8). b, Quantification of γH2Ax intensity (arbitrary units: a.u.; n = 75–91 cells). c, Representative images of γH2Ax. d, Quantification of BrdU staining of young (2–3 months, n = 5) versus geriatric satellite cells (28–31 months, n = 7). e, f, Expression levels for mouse Myog (e) and Myh3 mRNA (f) in young (2–3 months, n = 6) versus geriatric (28–31 months, n = 5) satellite cells after the indicated treatments. g, Experimental design. Tibialis anterior muscles were injected with cardiotoxin to induce damage and regeneration. Mice were treated with vehicle or 3DA daily (10 mg/kg, i.p.) and sacrificed at 4 days post muscle injury to assess SA-β-gal activity. h, Left, quantification of SA- β -gal+ cells in the damaged area (n = 4 mice per group). Right, representative images of SA- β-gal staining in cryosections of tibialis anterior muscle. Scale bars 10 μm in c and 50 μm in h. All error bars represent mean ± s.d; n represents number of mice unless otherwise stated. Statistical significances were calculated using two-tailed unpaired t test. This figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.
Extended Data Fig. 9 3DA treatment improves liver regeneration in aged mice.
a, Schematic representation of the experiment. Two-year old mice were treated 3 and 1 days before partial hepatectomy (PH) with 3DA or vehicle. The resected liver material was used for γH2AX staining and histopathology. 48 h post PH, the rest of the liver was harvested and proliferation level was determined by Ki67 staining. b, Right side shows representative photographs of IF staining with antibody against γH2AX and fluorescent DNA stain (DAPI). The inlay shows a magnification of positive nuclei from the respective main photograph. Left side shows the quantification. A significantly higher amount (p < 0.05) of γH2AX positive hepatocytes was detected in the control group (vehicle, n = 4) compared to experiment (3DA, n = 3), indicating a reduction in senescent cells. c, Right side shows representative photographs of IF staining with antibody against Ki67 and fluorescent DNA stain (DAPI). Left side shows the quantification. A significantly higher amount (p < 0.05) of Ki67 positive hepatocytes were detected in experimental group (3DA, n = 3) compared to control (vehicle, n = 3), indicating that a reduction in senescent hepatocytes is associated with improved proliferation. Statistical significance was calculated using the unpaired two-tailed Student’s t test. Error bars are represented as mean ± SEM; n represents number of mice. d-g, Pathological score (quantified blindly in a scale from 0–5) for the indicated parameters were assigned to H&E-stained liver sections from the experimental group (3DA, n = 4) and control group (vehicle, n = 4). Statistical significance was calculated using the unpaired two-tailed Student’s t test. Error bars are represented as mean ± SEM; n represents number of mice. This figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.
Extended Data Fig. 10 3DA improves the engraftment of umbilical cord blood cells.
a-d, GSEA signatures for the cord blood RNA-seq experiment. e, Cord blood-derived human hematopoietic stem and progenitor (CD34 + ) cells were treated at day 1, 4 and 7 with 10 μM 3DA or DMSO and analyzed before xenotransplantation at day nine. Absolute cell numbers were determined by manual cell counting. Data are represented as mean ± SD, n = 4 independent experiments. Statistical significance was calculated using one-tailed Student’s t test. f, 1.5 × 106 Cells from the experiment described in a were transplanted into NSG mice. The engraftment of human (CD45+) cells and the percentage of primitive cells (CD34 + CD38−) in the bone marrow was analyzed by flow cytometry. For e and f, each shape (open cicle, closed circle, star, square or triangle) represents a different cord blood sample. Each shape is the average of 2‐3 transplanted mice with that cord blood sample (n = 4 independent cord blood samples). Statistical significance was calculated using one-tailed Student’s t test. g, Gating strategy for the experiment shown in Fig. 7f. h, Gating strategy for the experiment shown in Fig. 7g.
Supplementary information
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Guerrero, A., Innes, A.J., Roux, PF. et al. 3-Deazaadenosine alleviates senescence to promote cellular fitness and cell therapy efficiency in mice. Nat Aging 2, 851–866 (2022). https://doi.org/10.1038/s43587-022-00279-9
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DOI: https://doi.org/10.1038/s43587-022-00279-9
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