Sapere Aude project: Deciphering the Signaling Networks of PARP Inhibitors by Quantitative Mass Spectrometry – University of Copenhagen

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In June 2014 Michael L. Nielsen was awarded the prestigious Sapere Aude grant from The Danish Council for Independent Research (Research Manager) for his pioneering research project, focusing on deciphering the signaling networks of PARP inhibitors by quantitative mass spectrometry.

Abstract

Mammalian cells are constantly exposed to genotoxic stress arising from various intracellular and extracellular sources, and cells have therefore developed sophisticated mechanisms to detect and deal with cellular damage1,2. PTMs significantly contribute to the regulation of these processes, and my research group at the Center for Protein Research (CPR) has actively been engaged in developing proteomics technologies for comprehensive analysis of PTM patterns in human cells3,4. Poly(ADP-ribosyl)ation (PARylation), which is catalyzed by several members of the PARP protein family5, is a PTM that plays a pivotal role in the cellular stress response. In the presence of DNA damage the catalytic activity of PARP1 rapidly increases 10- to 500-fold over that of normal cells leading to the synthesis of poly(ADP-ribose) (PAR) chains on acceptor proteins within only a few minutes6. Despite that PARylation has been known for 50 years, until recently, surprisingly little was known about the molecular protein targets of the modification, mainly due to the lack of ‘unbiased’ technologies for detecting PARP substrates on a global scale7.

Since DNA repair defects often are associated with cancer, PARylation has received much attention as a potential target in cancer therapy. With the discovery that PARP inhibitors are toxic in certain cancer cell lines and human tumors, PARP inhibitors showed promising results in early clinical trials within two therapeutic cancer applications: (i) as chemo/radio-potentiator and (ii) as a stand-alone therapy for tumor types that are already deficient in certain types of DNA repair mechanisms6. While substantial progress has been made over recent years in the development of applicable, small molecule PARP inhibitors that show only little side effects (Table 1)8,9, the molecular details surrounding PARP inhibitor treatment still remain elusive 6. In particular, current knowledge on the intrinsic protein targets and amino acids affected by a specific PARP inhibitor treatment is lacking. Moreover, pivotal down-stream effects of PARP inhibitors on other DNA damage-related PTMs, such as lysine ubiquitylation, have only recently started to emerge10. Considering that the molecular actions of PARP inhibitors are likely to be pleiotropic and extend beyond DNA repair, the molecular mechanisms of PARP inhibitors, their effect on DNA repair and other biological processes, would be pertinent to decipher. This is particularly relevant with regard to long-term administration of PARP inhibitors that can cause changes in gene expression, or PTM pattern which may have lasting impacts on the cell and organism. Moreover, a thorough understanding of the molecular mechanisms underlying clinical resistance developed to PARP inhibitor treatment is urgently needed in order to design better therapeutic strategies11,12. This proposal aims at establishing advanced proteomics technologies based upon high-resolution mass spectrometry for unbiased and proteome-wide characterization of the molecular functions of PARP inhibitors. Collectively, such comprehensive studies will significantly improve our understanding of the role that PARylation and PARP inhibitors play in human cells.

References

  1. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Molecular cell 40, 179-204, (2010).
  2. Huen, M. S. & Chen, J. The DNA damage response pathways: at the crossroad of protein modifications. Cell Res 18, 8-16, (2008).
  3. Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol Cell Proteomics 10, M110 003590, (2011).
  4. Wagner, S. A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10, M111 013284, (2011).
  5. Hassa, P. O. & Hottiger, M. O. The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front Biosci 13, 3046-3082, (2008).
  6. Rouleau, M., Patel, A., Hendzel, M. J., Kaufmann, S. H. & Poirier, G. G. PARP inhibition: PARP1 and beyond. Nat Rev Cancer 10, 293-301, (2010).
  7. Jungmichel, S. et al. Proteome-wide identification of poly(ADP-Ribosyl)ation targets in different genotoxic stress responses. Molecular cell 52, 272-285, (2013).
  8. Banerjee, S., Kaye, S. B. & Ashworth, A. Making the best of PARP inhibitors in ovarian cancer. Nat Rev Clin Oncol 7, 508-519, (2010).
  9. Chalmers, A. J., Lakshman, M., Chan, N. & Bristow, R. G. Poly(ADP-ribose) polymerase inhibition as a model for synthetic lethality in developing radiation oncology targets. Semin Radiat Oncol 20, 274-281, (2010).
  10. Altmeyer, M. et al. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling. Molecular cell, (2013).
  11. Montoni, A., Robu, M., Pouliot, E. & Shah, G. M. Resistance to PARP-Inhibitors in Cancer Therapy. Frontiers in pharmacology 4, 18, (2013).
  12. Turner, N. C. & Ashworth, A. Biomarkers of PARP inhibitor sensitivity. Breast cancer research and treatment 127, 283-286, (2011).