Mutations in p53 and attendant apoptotic pathways impair tumor cell responses to radiation and chemotherapy in many human malignancies. Combining genetics, functional genomics and proteomics in mammalian cells and zebrafish embryos these past 6 years, we identified a novel apoptotic pathway that bypasses p53-dependent pathways altogether via activation of the PIDDosome (PIDD-RAIDD- caspase-2) complex (Sidi et al., Cell 2008; Ando et al., Mol Cell 2012; Thompson et al., Mol Cell 2015; Ando et al., J Cell Biol 2017). Unlike the mitochondrial apoptosome (cytc-Apaf1-caspase-9) and death receptor complex (FAS-FADD-caspase-8), the PIDDosome does not require p53 for activation or function. PIDDosome assembly can be activated by inhibiting its negative regulator, Chk1 kinase. As such, Chk1 inhibitors restore radiosensitivity in p53 mutant zebrafish embryos, MEF, and human cancer cell lines. The PIDDosome is also responsive to DNA damaging chemotherapies such as topoisomerase inhibitors. Altogether, the PIDDosome pathway defines both a novel apoptotic axis and a promising targeted strategy for overcoming treatment resistance in cancer. However, our molecular understanding of the PIDDosome remains very limited. To expand our knowledge of the pathway and identify novel diagnostic tools and drug targets therein, this proposal will focus on the mechanisms by which DNA damage triggers PIDDosome assembly in vertebrate cells. Thus far, we have shown that DNA damage triggers PIDDosome formation via (i) ATM/ATR-mediated phosphorylation of PIDD, which enables RAIDD recruitment to the platform (Mol Cell 2012); and (ii) the binding of PIDD to nucleophosmin (NPM1), which provides a scaffold for PIDDosome assembly (JCB 2017).
In Aim 1, we will elucidate the mechanism by which a newly identified PIDD interactor, the DNA repair protein FANCI, recruits PIDD to DNA crosslinks and enables its phosphorylation by ATM at these lesions. Notably, these experiments may identify FANCI as the first biochemically described ?repair/death? switch in vertebrates.
In Aim 2, we will elucidate the mechanism by which NPM1 and two newly identified nucleolar PIDD-binding proteins, NOLC1 and NCL, coordinately orchestrate PIDDosome formation in response to IR. These experiments may ultimately outline the major apoptotic branch in the nucleolar DNA damage response. Finally, using xenograft models of intrinsic tumor radioresistance (Liu et al., Nat Cell Biol, accepted in principle), we will assess for the first time the potential of PIDDosome targeting as a strategy to overcome radioresistance in TP53 mutant cancers. Altogether, these studies integrate the PIDDosome in the cellular responses to DNA repair failure, replication stress and nucleolar stress. Our proposal is thus ideally positioned to reveal the role of the PIDDosome in cancer etiology, one of the most hotly debated questions in the field of apoptosis.

Public Health Relevance

The PIDDosome?PIDD-RAIDD-caspase-2 complex?defines a novel apoptotic pathway that does not require p53 for activation or function in response to DNA damage. As such, the pathway might restore treatment sensitivity in the many human tumors that evade radiation or chemotherapy via mutation of TP53. The systematic molecular dissection of the PIDDosome pathway will elucidate its role(s) in the DNA damage response and tumor suppression, and identify druggable targets and diagnostic tools for triggering PIDDosome signaling in TP53 mutant tumors.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Johnson, Ronald L
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Icahn School of Medicine at Mount Sinai
Internal Medicine/Medicine
Schools of Medicine
New York
United States
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Ando, Kiyohiro; Parsons, Melissa J; Shah, Richa B et al. (2017) NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus. J Cell Biol 216:1795-1810
Sidi, Samuel; Bouchier-Hayes, Lisa (2017) Direct pro-apoptotic role for NPM1 as a regulator of PIDDosome formation. Mol Cell Oncol 4:e1348325
Bouchier-Hayes, Lisa; Sidi, Samuel (2017) The nucleolus: A new home for the PIDDosome. Cell Cycle 16:1562-1563
Thompson, Ruth; Shah, Richa B; Liu, Peter H et al. (2015) An Inhibitor of PIDDosome Formation. Mol Cell 58:767-79