The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63 being responsible for DNA-damage-induced apoptosis of resting oocytes. Here, we provide the first detailed mechanistic insight into the activation of p63, a process that requires phosphorylation by both the priming kinase CHK2 and the executioner kinase CK1 in mouse primordial follicles. We further describe the structural changes induced by phosphorylation that enable p63 to adopt its active tetrameric conformation and demonstrate that previously discussed phosphorylation by c-Abl is not involved in this process. Inhibition of CK1 rescues primary oocytes from doxorubicin and cisplatin-induced apoptosis, thus uncovering a new target for the development of fertoprotective therapies.
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Fuzzy interactions between the auto-phosphorylated C-terminus and the kinase domain of CK1δ inhibits activation of TAp63α
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Kerr, J. B., Myers, M. & Anderson, R. A. The dynamics of the primordial follicle reserve. Reproduction 146, R205–R215 (2013).
Klinger, F. G., Rossi, V. & De Felici, M. Multifaceted programmed cell death in the mammalian fetal ovary. Int. J. Dev. Biol. 59, 51–54 (2015).
Johnston, R. J. & Wallace, W. H. Normal ovarian function and assessment of ovarian reserve in the survivor of childhood cancer. Pediatr. Blood Cancer 53, 296–302 (2009).
Practice Committee of American Society for Reproductive Medicine. Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion. Fertil. Steril. 100, 1214–1223 (2013).
De Vos, M., Smitz, J. & Woodruff, T. K. Fertility preservation in women with cancer. Lancet 384, 1302–1310 (2014).
Rose, S. R. et al. Late endocrine effects of childhood cancer. Nat. Rev. Endocrinol. 12, 319–336 (2016).
Laronda, M. M. et al. A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nat. Commun. 8, 15261 (2017).
Suh, E. K. et al. p63 protects the female germ line during meiotic arrest. Nature 444, 624–628 (2006).
Livera, G. et al. p63 null mutation protects mouse oocytes from radio-induced apoptosis. Reproduction 135, 3–12 (2008).
Deutsch, G. B. et al. DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer. Cell 144, 566–576 (2011).
Kerr, J. B. et al. DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa. Mol. Cell 48, 343–352 (2012).
Peters, H. & Levy, E. Effect of irradiation in infancy on the mouse ovary; a quantitative study of oocyte sensitivity. J. Reprod. Fertil. 7, 37–45 (1964).
Jeruss, J. S. & Woodruff, T. K. Preservation of fertility in patients with cancer. N. Engl. J. Med. 360, 902–911 (2009).
Woodard, T. L. & Bolcun-Filas, E. Prolonging reproductive life after cancer: the need for fertoprotective therapies. Trends Cancer 2, 222–233 (2016).
Coutandin, D. et al. Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level. eLife 5, e13909 (2016).
Gebel, J. et al. Control mechanisms in germ cells mediated by p53 family proteins. J. Cell Sci. 130, 2663–2671 (2017).
Bolcun-Filas, E., Rinaldi, V. D., White, M. E. & Schimenti, J. C. Reversal of female infertility by Chk2 ablation reveals the oocyte DNA damage checkpoint pathway. Science 343, 533–536 (2014).
Reinhardt, H. C. & Yaffe, M. B. Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr. Opin. Cell Biol. 21, 245–255 (2009).
Serber, Z. et al. A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism. Mol. Cell. Biol. 22, 8601–8611 (2002).
Flotow, H. et al. Phosphate groups as substrate determinants for casein kinase I action. J. Biol. Chem. 265, 14264–14269 (1990).
Kim, D. A. & Suh, E. K. Defying DNA double-strand break-induced death during prophase I meiosis by temporal TAp63α phosphorylation regulation in developing mouse oocytes. Mol. Cell. Biol. 34, 1460–1473 (2014).
Gonfloni, S. et al. Inhibition of the c-Abl-TAp63 pathway protects mouse oocytes from chemotherapy-induced death. Nat. Med. 15, 1179–1185 (2009).
Kerr, J. B. et al. Cisplatin-induced primordial follicle oocyte killing and loss of fertility are not prevented by imatinib. Nat. Med. 18, 1170–1172 (2012). Author reply 1172–1174.
Maiani, E. et al. Reply to: cisplatin-induced primordial follicle oocyte killing and loss of fertility are not prevented by imatinib. Nat. Med. 18, 1172–1174 (2012).
Morgan, S., Lopes, F., Gourley, C., Anderson, R. A. & Spears, N. Cisplatin and doxorubicin induce distinct mechanisms of ovarian follicle loss; imatinib provides selective protection only against cisplatin. PLoS One 8, e70117 (2013).
Yuan, M., Luong, P., Hudson, C., Gudmundsdottir, K. & Basu, S. c-Abl phosphorylation of ΔNp63α is critical for cell viability. Cell Death Dis. 1, e16 (2010).
Yang, F., Kemp, C. J. & Henikoff, S. Anthracyclines induce double-strand DNA breaks at active gene promoters. Mutat. Res. 773, 9–15 (2015).
Wakasugi, M. et al. Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells. J. Biol. Chem. 289, 28730–28737 (2014).
Rossi, V. et al. LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse. Cell Death Differ 24, 72–82 (2017).
Carlsson, I. B. et al. Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture. Reproduction 131, 641–649 (2006).
Jang, H., Hong, K. & Choi, Y. Melatonin and fertoprotective adjuvants: prevention against premature ovarian failure during chemotherapy. Int. J. Mol. Sci. 18, E1221 (2017).
Hancke, K. et al. Sphingosine 1-phosphate protects ovaries from chemotherapy-induced damage in vivo. Fertil. Steril. 87, 172–177 (2007).
Zelinski, M. B. et al. In vivo delivery of FTY720 prevents radiation-induced ovarian failure and infertility in adult female nonhuman primates. Fertil. Steril. 95, 1440–1445.e7 (2011).
Salih, S. M., Ringelstetter, A. K., Elsarrag, M. Z., Abbott, D. H. & Roti, E. C. Dexrazoxane abrogates acute doxorubicin toxicity in marmoset ovary. Biol. Reprod. 92, 73 (2015).
Ting, A. Y. & Petroff, B. K. Challenges and potential for ovarian preservation with SERMs. Biol. Reprod. 92, 133 (2015).
Codacci-Pisanelli, G., Del Pup, L., Del Grande, M. & Peccatori, F. A. Mechanisms of chemotherapy-induced ovarian damage in breast cancer patients. Crit. Rev. Oncol. Hematol. 113, 90–96 (2017).
Soleimani, R., Heytens, E., Darzynkiewicz, Z. & Oktay, K. Mechanisms of chemotherapy-induced human ovarian aging: double strand DNA breaks and microvascular compromise. Aging (Albany NY) 3, 782–793 (2011).
Rinaldi, V. D., Hsieh, K., Munroe, R., Bolcun-Filas, E. & Schimenti, J. C. Pharmacological inhibition of the DNA damage checkpoint prevents radiation-induced oocyte death. Genetics 206, 1823–1828 (2017).
Garrett, M. D. & Collins, I. Anticancer therapy with checkpoint inhibitors: what, where and when? Trends Pharmacol. Sci. 32, 308–316 (2011).
Gokare, P. et al. Targeting of Chk2 as a countermeasure to dose-limiting toxicity triggered by topoisomerase-II (TOP2) poisons. Oncotarget 7, 29520–29530 (2016).
Winkler, B. S. et al. CK1δ in lymphoma: gene expression and mutation analyses and validation of CK1δ kinase activity for therapeutic application. Front. Cell Dev. Biol 3, 9 (2015).
Richter, J. et al. Decreased CK1δ expression predicts prolonged survival in colorectal cancer patients. Tumour Biol. 37, 8731–8739 (2016).
Rosenberg, L. H. et al. Therapeutic targeting of casein kinase 1δ in breast cancer. Sci. Transl. Med. 7, 318ra202 (2015).
Schittek, B. & Sinnberg, T. Biological functions of casein kinase 1 isoforms and putative roles in tumorigenesis. Mol. Cancer 13, 231 (2014).
Straub, W. E. et al. The C-terminus of p63 contains multiple regulatory elements with different functions. Cell Death Dis. 1, e5 (2010).
Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014).
Johannessen, C. M. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010).
Raingeaud, J., Whitmarsh, A. J., Barrett, T., Dérijard, B. & Davis, R. J. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol. Cell. Biol. 16, 1247–1255 (1996).
Smyrek, I. & Stelzer, E. H. Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy. Biomed. Opt. Express 8, 484–499 (2017).
Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).
The authors thank M. B. Gill, T. J. Wu and J. S. Carroll for their review of and input into this manuscript. Further, we thank F. Pampaloni (Goethe University) for providing the FEP foil holders. The research was funded by the DFG (DO 545/8-1), the Centre for Biomolecular Magnetic Resonance (BMRZ) and the Cluster of Excellence Frankfurt (Macromolecular Complexes). M.T. was supported by a Fellowship from the Funds of the Chemical Industry and Boehringer Ingelheim travel grant. V.R. was funded by Covert Italia S.p.A. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.
The authors declare that they have no competing interests.
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Integrated supplementary information
Supplementary Figure 1 In vitro phosphorylation of TAp63α wt and TAp63α S582A S583A using preactivated MK2 and MS analysis of DNA-damage- induced phosphorlyation of TAp63α in H1299 cells
a, Purified TAp63α wt and the S582A/S583A mutant were incubated with pre-activated MK2 for indicated periods. Oligomeric state was monitored by BN-PAGE. b, The same BN-PAGE from a was also analyzed using the α-pS582 antibody. c, SDS PAGE analysis of the in vitro reaction from a and b demonstrating that the α-pS582 antibody is specific for phosphorylation at S582 in TAp63α. d, Separate, extended time course of experiment shown in a. e, Summary of combined sequence coverage (bold characters) and identified phospho-sites (yellow characters) obtained from two sets of samples (Trypsin, AspN and Trypsin + AspN) and (Trypsin-low, Trypsin-high and Trypsin + LysC). f, Annotated MS/MS spectrum of the TI peptide (AA580-595) harboring phosphor-groups at the CHK2 site S582 and along the CK1 pattern (S585, T586 and S588).
Supplementary Figure 2 Most of the DNA-damage-induced phosphorylation events identified by MS are dispensable for TAp63α tetramerization, while inhibition of CK1δ abrogates TAp63α tetramerization, and in vitro phosphorylation of TAp63α pS582 by CK1 is sufficient to induce tetramerization
TAp63α wt and various Ser or Thr to Ala and Tyr to Phe mutants as well as deletion constructs covering the TAD (a), the linker between TD and SAM (b), phosphorylated residues directly preceding the CHK2 site (AA572-580) (c), T610 and S612 in the TID (d) as well as several individual sites identified throughout this study (T178, S233, S356, S381 and Y387) (e) were transiently expressed in U2OS cells and tetramerization was induced by Dox treatment (10 µM, 6 h). Oligomeric state and phosphorylation induced electrophoretic mobility shift were monitored by BN-PAGE and SDS-PAGE, respectively. f, TAp63α stable expressing H1299 cells and cultured mouse ovaries (g) were treated with increasing concentrations (5, 25 and 50 µM) of the CK1 inhibitors PF5006739 (IC50(CK1δ) = 3.9 nM, IC50(CK1ε) = 17 nM) and PF4800567 (IC50(CK1δ) = 711 nM, IC50(CK1ε) = 32 nM) 1 h prior to induction of tetramerization with Dox (10 µM, 6 h). Oligomeric state and phosphorylation induced electrophoretic mobility shift were monitored by BN-PAGE and SDS-PAGE, respectively. h, U2OS cells were co-transfected with TAp63α and constitutive active or dominant negative mutants of CK1 AA1-319 and tetramerization was induced with 10 µM Dox for 6 h. Oligomeric state was monitored by BN-PAGE. i, Bacterially expressed TAp63α was successively in vitro phosphorylated by pre-activated MK2 and in a second step by constitutively active CK1 (black line) and analyzed using size exclusion chromatography (Superose 6 10/300GL). Purified dimeric, MK2 phosphorylated TAp63α (red line) served as a retention time reference.
Supplementary Figure 3 Flowchart of the CHK2- and CK1-mediated disruption of the autoinhibitory structure of TAp63α
a, Schematic representation of the structural model of the autoinhibitory state formed by TAp63α. Within a monomer of the dimer the TA2A and TA2B domains together with the TID form an anti-parallel three-stranded β-sheet. Together with the second monomer of the dimer the anti-parallel six-stranded β-sheet blocks the tetramerization interface of the TD (Cyan). This closed conformation is further locked by the α-helical TA1 domain occupying the binding site of the second helix (H2) of the TD that only forms upon tetramerization. In close proximity of the CK1 phosphorylation sites, which are N-terminal to the TID, is a stretch of three negative charged aspartates (D61, D63, D66) C-terminal to the TA2B within each monomer. Color code of the core structure of the autoinhibitory conformation of TAp63α according to schematic domain representation in Figure 1c. b, Upon DNA damage active CHK2 phosphorylates TAp63α on S582. c, Phosphorylation on S582 turns TAp63α into a substrate for CK1, which subsequently phosphorylates S585. d, Phosphorylation of S585 generates again a consensus motif for CK1 to phosphorylate S588. e, As in c and d phosphorylation of S588 triggers the phosphorylation of S591 by CK1. f, Finally CK1 phosphorylates T594 without generating a new consensus motif. g, Thus the DNA damage induced phosphorylation events lead to an accumulation of negative charges in close proximity to the intrinsically negatively charged sequence harboring D61, D63 and D66. h and i, The generated forces due to charge repulsion overcome the energy barrier of the kinetically trapped dimer and break up the anti-parallel six-stranded β-sheet. j, Two open dimers will subsequently form an open tetramer. The tetrameric TAp63α is then capable to bind DNA and induce target gene expression that ultimately leads to death of the primordial follicle.
Supplementary Figure 4 c-Abl is not required for TAp63α tetramerization, and activation of TAp63α follows a slower kinetics after cisplatin compared to doxorubicin treatment and LH treatment does not abrogate Cs induced tetramerization in oocytes
a, TAp63α stable expressing H1299 cells were treated with increasing concentrations (5, 25 and 50 µM) of the c-Abl inhibitor imatinib 1 h prior to induction of tetramerization with Dox (10 µM, 6 h). Oligomeric state and phosphorylation induced electrophoretic mobility shift were monitored by BN-PAGE and SDS-PAGE, respectively. b, CD-1 P8 ovaries were cultured with or without 10 µM Dox or Cs for the indicated periods. Oligomeric state was monitored by BN-PAGE and phosphorylation induced electrophoretic mobility shift were SDS-PAGE, respectively. ATM total protein abundance as well as pATM levels were assessed by Western blotting. Ovaries were further subjected to 10 µM Cs in the presence of 200 mIU/ml LH.
a, Ovarian fragments isolated from P4 ovaries of p-18 c-Kit/GFP mice cultured for 4 days (0 h) before beginning of indicated treatment for further 24 h. Oocytes were imaged at 0 h, 14 h and 24 h. 0 h and 24 h time points of the untreated, Dox and Dox + 25 µM inhibitor treated are shown in Figure 4c. b, Oocytes were imaged at 0 h, 14 h and 24 h. 0 h and 24 h time points of the untreated, Cs and Cs + 25 µM inhibitor treated are shown in Figure 4d. Scale bar, 75 μm.
Supplementary Figure 6 Imatinib reduces only cisplatin induced oocyte death and shows no effect against doxorubicin
I Ovarian fragments isolated from P4 ovaries of p-18 c-Kit/GFP mice cultured for 4 days (0 h) before beginning of indicated treatment for further 24 h. Oocytes were imaged at 0 h, 14 h and 24 h. Scale bar, 75 μm. 0 h and 24 h time points of the untreated, Dox, Dox + 25 µM Imatinib, Cs, and Cs + 25 µM inhibitor, treated are shown in Figure 4b & d.
Supplementary Figure 7 Inhibition of CK1 markedly reduce cisplatin- and doxorubicin-induced oocyte death
a, Ovarian fragments isolated from P4 ovaries of p-18 c-Kit/GFP mice cultured for 4 days (0 h) before treatment for further 24 h (Ctrl – untreated, Dox – 0.4 µM, Cs – 10 µM, PF5006739 5, 25 and 50 µM. Scale bar, 75 μm. b, Percentage of healthy POs scored after 24 h culture with or without Cs or Dox and increasing concentrations (5, 25 and 50 µM) of PF5006739. Data presented as mean ± SEM (3 fragments each). n.s., not significant. **P < 0.01, ***P < 0.001, ****P < 0.001 two-tailed t-test.
3D staining of whole ovaries after doxorubicin treatment. Upper left, untreated ovary and stained using the oocyte marker DDX-4. Upper right, ovary treated 48 h with 0.4 µM Dox. Lower right, ovary treated 48 h with 0.4 µM Dox and 25 µM CK1 inhibitor. Lower left, ovary treated 48 h with 0.4 µM Dox and 25 µM CHK2 inhibitor. 360° rotation, increments set to 1°.
3D staining of whole ovaries after cisplatin treatment Upper left, untreated ovary and stainedusing the oocyte marker DDX-4. Upper right, ovary treated 48 h with 10 µM Cs. Lower right, ovary treated 48 h with 10 µM Dox and 25 µM CK1 inhibitor. Lower left, ovary treated 48 h with 10 µM Dox and 25 µM CHK2 inhibitor. 360° rotation, increments set to 1°.
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Tuppi, M., Kehrloesser, S., Coutandin, D.W. et al. Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63. Nat Struct Mol Biol 25, 261–269 (2018). https://doi.org/10.1038/s41594-018-0035-7
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