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Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage

Nature Cell Biologyvolume 20pages413421 (2018) | Download Citation

Abstract

Ageing of haematopoietic stem cells (HSCs) contributes to deficits in the aged haematopoietic system. HSC decline is driven in part by DNA damage accumulation; yet, how ageing impacts the acute DNA damage response (DDR) of HSCs is poorly understood. We show that old HSCs exhibit diminished ATM activity and attenuated DDR, leading to elevated clonal survival in response to a range of genotoxins that was underwritten by diminished apoptotic priming. Distinct HSC subsets exhibited ageing-dependent and subtype-dependent differences in apoptotic priming and survival in response to DNA damage. The defective DDR of old HSCs was non-cell autonomous, as ATM signalling and clonal survival in response to DNA damage could be restored to levels observed in young HSCs post-transplantated into young recipients. These data indicate that defective DDR and diminished apoptotic priming provide a selective advantage to old HSCs that may contribute to mutation accrual and disease predisposition.

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References

  1. 1.

    Rossi, D. J., Jamieson, C. H. & Weissman, I. L. Stems cells and the pathways to aging and cancer. Cell 132, 681–696 (2008).

  2. 2.

    Geiger, H., de Haan, G. & Florian, M. C. The ageing haematopoietic stem cell compartment. Nat. Rev. Immunol. 13, 376–389 (2013).

  3. 3.

    Beerman, I., Maloney, W. J., Weissmann, I. L. & Rossi, D. J. Stem cells and the aging hematopoietic system. Curr. Opin. Immunol. 22, 500–506 (2010).

  4. 4.

    Beerman, I., Seita, J., Inlay, M. A., Weissman, I. L. & Rossi, D. J. Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle. Cell Stem Cell 15, 37–50 (2014).

  5. 5.

    Rossi, D. J. et al. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447, 725–729 (2007).

  6. 6.

    Nijnik, A. et al. DNA repair is limiting for haematopoietic stem cells during ageing. Nature 447, 686–690 (2007).

  7. 7.

    Walter, D. et al. Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature 520, 549–552 (2015).

  8. 8.

    Rube, C. E. et al. Accumulation of DNA damage in hematopoietic stem and progenitor cells during human aging. PLoS ONE 6, e17487 (2011).

  9. 9.

    Rossi, D. J. et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc. Natl Acad. Sci. USA 102, 9194–9199 (2005).

  10. 10.

    Chambers, S. M. et al. Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation. PLoS Biol. 5, e201 (2007).

  11. 11.

    Beerman, I. et al. Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging. Cell Stem Cell 12, 413–425 (2013).

  12. 12.

    Sun, D. et al. Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal. Cell Stem Cell 14, 673–688 (2014).

  13. 13.

    Florian, M. C. et al. Cdc42 activity regulates hematopoietic stem cell aging and rejuvenation. Cell Stem Cell 10, 520–530 (2012).

  14. 14.

    Beerman, I. et al. Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc. Natl Acad. Sci. USA 107, 5465–5470 (2010).

  15. 15.

    Kim, M., Moon, H. B. & Spangrude, G. J. Major age-related changes of mouse hematopoietic stem/progenitor cells. Ann. NY Acad. Sci. 996, 195–208 (2003).

  16. 16.

    Morrison, S. J., Wandycz, A. M., Akashi, K., Globerson, A. & Weissman, I. L. The aging of hematopoietic stem cells. Nat. Med. 2, 1011–1016 (1996).

  17. 17.

    Sudo, K., Ema, H., Morita, Y. & Nakauchi, H. Age-associated characteristics of murine hematopoietic stem cells. J. Exp. Med. 192, 1273–1280 (2000).

  18. 18.

    Baguley, B. C. & Kerr, D. J. Anticancer drug development. Brit. J. Cancer 86, 1665–1666 (2002).

  19. 19.

    Brunelle, J. K. & Letai, A. Control of mitochondrial apoptosis by the Bcl-2 family. J. Cell. Sci. 122, 437–441 (2009).

  20. 20.

    Cory, S. & Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer 2, 647–656 (2002).

  21. 21.

    Llambi, F. et al. A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol. Cell. 44, 517–531 (2011).

  22. 22.

    Letai, A. et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183–192 (2002).

  23. 23.

    Ren, D. et al. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 330, 1390–1393 (2010).

  24. 24.

    Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R. D. & Korsmeyer, S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334–336 (1990).

  25. 25.

    Oda, E. et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053–1058 (2000).

  26. 26.

    Boyd, J. M. et al. Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. Oncogene 11, 1921–1928 (1995).

  27. 27.

    Kutuk, O. & Letai, A. Regulation of Bcl-2 family proteins by posttranslational modifications. Curr. Mol. Med. 8, 102–118 (2008).

  28. 28.

    Del Gaizo Moore, V. & Letai, A. BH3 profiling—measuring integrated function of the mitochondrial apoptotic pathway to predict cell fate decisions. Cancer Lett. 332, 202–205 (2013).

  29. 29.

    Ni Chonghaile, T. et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 334, 1129–1133 (2011).

  30. 30.

    Ryan, J. A., Brunelle, J. K. & Letai, A. Heightened mitochondrial priming is the basis for apoptotic hypersensitivity of CD4+ CD8+ thymocytes. Proc. Natl Acad. Sci. USA 107, 12895–12900 (2010).

  31. 31.

    Vo, T. T. et al. Relative mitochondrial priming of myeloblasts and normal HSCs determines chemotherapeutic success in AML. Cell 151, 344–355 (2012).

  32. 32.

    Sarosiek, K. A. et al. Developmental regulation of mitochondrial apoptosis by c-Myc governs age- and tissue-specific sensitivity to cancer therapeutics. Cancer Cell 31, 142–156 (2017).

  33. 33.

    Sarosiek, K. A. et al. BID preferentially activates BAK while BIM preferentially activates BAX, affecting chemotherapy response. Mol. Cell 51, 751–765 (2013).

  34. 34.

    Opferman, J. T. et al. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 307, 1101–1104 (2005).

  35. 35.

    Campbell, C. J. et al. The human stem cell hierarchy is defined by a functional dependence on Mcl-1 for self-renewal capacity. Blood 116, 1433–1442 (2010).

  36. 36.

    Villunger, A. et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins Puma and Noxa. Science 302, 1036–1038 (2003).

  37. 37.

    Dykstra, B., Olthof, S., Schreuder, J., Ritsema, M. & de Haan, G. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells. J. Exp. Med. 208, 2691–2703 (2011).

  38. 38.

    Muller-Sieburg, C. E., Cho, R. H., Karlsson, L., Huang, J. F. & Sieburg, H. B. Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness. Blood 103, 4111–4118 (2004).

  39. 39.

    Benz, C. et al. Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs. Cell Stem Cell 10, 273–283 (2012).

  40. 40.

    Challen, G. A., Boles, N. C., Chambers, S. M. & Goodell, M. A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-β1. Cell Stem Cell 6, 265–278 (2010).

  41. 41.

    Morita, Y., Ema, H. & Nakauchi, H. Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J. Exp. Med. 207, 1173–1182 (2010).

  42. 42.

    Beerman, I. & Rossi, D. J. Epigenetic control of stem cell potential during homeostasis, aging, and disease. Cell Stem Cell 16, 613–625 (2015).

  43. 43.

    Seita, J. et al. Gene Expression Commons: an open platform for absolute gene expression profiling. PLoS ONE 7, e40321 (2012).

  44. 44.

    Lukas, C. et al. Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J. 23, 2674–2683 (2004).

  45. 45.

    Bakkenist, C. J. & Kastan, M. B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

  46. 46.

    D’Amours, D. & Jackson, S. P. The Mre11 complex: at the crossroads of DNA repair and checkpoint signalling. Nat. Rev. Mol. Cell Biol. 3, 317–327 (2002).

  47. 47.

    Ito, K. et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature 431, 997–1002 (2004).

  48. 48.

    Maryanovich, M. et al. The ATM–BID pathway regulates quiescence and survival of haematopoietic stem cells. Nat. Cell Biol 14, 535–541 (2012).

  49. 49.

    Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. & Chen, D. J. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276, 42462–42467 (2001).

  50. 50.

    Flach, J. et al. Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature 512, 198–202 (2014).

  51. 51.

    Schultz, L. B., Chehab, N. H., Malikzay, A. & Halazonetis, T. D. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J. Cell. Biol. 151, 1381–1390 (2000).

  52. 52.

    Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159 (2004).

  53. 53.

    Bondar, T. & Medzhitov, R. p53-mediated hematopoietic stem and progenitor cell competition. Cell Stem Cell 6, 309–322 (2010).

  54. 54.

    Pang, W. W. et al. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age. Proc. Natl Acad. Sci. USA 108, 20012–20017 (2011).

  55. 55.

    Jan, M. et al. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci. Transl. Med. 4, 149ra118 (2012).

  56. 56.

    Ley, T. J. et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010).

  57. 57.

    Jaiswal, S. et al. Age-related clonal hematopoiesis associated with adverse outcomes. N. Engl. J. Med. 371, 2488–2498 (2014).

  58. 58.

    Busque, L. et al. Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis. Nat. Genet. 44, 1179–1181 (2012).

  59. 59.

    Xie, M. et al. Age-related mutations associated with clonal hematopoietic expansion and malignancies. Nat. Med. 20, 1472–1478 (2014).

  60. 60.

    Ryan, J. & Letai, A. BH3 profiling in whole cells by flourimeter or FACS. Methods 61, 156–164 (2013).

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Acknowledgements

We thank E. Shlevkov for help with statistics, U. Rajarajacholan for help with dot blots and all the members of the D.J.R. laboratory for help. The A.N. laboratory was supported by the Intramural Research Program of the NIH, the National Cancer Institute, the Center for Cancer Research and the Alex Lemonade Stand Foundation Award. L.H. was supported by the NIH fellowship F31CA186301. The A.L. laboratory was supported by the NIH grant P01 CA066996 and the Leukemia and Lymphoma Society Grant TRP6387-13. D.J.R. is supported by grants from the NIH (RO1HL107630, R00AG029760 and UO1DK072473-01) as well as grants from The Leona M. and Harry B. Helmsley Charitable Trust, The New York Stem Cell Foundation, The Harvard Stem Cell Institute and the American Federation for Aging Research.

Author information

Author notes

  1. These authors contributed equally: Paula Gutierrez-Martinez, Leah Hogdal.

Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

    • Paula Gutierrez-Martinez
    • , Manavi Nagai
    • , Miriama Kruta
    •  & Derrick J. Rossi
  2. Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA

    • Paula Gutierrez-Martinez
    • , Manavi Nagai
    • , Miriama Kruta
    •  & Derrick J. Rossi
  3. Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    • Leah Hogdal
    •  & Anthony Letai
  4. John B. Little Center for Radiation Sciences, Department of Environmental Health, Harvard T. H. Chan School of Public Health, Boston, MA, USA

    • Rumani Singh
    •  & Kristopher Sarosiek
  5. Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA

    • Andre Nussenzweig
  6. Translational Gerontology Branch, National Institute on Aging, NIH, Baltimore, MD, USA

    • Isabel Beerman
  7. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    • Derrick J. Rossi
  8. Harvard Stem Cell Institute, Cambridge, MA, USA

    • Derrick J. Rossi

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Contributions

P.G.-M. designed, performed and analysed most of the experiments and wrote the manuscript. L.H. designed and performed the BH3 profiling experiments. M.N. provided technical help. M.K. performed the immunostainings. I.B. performed the comet assays, transplantation of the CD150 HSC subsets and provided critical guidance. R.S. and K.S. provided Bax–/– and Bak–/– mice. A.N. provided Atm–/– mice and ATM guidance. A.L. and D.J.R. designed the experiments and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Anthony Letai or Derrick J. Rossi.

Integrated supplementary information

  1. Supplementary Figure 1 Inclusion of CD48 did not alter purity of HSCs gated on LSKFlk2-CD34- in either young or old mice.

    a) Quantification and b) representative flow plots of CD48 expression levels in young and old HSCs (LSKFlk2-CD34-/lo), MPP1s (LSKFlk2-CD34+) and MPP2s (LSKFlk2+CD34+). Source data are included in Supplementary Table 1.

  2. Supplementary Figure 2 Clonal colony size is similarly affected in young and old HSCs upon DNA damage induction.

    a) Representative FACS plots showing HSC gating strategy and post-sort purity from young and old mice. b) Representative cell cycle profile and quantification of G0/G1 and S/G2M in young and old HSCs and steady state and upon 18 hours of culture. Data pooled from 4 independent experiments. (yHSC n = 2, oHSC n = 4, yHSC 18h n = 2, oHSC 18h n = 5) c) Frequency of live young and old HSCs that have divided at the indicated days. Data pooled from 6 independent experiments. d–g) Quantification of colony size upon exposure to the indicated treatments at day 10 measured as diameter of the colony in d, e) young and old HSCs and f, g) young and old MPs. Gamma irradiation (IR), ethyl-nitrosourea (ENU), ethyl-methanesulfonate (EMS), hydroxyurea (HU) doxorubicin (Doxo). Cell numbers (n) between 7–490, exact cell number per condition indicated in Supplementary Table 1. For d–g) each dot represents individual cells, data pooled from 2 independent experiments. P<0.05 (*), P<0.005 (**), P<0.0005 (***). Two-tailed Student’s t-test. Centre bar represents mean and error bars represent SEM. Source data are included in Supplementary Table 1.

  3. Supplementary Figure 3 Clonal colony survival and growth dynamics is differentially affected in young and old HSCs upon DNA damage induction.

    Cell number and viability of single untreated or irradiated young and old HSCs during 8 days of clonal culturing. Each column depicts a day and each row indicates a cell. 288 single HSCs per condition were analysed.

  4. Supplementary Figure 4 BH3 profiling in stem and progenitor cells.

    a) Dots plots and histograms showing gating strategy and TMRE intensity for each of the indicated populations in young and old bone marrow in response to BIM 3 μM and 8 μM. CLP: common lymphoid progenitors, MP: myeloid progenitors, LSK: lineage-Sca1+c-kit+, GMP: granulocyte monocyte progenitor, CMP: common myeloid progenitor, MEP: megakaryocyte erythroid progenitor, MPP1: multi-potent progenitor 1, MPP2: multi-potent progenitor 2, HSC: haematopoietic stem cells. b, c) Apoptotic priming of Bax-/- and Bak-/- HSCs in response to b) BIM 80 μM and c) BID 80 μM. Each dot represents an individual mouse (n = 3-5). Data pooled from 4 independent experiments. d) Clonal survival of young and old Bax-/- and Bak-/- HSCs measured as a percentage of viable clones in response to IR. Each dot represents percent survival of 24 to 48 single HSCs (LSK CD34-/lo Flk2- and LSK CD34-/lo CD150+) purified from individual mice (n = 2–4 mice per group). Numbers above the graphs indicate total number of surviving clones (black) vs total number of clones analysed (grey). Data pooled from 4 independent experiments. e) Apoptotic priming of young and old MPs in response to BAD 80 μM, HRK 80 μM and NOXA 80 μM. Each dot represents individual mice. Data pooled from 2 independent experiments. f) Frequency of CD150 HSCs subsets in young and old bone marrow (n = 5). Data pooled from 6 independent experiments. g) Apoptotic priming of young and old HSC subsets in response to BIM (8 μM) and BID (3 μM). Same data as in Fig 3f. Numbers above the graphs indicate total number of surviving clones (black) vs total number of clones analysed (grey). *, P< 0.05, **, P< 0.005, ***, P< 0.0005 (Two-tailed Student’s t-test). Centre bar represents mean and error bars represent SEM. Source data are included in Supplementary Table 1.

  5. Supplementary Figure 5 Apoptotic priming and survival upon DNA damage induction is equalized upon transplantation.

    a-b) Non-competitive transplantation of 5x106 young and old bone marrow showing a) peripheral blood chimaerism of recipient mice over the time course of the experiment, and b) lineage contribution of donor cells in recipients shown at week 16. Data from 1 experiment, n = 9 individual young recipient mice. c) Apoptotic priming of MP in response to BIM (8 μM), BID (3 μM) and BAD (80 μM) in steady-state young and old bone marrow and in bone marrow transplant-derived young and old LSK and MP (21–23 weeks post-transplantation). Data pooled from 5 independent experiments, each dot represents an individual mouse (n = 3, 4 or 5 mice per group). d-e) Non-competitive transplantation of 5×106 young bone marrow showing d) peripheral blood chimaerism of recipient mice over the time course of the experiment and e) lineage contribution of donor cells in recipients shown at week 16. Data from 1 experiment, n = 5 individual 17- month old recipient mice. ***, P< 0.0005 (Two-tailed Student’s t-test). Centre bar represents mean and error bars represent SEM. Source data are included in Supplementary Table 1.

  6. Supplementary Figure 6 ATM levels and activity are diminished in old HSCs.

    a-b) Expression of a) DNA damage response and b) DNA damage sensor genes in young and old HSCs, (n = 3–5 mice per group, as indicated with dots). Data represents normalized signal intensity (data from Gene Expression Commons4,43, Gene Expression Omnibus (Series GSE55525). c) Olive tail moment of young and old freshly isolated HSCs or upon 1 hour in culture post irradiation with 2Gy. Data represent 1 experiment, n numbers represent cells and are indicated in the Figure. d) Dot blot showing γH2AX and actin levels in LSKs non-treated or irradiated (1 hour), with and without ATM inhibitor (ATMi, 10 μM). Experiment was performed once. e) Quantification of γH2AX levels in c-kit enriched bone marrow upon irradiation and 4h of culture with or ATMi. n = 3 mice. *, P< 0.05, **, P< 0.005, ***, P< 0.0005 (Two-tailed Student’s t-test). Centre bar represents mean and error bars represent SEM. Source data are included in Supplementary Table 1.

  7. Supplementary Figure 7 Unprocessed dot blots for Supplementary Figure 6d.

    Please note that in blot 1 the samples for NT 25 μM and 2Gy 25 μM are switched and do not correspond to the labels and NT 50 μM and 2Gy 50 μΜ samples were not treated with ATMi (KU55933). Experiment performed once.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7 and Supplementary Table legend.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Statistics source data.

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https://doi.org/10.1038/s41556-018-0054-y