© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 3779-3787 doi:10.1242/jcs.203448

REVIEW The resurrection of the PIDDosome – emerging roles in the DNA-damage response and centrosome surveillance Valentina Sladky1,*, Fabian Schuler1,*, Luca L. Fava1,2 and Andreas Villunger1,‡

ABSTRACT cellular physiology and aim to reconcile some of the remaining The PIDDosome is often used as the alias for a multi-protein complex controversy that surrounds the PIDDosome, first described as a that includes the p53-induced death domain protein 1 (PIDD1), the mediator of cell death in the DNA-damage response, containing bipartite linker protein CRADD (also known as RAIDD) and the pro- PIDD1, CASP2- and RIPK1-domain-containing adaptor with death form of an endopeptidase belonging to the caspase family, i.e. domain (CRADD, hereafter referred to as RAIDD) and CASP2 caspase-2. Yet, PIDD1 variants can also interact with a number of (Bock et al., 2012; Janssens and Tinel, 2012). other proteins that include RIPK1 (also known as RIP1) and IKBKG (also known as NEMO), PCNA and RFC5, as well as nucleolar The discovery of PIDD1 components such as NPM1 or NCL. This promiscuity in protein The official nomenclature for PIDD is now PIDD1 (p53-induced binding is facilitated mainly by autoprocessing of the full-length death domain protein 1). This was deemed necessary to avoid protein into various fragments that contain different structural confusion with primary immune deficiency disorders, often domains. As a result, multiple responses can be mediated by abbreviated the same way in the literature. Please note that there protein complexes that contain a PIDD1 domain. This suggests that are no reported PIDD1 orthologues in non-vertebrates, neither have PIDD1 acts as an integrator for multiple types of stress that need PIDD1 paralogues been found in vertebrates. PIDD1 was originally instant attention. Examples are various types of DNA lesion but also also known as leucine-rich repeat and death-domain-containing the presence of extra centrosomes that can foster aneuploidy and, protein (LRDD) and had been independently described by two ultimately, promote DNA damage. Here, we review the role of PIDD1 groups in the year 2000 (Telliez et al., 2000; Lin et al., 2000). In a in response to DNA damage and also highlight novel functions of bioinformatics screen for proteins containing a death domain PIDD1, such as in centrosome surveillance and scheduled (Box 1) similar to the one found in human receptor-interacting polyploidisation as part of a cellular differentiation program during serine/threonine kinase 1 (RIPK1, hereafter referred to as RIP1), organogenesis. Telliez et al. identified a protein and named it, according to its structural features, LRDD (Telliez et al., 2000). The characterisation KEY WORDS: Apoptosis, Caspase-2, Centrosomes, PIDD1, p53 of its sequence revealed leucine-rich repeats (LRRs) at the N- terminus, ZU5 domains (i.e. domains present in ZO-1 and Unc5- Introduction like netrin receptors) in the intermediate region, as well as a death The PIDDosome was described in 2004 by Antoine Tinel and the domain (DD) at the C-terminal end (Fig. 1A-C). Another structural late Jürg Tschopp, who spearheaded research in the field of cell domain called uncharacterised protein domain in UNC5, PIDD and death and inflammation for many years. Their initial findings ankyrins (UPA) was later defined between the ZU5 and the DD provided evidence for a long-sought function of a highly conserved (Wang et al., 2009). Moreover, the authors observed evidence for member of the caspase family caspase-2 (CASP2) as a cell death processing of PIDD1 as they detected truncated forms of the protein effector in the DNA-damage response (Tinel and Tschopp, 2004). in mammalian cells when overexpressing LRDD cDNA. Based on Yet, after a short period of excitement, interest in the PIDDosome as structural features, they speculated that LRDD functions as an a regulator of CASP2 ceased. This was mostly because of the lack of adapter for small G-proteins that had been shown to interact with phenotypes in mice depleted of individual PIDDosome components LRR sequences (Telliez et al., 2000). (Berube et al., 2005; Kim et al., 2009; Manzl et al., 2009; O’Reilly Lin and colleagues identified PIDD1 as a direct transcriptional et al., 2002); further, CASP2 could still become activated in the target of p53 by differential display analysis in an erythroleukemia absence of p53-induced death domain protein 1 (PIDD1), e.g. in cell cell line (Lin et al., 2000). Consistently, the sequence of the Pidd1 extracts in vitro, but also in dying neurons (Manzl et al., 2009; Ribe gene locus contained a non-canonical p53-responsive element in the et al., 2012). As the biology of CASP2 has been extensively Pidd1 promoter and upon γ-irradiation of mouse embryonic reviewed elsewhere (Bouchier-Hayes and Green, 2012; Fava et al., fibroblasts its mRNA was induced at the transcriptional level in a 2012; Puccini et al., 2013), it will not be the focus of this review. p53-dependent manner to an extent similar to that of cyclin- Here, we aim to give an overview on the biology of PIDD1. We dependent kinase inhibitor 1 (p21, officially known as CDKN1A). discuss potential roles in DNA damage, inflammation and normal Similar findings were made in MCF7 breast cancer and AML-4 leukaemia cells (Lin et al., 2000). Also, overexpression of PIDD1 suppressed cell growth by inducing apoptosis in p53-deficient cells, 1Division of Developmental Immunology, Biocenter, Medical University of and this effect was reversed by PIDD1 knockdown; PIDD1 was, Innsbruck, Innrain 80, 6020 Innsbruck, Austria. 2Center for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Povo, Italy. thus, assumed to be an essential component of the apoptotic arm of *These authors contributed equally to this work p53 (Lin et al., 2000). Around the same time, alternative cell death regulators that are induced by p53, such as phorbol-12-myristate- ‡Author for correspondence ([email protected]) 13-acetate-induced protein 1 (PMAIP1, hereafter referred to as

A.V., 0000-0001-8259-4153 NOXA) (Oda et al., 2000) and Bcl-2-binding component 3 (BBC3, Journal of Cell Science

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difficult to scrutinize which PIDD1 isoforms are expressed in a Box 1. Death domain proteins given cell type at protein level. Proteins that contain a death domain (DD), such as PIDD1, are PIDD1 autoprocessing requires binding of the chaperone heat characterised by a structural motif that contains six α-helical bundles shock protein 90 (Hsp90) and the co-chaperone p23 to facilitate the that make up a so-called ‘death fold’. This tertiary structure is also found optimal conformation needed for self-cleavage (Table 1). Hsp90 in other proteins that harbour either a caspase-recruitment domain interacts directly with FL-PIDD1 and recruits p23, a HSP90 co- (CARD), a death effector domain (DED), a pyrin domain (PYD) or, sometimes, a combination of such motifs (DD/CARD; DD/DED; PYRIN/ chaperone (Weaver et al., 2000; Tinel et al., 2011). Yet, another heat CARD), which links different death fold proteins with each other. Death shock protein, Hsp70, binds FL-PIDD1 as well as PIDD-N and folds generally allow for homotypic protein−protein interactions (DD/DD; PIDD-C; however, the role of this interaction remains to be CARD/CARD) that foster assembly of large multi-protein signalling investigated. Alongside autoproteolysis, the stability and function of complexes. Examples are the apoptotic protease-activating factor 1 PIDD1 also depends on Hsp90, which indicates an essential role for − (APAF1) caspase-9-containing apoptosome (i.e. a large quaternary chaperones in the regulation of PIDD1 self-processing and its protein structure that is formed during apoptosis), the caspase-8- containing death-inducing signalling complex (DISC) that comprises protein abundance (Table 1). Inhibition of Hsp90 allows rapid members of the tumour necrosis factor receptor (TNFR) superfamily degradation of PIDD1 by the E3 ubiquitin-protein ligase CHIP (also (Langlais et al., 2015), or different inflammasomes (i.e. oligomers known as STUB1), another co-chaperone that appears to comprising CASP1, PYCARD, NRLPs and sometimes CASP5), that preferentially ubiquitylate PIDD-C over PIDD-CC (Tinel et al., control activation of caspase-1 (Lamkanfi and Dixit, 2014). Common to 2011). CHIP directly interacts with PIDD1, but also with Hsp70, all these complexes is that they are engaged in response to different which might explain the role of Hsp70 in the regulation of PIDD1. developmental or environmental cues to control cell death and inflammatory responses, a feature conserved from invertebrates to Although the PIDDosome can form upon a temperature shift in vitro mammals. and dissociation of HSP90 is needed for its formation, the initial binding appears to be required for PIDD1 function: disruption of the interaction of Hsp90 with PIDD1 impairs its autoprocessing and binding to different effector proteins (Tinel et al., 2011). Yet, it is hereafter referred to as PUMA) (Han et al., 2001; Nakano and tempting to speculate that these effects are secondary to impaired Vousden, 2001; Yu et al., 2001), BH3-only members of the B-cell PIDD1 autoprocessing when Hsp90 is absent. Given the complex CLL/Lymphoma 2 (BCL2) family, were also described. Notably, regulation of the autoprocessing mechanism and the stability of subsequent loss-of-function analyses in mice confirmed roles for PIDD1, we anticipate that the stoichiometry and localisation of these two BH3-only proteins in the regulation of p53-induced cell protein complexes that contain PIDD1 domains are tightly regulated death; however, they failed to provide equally compelling evidence in order to elicit the desired biological response. for a role for PIDD1 in this process (Kim et al., 2009; Manzl et al., 2009; Shibue et al., 2003; Villunger et al., 2003). Certainly, a basal PIDD1-containing multiprotein complexes expression of PIDD1 was detectable in p53-deficient HCT116 and PIDD-CC nucleates a complex with the dual adapter RAIDD that is HEK-293 cells that express the large T antigen, which supported the needed for the recruitment and activation of CASP2 and, notion that PIDD1 also has roles outside the canonical p53 response potentially, cell death initiation. This complex is commonly to DNA damage (Cuenin et al., 2008; Tinel et al., 2007). referred to as the PIDDosome. We propose the term ‘Caspase-2 −PIDDosome’ for historic reasons, complex-I, to discriminate it Regulation of PIDD1 autoprocessing from other PIDD1-containing complexes discussed below (Fig. 1C, In humans, PIDD1 mRNA transcript variant 1 gives rise to a full- Table 1). In contrast, signalling that involves PIDD-C has been length (FL)-PIDD1 protein that is 910 aa in length (∼100 kDa) and associated with NF-κB activation and cell survival (Fig. 2B). DNA can be processed into three fragments (Box 2): the N-terminal damage can cause translocation of PIDD-C to the nucleus, where it 48 kDa fragment PIDD-N, and the two C-terminal fragments PIDD- forms a complex with RIP1 and inhibitor of NF-kappa-B kinase C and PIDD-CC that have a molecular mass of 51 kDa and 37 kDa, subunit gamma (NEMO, officially known as IKBKG), the ‘NEMO- respectively (Fig. 1A,B). Cleavage occurs at S446 and S588 by an PIDDosome’ (Janssens et al., 2005; Tinel et al., 2007). Subsequent autoproteolytic process that is similar to the mechanism described sumoylation, phosphorylation and ubiquitylation of NEMO leads to for self-cleaving protein segments, such as inteins, or for other its export and to activation of NF-κB signalling in the cytoplasm, proteins, such as nucleoporin Nup98 (Hodel et al., 2002). These which is considered an anti-apoptotic signal (Janssens et al., 2005; proteins contain a conserved HSF tripeptide motif that enables a Tinel et al., 2007). Although no protein complex that contains hydrophilic attack of the hydroxyl-group within the serine residue PIDD-N has been described, it might be a negative regulator of on the preceding peptide bond, converting it into an ester bond that PIDD-C, as its overexpression appears to dampen NF-κB activation is vulnerable to breakage through a second nucleophile (Hodel (Janssens et al., 2005; Tinel et al., 2011). et al., 2002; Mills et al., 2014; Tinel et al., 2007). The processing of Of note, these studies nicely document a role for PIDD1 in NF- FL-PIDD1 into either PIDD-C or PIDD-CC appears to be a κB activation that is induced by DNA damage; the evidence that constitutive event and, as a result, FL-PIDD1 is barely detectable PIDD1 activation, indeed, promotes cell survival under these even upon induction of p53 (Tinel et al., 2011, 2007). Notably, conditions is sparse (Bock et al., 2012). Whereas we were able to PIDD-CC can be generated by autoproteolysis of FL-PIDD1(Tinel confirm a deficit in NF-κB activation in response to DNA damage in et al., 2011, 2007) or PIDD-C (our unpublished observations). Yet, the absence of PIDD1, comparable to that caused by loss of RIP1 or the fact that accumulation of endogenous PIDD-C in response to PARP1, we were unable to document a role for PIDD1 in cell DNA damage precedes the detection of PIDD-CC is consistent with survival under the conditions tested (Bock et al., 2013). Primary PIDD-C being the most-prominent source for PIDD-CC (Tinel and cells derived from Pidd1−/− mice showed normal cell death Tschopp, 2004: Tinel et al., 2007). Furthermore, alternative splicing responses, similar to those we noticed upon lack of Raidd or and protein autoprocessing may act redundantly to generate PIDD1 Casp2 (Manzl et al., 2009). Furthermore, the DNA-damage protein variants (Box 2). However, with the current tools it is repair potential of mouse hematopoietic stem cells exposed to Journal of Cell Science

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ABPIDD1 mRNA transcript variants PIDD1 protein autoprocessing products Autoprocessing sites

100 kDa Variant 1 LRR ZU5 ZU5 UPA DD FL-PIDD1 LRR ZU5 ZU5 UPA DD 1 S446 S588 910 S446 S588

Variant 2 LRR ZU5 ZU5 UPA DD PIDD-N LRR ZU5 48 kDa 147 S446 Δ 580-590 910 F445

Variant 3 LRR ZU5 ZU5 UPA DD PIDD-C ZU5 UPA DD 51 kDa 1 S446 S588 Δ 705-721 910 S446 S588

Variant 4 ZU5 ZU5 UPA DD PIDD-CC UPA DD 37 kDa 314 S446 S588 910 S588

Variant 5 ZU5 ZU5 314 S446 584

C Caspase-2−PIDDosome NEMO-PIDDosome PCNA-PIDDosome

PIDD-CC DD ZU5 PIDD-C DD ZU5 PIDD-C DD

CARD RAIDD DD KD RIP1 DD N-term PCNA C-term

CARD Caspase-2 NEMO

Fig. 1. PIDD1 isoforms, autoprocessing products and protein complexes. (A) mRNA transcript variants reported to encode for different isoforms of PIDD1. Transcript variant 1 was first characterised by Lin et al. (2000). Alternative transcription start-site usage or splicing can create alternative transcript variant 2 and 3, respectively. Isoform 4 is also generated by splicing and translation is initiated in exon 5. Isoform 5 has only been detected in silico (Cuenin et al., 2008). Transcript variant 1 encodes seven leucine-rich repeats (LRR; dark blue), two ZU5 domains (present in ZO-1 and Unc5-like netrin receptors; purple), an UPA domain (conserved in UNC5, PIDD1 and ankyrins; blue), as well as the C-terminal death domain (DD; orange). (B) Autoprocessing of full-length (FL)-PIDD1 at positions S446 and S588 can give rise to three protein fragments of indicated length that contain different structural motifs relevant for protein-protein interaction and complex formation, as described above. (C) Three different protein complexes that contain PIDD-1-derived fragments in combination with different interacting proteins have been reported. (CARD, caspase activation and recruitment domain; KD, kinase domain).

γ-irradiation, as read out by colony formation, appeared unaffected different locations and amounts. It is also not entirely clear whether by loss of PIDD. Yet, we observed a clear deficit in cytokine autoprocessing of PIDD-CC is prevented once PIDD-C enters the production in response to DNA damage in bone marrow-derived nucleus or upon its binding to RIP1. macrophages and mouse embryonic fibroblasts (MEFs) of these Nuclear localisation of PIDD-C is needed for NF-κB activation; mice (Bock et al., 2013). Also, production of tumour necrosis factor but PIDD-CC has also been detected in the nucleolus (Fig. 2C), an (TNF) in the gastrointestinal tract after γ-irradiation was found to be organelle with various functions that range from ribosome reduced in PIDD-deficient animals. Moreover, survival and tumour biogenesis to DNA repair (Ogawa and Baserga, 2017). A yeast- transformation potential of these mice were unaffected, which two-hybrid screen using a mouse thymoma cDNA library and suggested that the role of the ‘NEMO-PIDDosome’ is to promote PIDD1 isoform 2 as a bait identified nucleolin (NCL) as a potential inflammation, rather than to control cell survival or DNA repair binding partner for PIDD1 (Table 1). The subsequent analysis (Bock et al., 2013). documented that GFP-tagged PIDD-C is constitutively targeted to Formation of the Caspase–2–PIDDosome (Fig. 1C) involves the nucleolus (Pick et al., 2006). Interestingly, whereas the interaction with the adapter molecule RAIDD, which contains a DD cytoplasmic pool of this fusion protein was rapidly degraded after as well as a caspase-recruitment domain (CARD) (Duan and Dixit, UV-treatment, potentially through CHIP-mediated ubiquitylation, 1997). RAIDD and PIDD-CC associate through their DD to form a the nucleolar fraction of the protein remained stable, which indicates high molecular weight complex, whereas the N-terminal CARD in that nucleolar PIDD-C is protected from degradation. As PIDD1 RAIDD acts as a docking site for the pro-form of CASP2. protein isoform 2 cannot be autoprocessed into PIDD-CC, it Interaction with RAIDD is restricted to PIDD-CC, which is remains unclear whether the latter can also localize to or might even generated from the human PIDD1 transcript variant 1, because the be generated inside the nucleoli (Pick et al., 2006). Recent work, minor deletion in transcript variant 3 appears to be sufficient to however, documents the localisation of PIDD-CC in the nucleolus abrogate RAIDD binding (Tinel et al., 2007; Tinel and Tschopp, (Ando et al., 2017). Here, mass spectrometry (MS) revealed the 2004) (Fig. 1A,B). Whereas RIP1 and NEMO are immediately association of PIDD1 with nucleophosmin 1 (NPM1), a key recruited to PIDD1, the interaction with RAIDD occurs later. This is nucleolar protein and established tumour suppressor that is most in line with the observation that PIDD-C and PIDD-CC are frequently mutated in acute myeloid leukaemia (Heath et al., 2017). generated in a sequential manner (Janssens et al., 2005; Tinel et al., Upon DNA damage, and most prominently in response to inhibition 2007). Yet, given the overlapping kinetics with which the different of topoisomerase I, NPM1 binds to the LRRs in PIDD-N as well complexes are formed, we speculate that both types, the Caspase–2– as to PIDD-CC; the latter directs activation of caspase-2 in the and NEMO-PIDDosome, can be present in the cell at a given time in nucleolus in a PIDD1-dependent manner, thereby promoting cell Journal of Cell Science

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Table 1. Reported protein-protein interactions involving FL-PIDD1 or its Box 2. PIDD1 mRNA transcript variants and their autoprocessing products expression patterns PIDD1- Reported function(#) or Method The mouse and human PIDD1 gene (Pidd1 and PIDD1, respectively) is PIDD1 fragment proposed role(*) with used for Reference encoded in 16 exons located on chromosome 7 and 11, respectively. interactor interacting PIDD1 detection** first reporting Five potential human PIDD1 mRNA transcript variants have been FADD n.d. Cell death# WB Telliez et al., reported (Fig. 1A). PIDD1 mRNA transcript variant 1 encodes a protein of 2000 910 amino acids (aa) that contains seven LRRs, two ZU5 domains, the MADD n.d. MAPK signalling# WB UPA domain and the C-terminal DD (Lin et al., 2000). Transcript variant 2 RAIDD PIDD-CC Cell death* WB, MS Tinel et al., lacks the genomic information that encodes the first 147 N-terminal aa Cell cycle arrest* 2004 (Telliez et al., 2000), which includes one LRR, as well as 11 aa at position 580 that abrogate the second auto-processing site. Therefore, the RIPK1 PIDD-C NF-κB signalling* WB Janssens derived PIDD1 protein can only generate FL-PIDD1 and PIDD-C. In et al., 2005 contrast, mRNA transcript variant 3 encodes a protein that is able to fully NCL PIDD-C Ribosome biogenesis# Y2H Pick et al., autoprocess, but the PIDD-CC version it generates lacks 17 aa at 2006 position 705 and has lost the ability to interact with RAIDD (Cuenin et al., HSP90 FL-PIDD1 Autoprocessing* WB, Y2H Tinel et al., 2008). Both mRNA variant 4 and 5 lack the N-terminal LRRs (314 aa) 2011 and, in addition, variant 5 does not encode a DD owing to a frame shift HSP70 FL-PIDD1, Autoprocessing* WB, Y2H that is caused by alternative splicing (Fig. 1A). Hence, PIDD1 isoform 5 PIDD-N, only consists of the two ZU5 domains. Currently, it is unclear whether PIDD-C these transcript variants are regularly generated in cells through differential splicing or the usage of alternative transcription start sites p23 FL-PIDD1 Autoprocessing* WB, Y2H and, if this is the case, whether they are translated into proteins (Cuenin CHIP FL-PIDD1, PIDD1 ubiquitylation* WB, Y2H et al., 2008; Huang et al., 2011). Anti-PIDD1 antibodies are of limited PIDD-C quality and fail to discriminate between the isoforms, as they are HOP n.d. Co-chaperone# Y2H generated against the DD within PIDD1. Thus, they recognize FL-PIDD1 DNAJ n.d. HSP70 co-chaperone# Y2H and all PIDD1-derived fragments arising from it, but not PIDD-N (see BAG1 n.d. HSP70 co-chaperone# Y2H main text). Yet, western analysis using several cell lines revealed a PCNA FL-PIDD1, Translesion synthesis* WB, MS Logette et al., minimum of two - sometimes even more - protein bands that represent PIDD-C 2011 different versions of PIDD-C. In principle, these can be generated from RCF4 PIDD-C Translesion synthesis* WB, MS PIDD1-encoding transcript variants 1, 2 or 3, potentially even variant 4 RCF5 PIDD-C Translesion synthesis* WB, MS (Cuenin et al., 2008). Interestingly, under these conditions only a single Polδ n.d. Translesion synthesis* MS band representing PIDD-CC is usually detected, in line with the finding Tel2 n.d. DDR# MS that the second cleavage event − the one that generates PIDD-CC – homolog occurs at a location that is distal to all splicing events that affect MCM7 n.d. DNA replication# MS composition of the mRNAs for PIDD1 in a cell. Endogenous FL-PIDD1 RPB2 n.d. Transcription# MS protein is difficult to detect and the size-resolution is too low to define the RPAP3 n.d. Transcription# MS presence of one or more FL-PIDD1 isoforms derived from transcript PP6 n.d. Translation# MS variants 1−3 (Cuenin et al., 2008; Logette et al., 2011; Tinel et al., 2007). DDB1 n.d. DNA repair# MS In mice and rats, only one transcript variant is found, and mouse PIDD1, FANCI n.d. DNA repair# MS 915 aa in size, shows 81% identity with its human counterpart (Cuenin DNA-PKs n.d. DNA repair# MS et al., 2008). XPD n.d. DNA repair# MS XPG n.d. DNA repair# MS ANP32B n.d. Histone modification# MS TRIM32 n.d. Ubiquitylation# MS # death (Ando et al., 2017). Currently, it remains unclear what drives SENP3 n.d. Sumoylation MS NUP93 n.d. Nuclear pore protein# MS the interaction of PIDD1 and NPM1 in response to DNA damage PRDX1 n.d. REDOX regulation# MS and whether or how, PIDD-N is involved in PIDDosome assembly, AIF n.d. NADH oxido- MS as it has never been reported to shuttle to the nucleus. Remarkably, reductase# NPM1 can also localise to the cytoplasm (Maggi et al., 2008), HAX1 n.d. Actin polymerisation# MS which raises the possibility that it there interacts with FL-PIDD1 and NPM1 PIDD-CC, Ribosome biogenesis# WB, MS Ando et al., shuttles it to the nucleolus where autoprocessing into PIDD-CC is PIDD-N Cell death* 2017 completed. The study by Maggi and colleagues also raises the **Methods used to identify/confirm interactions: WB, western blot; question whether the mutated form of NPM1 (NPM1c+), which is MS, mass-spectrometry; Y2H, yeast-two-hybrid screen. preferentially found in the cytoplasm (Heath et al., 2017), promotes n.d., not defined. disease, in part, by preventing PIDD1 from entering the nucleolus, thereby reducing proficiency of CASP2 activation. needed to load DNA polymerases for replication (Logette et al., Independently of the signalling pathways described above, 2011). Moreover, it exerts critical roles in DNA repair. Importantly, PIDD1 participates in translesion DNA synthesis (TLS), i.e. DNA the interactions between PIDD1, PCNA and RFC5 are mediated by extension across a lesion, in response to UV-radiation (Fig. 2D). In their ZU5 domains; hence, the complex can only be formed by FL- the nucleus, PIDD1 was reported to form yet another complex with PIDD1, or − given the low abundance of FL-PIDD1 and the key components of the replication machinery, proliferating cell preferred nuclear localisation of its first autoprocessing product − nuclear antigen (PCNA), replication factor C subunit 5 (RFC5) and by PIDD-C (Logette et al., 2011). A role for PIDD1 isoform 4 or RFC4 referred to as PCNA-PIDDosome (Table 1). These proteins isoform 5 (only the latter encodes the two ZU5 domains) (Fig. 1A) were identified by MS-analysis as binding partners of overexpressed remains unexplored. Yet, when PIDD1 binds the C-terminal part of PIDD1 (Logette et al., 2011). PCNA acts as a DNA-sliding clamp PCNA, p21 dissociates from PCNA and is degraded, which allows that requires the RFC for its positioning onto DNA and is itself PCNA mono-ubiquitylation of lysine 164 (Bruning and Shamoo, Journal of Cell Science

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A B

PIDD-C Extra centrosomes PIDD-CC RAIDD MDM2 PIDD-C PIDD-C ATM P RIP-1 RIP-1 NEMO NEMO Caspase-2 SUMO Ub Ub NEMO NEMO p53

DNA Cell cycle damage NF-κB NF-κB p53 p21 arrest Survival IκB Ub Ub Ub IκB

Nucleus Nucleus Cytoplasm Cytoplasm

C D

BubR1 CHK1 CHK1

P PIDD-CC ATM PIDD-CC RAIDD RAIDD Ub Caspase-2 UV Ub Caspase-2 Ub p21 Translesion p21 PIDD-C DNA synthesis NPM1 Apoptosis DNA Ub PIDD-CC damage PCNA PCNA RAIDD UbU Caspase-2 DNA Pol η Nucleolus DNA Pol η Ub

Nucleus Nucleus Cytoplasm Cytoplasm

Fig. 2. Cues for the formation of distinct PIDDosome signalling complexes. (A) Cytokinesis failure causes accumulation of extra centrosomes (blue). PIDD1 localises to the mature centriole within the centrosome that is characterised by appendage proteins (triangles). The presence of more than one mature centriole causes formation of the Caspase-2−PIDDosome and CASP2 activation. Subsequent MDM2 cleavage by CASP2 generates an N-terminal MDM2-fragment that binds to and stabilises p53. This results in p21-dependent cell cycle arrest. (B) In response to genotoxic stress that causes double-strand breaks, PIDD-C localises to the nucleus. There, it interacts with RIP-1 to form the NEMO−PIDDosome, thereby facilitating NEMO sumoylation that is, in turn, required for its phosphorylation by ATM and its subsequent mono-ubiquitylation. The latter acts as the nuclear export signal for NEMO. In the cytoplasm, mono-ubiquitylated NEMO targets IκB for proteasomal (grey barrel) degradation, which results in NF-κB activation and transcription of survival and inflammatory genes. (C) DNA damage triggers Caspase-2−PIDDosome formation in the nucleolus. This involves interaction with NPM1 to promote apoptosis. Similarly, ionising radiation allows ATM to phosphorylate PIDD-CC when CHK1 activity is suppressed; this, in turn, facilitates interaction with RAIDD and promotes CASP2-mediated cell death by so-far unknown effectors. BubR1 suppresses Caspase-2−PIDDosome assembly during mitosis in DNA-damaged cells, which delays initiation of cell death to the next G1 phase. (D) To prevent replicative arrest due to bulky DNA lesions, cells resort to translesion DNA synthesis (TLS), which depends on the PCNA-PIDDosome. Binding of PIDD-C to the homotrimeric PCNA in the nucleus causes dissociation and degradation of its inhibitor p21, which facilitates mono- ubiquitylation of each PCNA unit. This, in turn, enables recruitment of the low-stringency DNA polymerase η (DNA Pol η), which then commences TLS to avoid stalling of replication.

2004). This modification, in turn, facilitates chromatin association owing to an increased mutational load or the lack of survival signals of the low-fidelity DNA polymerase eta (Pol η), which then that depend on NF-κB remains unclear. Intriguingly, it has been performs TLS (Fig. 2D). In doing so, the cell overcomes unrepaired shown that NPM1c+ can delocalize Pol η to enhance its cytoplasmic DNA lesions in S-phase that are not resolved by nucleotide excision degradation (Ziv et al., 2014). Hence, it is tempting to speculate that repair and allows to replicate DNA in the presence of cyclobutan- interactions between PIDD1 and NPM1 are not only needed to pyrimidine-dimers (Logette et al., 2011). In this context, PIDD1 facilitate the activation of nucleolar CASP2, but also do affect TSL appears to act as a pro-survival factor in the ‘PCNA-PIDDosome’ efficiency by fine-tuning Pol η localisation and abundance. In (Fig. 1C). Consistently, cells of the human keratinocyte cell line summary, by forming different protein complexes, PIDD1 appears HaCaT that were depleted of PIDD1 through RNA interference critical to fine-tune a number of cellular responses that deal with the (RNAi) showed increased cell death upon UV-C treatment. consequences of DNA damage, including the maintenance of Moreover, skin from Pidd1-deficient mice that were exposed to replication capacity and inflammatory cytokine production. Yet, its UV-radiation showed higher numbers of apoptotic keratinocytes in role in another key response to DNA-damage, i.e. the induction of situ (Logette et al., 2011). Whether this increase in cell death was cell death, remains controversial. Journal of Cell Science

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The Caspase-2−PIDDosome as an initiator of cell death complex (MCC) that executes the spindle assembly checkpoint Motivated by the early observations made by Tinel and Tschopp, (SAC). The checkpoint is responsible for the inhibition of the many follow-up studies on PIDD1 aimed to position it as an initiator anaphase-promoting complex (APC/C), thereby preventing mitotic of CASP2-mediated cell death (Bock et al., 2012; Janssens and progression until all kinetochores have achieved proper microtubule Tinel, 2012). Interestingly, zebrafish embryos that lack p53 are attachment (Musacchio, 2015). Since small interfering RNA radio-resistant, similar to several human cancer cell lines that lack (siRNA) that targets BUBR1 triggers Caspase-2−PIDDosome functional p53. However, radiation sensitivity was restored when formation in response to IR alone, BUBR1 has been suggested to p53-deficient zebrafish embryos were treated with a morpholino that function either downstream or in parallel to CHK1 (Thompson et al., targets checkpoint kinase 1 (CHK1) or a chemical CHK1 inhibitor 2015). Of note, knockdown of BUBR1 phenocopied the effects that (CHK1i) (Sidi et al., 2008). Remarkably, this type of DNA-damage- are mediated by siRNA targeting CHK1, which leads to PIDDosome- induced cell death appears to be independent of CASP3, CASP8 or dependent apoptosis in response to IR. As siRNA against MAD2, CASP9, is not blocked by BCL2 overexpression − yet requires another key-component of the MCC, had no such effect on CASP2. This cell death pathway has, therefore, been dubbed PIDDosome assembly, a novel non-canonical function of BUBR1 ‘CHK1-suppressed’ (CS) pathway of cell death and was further outside the SAC has been proposed (Thompson et al., 2015). shown to depend on an ATM−ATR−CASP2 signalling axis (Sidi Intriguingly, phosphorylated PIDD1 colocalises with histone et al., 2008). However, the CS pathway does not depend on H2AX phosphorylated at Ser139 and ATM protein kinase canonical regulators of cell death, such as BCL2, PUMA, NOXA, phosphorylated at Ser1981 at DNA lesions in interphase, which p63, p73 or cytochrome c release. These findings suggest that positions it as a putative substrate of ATM, but was found at CHK1 prevents a cell death response that becomes activated upon kinetochores in early prophase and this localisation required mitotic entry in the presence of DNA damage or, equally possible, BUBR1 (Thompson et al., 2015). BUBR1 itself is recruited to after exit from erroneous mitosis (Sidi et al., 2008) (Fig. 2C). kinetochores to initiate the SAC; there, it competes with RAIDD to Importantly, this cell death pathway, which was nicely genetically bind to PIDD1, which has been suggested to avoid the induction of delineated in zebrafish embryos appeared to be conserved in human apoptosis during mitosis. The formation of the Caspase-2− cancer cells. However, it was impossible to recapitulate all features PIDDosome is, thus, only possible once the APC/C degrades of the CS pathway in mice (Manzl et al., 2013). Furthermore, it BUBR1 at the end of mitosis, an event that triggers formation of the remained unclear how ATM/ATR-signalling could activate CASP2. Caspase-2−PIDDosome for cell death in the following interphase In a follow-up study, the group of S. Sidi showed that ATM but, (Thompson et al., 2015). However, CASP2 is also phosphorylated somewhat surprisingly, not the CHK1 activator ATR binds to in the linker region at Ser340 through CDK1 in order to prevent its PIDD1 (Ando et al., 2012). It does so by engaging its LRR domains activation in mitosis (Andersen et al., 2009). Thus, BUBR1- and by phosphorylating PIDD1 at Thr788 in response to irradiation, mediated control of PIDDosome formation during mitosis appears most prominently when CHK1 function was blocked to serve here as an additional fail-safe mechanism. Hence, in the simultaneously (Ando et al., 2012). This binding of PIDD1 to absence of DNA damage, CASP2 might act as a sensor of the ATM resulted in CASP2-dependent cell death of HeLa cells. ATM- metabolic state in cells stuck in M-phase for extended periods of mediated phosphorylation of Thr788, which is located in the DD, time (Salazar-Roa and Malumbres, 2017). This idea finds support in was shown to be necessary and sufficient for PIDD1 to bind to observations that have been made in Xenopus oocytes that were RAIDD and to activate CASP2. At the same time, this prevents the arrested in meiosis II and experienced nutrient deprivation (Nutt binding of PIDD1 to RIP1, which would otherwise activate NEMO- et al., 2009). Such a role would be consistent with reports that claim sumoylation and NF-κB signalling (Ando et al., 2012). This that CASP2 can contribute to mitotic cell death of cells treated with suggests that ATM can convert PIDD1 from a survival factor to a anti-mitotic drugs (Ho et al., 2008). We propose that such a protein that promotes cell death. How CASP2 drives cell death in metabolic sensor function of CASP2, if it exists, does not require this setting remains to be defined. PIDD1 (Fig. 2) because its formation is actively prevented in M- It remains uncertain how selective this ATM-mediated phase (Thompson et al., 2015), and that p53-activation triggered in phosphorylation event is for the CS pathway. ATM appears to response to extended mitosis does not involve CASP2 (Fava et al., bind to PIDD1 also upon IR damage alone and the phosphorylation 2017). of Thr788 clearly affects the ability of cells to activate NF-κBin It remains unclear why the death of cells entering mitosis in the response to the topoisomerase IIa inhibitor etoposide, which presence of DNA damage should be postponed until the next induces DNA damage (Ando et al., 2012). This occurs in the interphase, unless the cell manages efficient DNA repair during absence of CHK1 inhibition, which suggests a role for PIDD1 in the mitotic traverse. However, DNA repair activity in mitosis appears to canonical DNA-damage response. Further, the need to force cells to be minimal and double-strand breaks are usually only marked for override the G2/M checkpoint complicates a clean epistatic analysis repair in the next interphase, whereas extensive DNA damage of events, as multiple additional signalling cascades are activated. activates the SAC (Heijink et al., 2013). Moreover, given the fact Moreover, only HeLa cells and SV40-immortalised MEFs were that canonical apoptosis can also be engaged by alternative routes in tested rigorously and both lack a functional p53 response. cells that lack p53, e.g. through the activation of PUMA or NOXA Regardless of this, IR − in combination with CHK1i treatment − by p73 (Flinterman et al., 2005; Melino et al., 2004), it seems was clearly more effective in driving phosphorylation of PIDD1 and unlikely to us that evolution has developed the PIDDosome solely to Caspase-2−PIDDosome assembly than the use of IR alone (Ando promote the death of p53-deficient cells in an unforeseeable et al., 2012), which suggests that entry into mitosis, or even mitotic artificial setting. The controversy was perpetuated by the fact traverse, is needed for efficient Caspase-2−PIDDosome activation that mouse genetics did not support a critical role for either and cell death. In line with this is the observation that the mitotic Caspase-2−PIDDosome component in DNA-damage-induced cell pseudo-kinase BUBR1 (also known as BUB1B) can prevent death; further, the CS pathway is poorly conserved (Kim et al., PIDDosome formation in mitotic cells (Thompson et al., 2015) 2009; Manzl et al., 2013, 2009). Evolutionary conservation was

(Fig. 2C). BUBR1 is a component of the mitotic checkpoint carefully evaluated in a range of primary haematopoietic cells, Journal of Cell Science

3784 REVIEW Journal of Cell Science (2017) 130, 3779-3787 doi:10.1242/jcs.203448 including stem cells, thymocytes, resting and mitogen-stimulated T number of mature mother centrioles that appears to be counted by and B cells, gastrointestinal epithelial cells, and primary as well as PIDD1, thereby employing a mechanism that still needs to be immortalised MEFs (Manzl et al., 2013). The latter were shown to discerned. Intriguingly, PIDD1 localises to the distal end of mature have a clear deficit in activating the Caspase-2−PIDDosome (Ando centrosomes in healthy cells (Fava et al., 2017). More than one mature et al., 2012; Thompson et al., 2015), but died similar to wild-type cells centriole with appendages can initiate Caspase-2−PIDDosome upon DNA damage (Manzl et al., 2013). Equally puzzling was the assembly, which promotes CASP2-mediated MDM2 cleavage, p53 phenomenon that all that seems to be required for PIDDosome accumulation and p21-mediated cell cycle arrest (Fig. 2A). All this assembly is exit from mitosis in the presence of DNA damage. Hence, occurs in the absence of appreciable cell death. Consistently, depletion we wondered: what might all these treatments have in common such of centrosomes abrogated the proficiency of the pathway upon that, eventually, the Caspase-2−PIDDosome is activated? cytokinesis failure, whereas the generation of extra centrosomes induced by overexpression of polo-like kinase 4 (PLK4) sufficed to Novel concepts for the activation and function of PIDD1 activate the pathway in the absence of cytokinesis failure (Fava et al., PIDD1, together with p21, has been shown to be upregulated in an 2017). Our findings, therefore, established the Caspase-2 Adeno-Cre inducible, KRASG12D-driven lung cancer mouse model −PIDDosome as the missing link that connects supernumerary after prolonged treatment with cisplatin that caused drug-resistance centrosomes to activation of p53. Intriguingly, anti-mitotic drugs, (Oliver et al., 2010). Interestingly, repeated long-term drug-treatment of DNA-damaging agents (Dodson et al., 2004; Sato et al., 2000), these mice failed to increase overall survival rates, despite intermittent impaired CHK1 function (Peddibhotla et al., 2009), inhibition of tumour regression. This was because the growth of more aggressive BubR1 (Oikawa et al., 2005) or inhibition of Aurora B kinase clones increased when mice did not receive any drugs. As a result these (Ditchfield et al., 2003) all have the strong potential to cause tumours presented with huge variations in chromosome copy numbers, cytokinesis failure or direct centrosome amplification. Hence, it seems indicative for high-grade aneuploidy (Oliver et al., 2010). Remarkably, highly likely that at least one component feeding into Caspase-2 when drug-resistant tumours were treated again with cisplatin, these −PIDDosome activation under all these different conditions is the aneuploid tumours showed a stronger induction of PIDD1, along with increase in centrosome number. Such an increase happens in a p21, compared to mice with euploid tumours that received drug- substantial fraction of cells exposed to these treatments, either in treatment for the very first time. Therefore, an enhanced up-regulation response to cytokinesis failure or centrosome amplification. of PIDD1 is linked to increased aneuploidy in KRASG12D-driven lung Given what has been discussed above, we propose that lagging cancer (Oliver et al., 2010). Similarly, cisplatin treatment induced up- chromosomes in aneuploid KRAS-driven lung tumours frequently regulation of PIDD1 in RAS-mutant but p53-proficient human non- result in failure of cytokinesis. This triggers centrosome accumulation, small-cell lung carcinoma and HCT116 colon cancer cells. Caspase-2−PIDDosome-dependent p53 stabilisation and p21 Remarkably, exogenous expression of PIDD1 resulted in induction induction and, in turn, may increase PIDD1 expression levels over of p21 and reduced growth rates, which was associated with an time because it is also a p53 target. The rise in PIDD1 expression is emerging drug resistance (Oliver et al., 2010, 2011). The reduced possibly owing to subsequent DNA damage in cells that fail to arrest growth and drug responses caused by PIDD1 overexpression were, properly in the presence of extra centrosomes and, again, activate p53 again, found to be abrogated in p53-null cells; this positions PIDD1 − either along the canonical DNA-damage response route or upon upstream of p53 in this setting (Oliver et al., 2010, 2011). Together, this delayed M-phase progression in response to problems of chromosome suggests that PIDD1 has an anti-proliferative and hence, potentially, alignment (Lambrus and Holland, 2017). This would secure p21- also a pro-survival function that is independent of NF-κB signalling, at checkpoint proficiency and the survival of these aneuploid cells. least in the presence of functional p53. Consistently, CASP2 deficiency facilitates tumour recurrence in this Oliver and colleagues also investigated the link between the fast cancer model after cisplatin treatment (Terry et al., 2015). In addition, recurrence after initial chemo therapy (‘platinum-resistance’) and reduced CASP2 expression that is linked to impaired function of the PIDD1, p21 and their common inducer of gene expression, p53. transcriptional regulator BCL9L contributes to aneuploidy tolerance They proposed a feed-forward loop in which, upon DNA damage, in colon cancer (López-García et al., 2017). This phenomenon, p53 induces PIDD1, in turn, contributing to p53 activation and potentially, also explains why p53-deficient KRAS tumours, which continued p21 expression that explains the reduced growth of cells are aneuploid early on and cannot trigger this response, show a relative overexpressing PIDD1. Importantly, this loop requires activation of survival benefit after cisplatin treatment that is not observed in mice caspase-2 that leads to selective cleavage of MDM2 at Asp367. The bearing p53-proficient tumours (Oliver et al., 2010). It remains to be N-terminal fragments of MDM2 that are generated by CASP2- seen whether such p53-deficient tumours are more prone to cell death mediated proteolysis lack the RING domain that is required for E3 when exposed to cisplatin or other drugs that promote G2/M failures, ligase activity. Hence, these fragments accumulate, bind to p53, and but it would fit nicely with observations made in p53-mutant zebrafish increase its stability and target gene transcription, which leads to or HeLa cells exposed to DNA damaging agents in combination p21-mediated cell cycle arrest (Oliver et al., 2011). with RNAi of CHK1i or BUBR1 (Thompson et al., 2015). Here, These findings, together with genetic data from mice that lack Caspase-2−PIDDosome assembly is most likely also triggered by PIDD1 (Kim et al., 2009; Manzl et al., 2013, 2009; Oliver et al., 2010) extra centrosomes in G1 cells that were forced to override the G2 and argue against a clear-cut pro-death role of PIDD1. How can these M checkpoints. However, in this situation the activation of CASP2 discrepancies be reconciled? Our own recent findings suggest that preferentially causes cell death as these cells cannot arrest (Thompson CASP2 activation that depends on PIDD1 does neither contribute to et al., 2015). This type of cell death does not seem to depend on cell death, nor to p53 activation induced by DNA damage (e.g. upon components of the canonical mitochondrial apoptosis pathway (Sidi treatment with doxorubicin) or prolonged mitotic arrest (e.g. upon et al., 2008). However, it also largely excludes a role for the BH3- treatment with taxol). Yet, p53 activation that is triggered by interacting domain death agonist (BID), a bona fide CASP2 substrate centrosome amplification – a phenomenon that can occur as a and potent apoptosis effector (Guo et al., 2002); thus, it will be very consequence of cytokinesis failure – does clearly depend on the interesting to find out how CASP2 actually kills these cells. Given its

Caspase-2−PIDDosome (Fava et al., 2017). Remarkably, it is the high homology with C. elegans Ced3−the one and only caspase in the Journal of Cell Science

3785 REVIEW Journal of Cell Science (2017) 130, 3779-3787 doi:10.1242/jcs.203448 worm−one is tempted to speculate that it acts as an initiator and Ando, K., Parsons, M. J., Shah, R. B., Charendoff, C. I., Paris, S. L., Liu, P. H., effector at the same time. If this were the case, it would raise the Fassio, S. R., Rohrman, B. A., Thompson, R., Oberst, A. et al. (2017). NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus. J. Cell Biol. question regarding potential CASP2 substrates that cause cell death 216, 1795-1810. −possibly in a fashion similar to that of gasdermin D, which is Berube, C., Boucher, L.-M., Ma, W., Wakeham, A., Salmena, L., Hakem, R., Yeh, activated by caspase-1 (Shi et al., 2017). W.-C., Mak, T. W. and Benchimol, S. (2005). Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD. All these observations led us to assign a potentially oncogenic Proc. Natl. Acad. Sci. USA 102, 14314-14320. function to PIDD1, as it might increase the selection pressure in Bock, F. J., Peintner, L., Tanzer, M., Manzl, C. and Villunger, A. (2012). P53- aneuploid, p53-proficient cells to overcome this anti-proliferative induced protein with a death domain (PIDD): master of puppets? Oncogene 31, 4733-4739. barrier. This possibility is supported by the delayed outgrowth of Bock, F. J., Krumschnabel, G., Manzl, C., Peintner, L., Tanzer, M. C., Hermann- −/− MYC-driven lymphomas in Pidd1 mice (Manzl et al., 2012), a Kleiter, N., Baier, G., Llacuna, L., Yelamos, J. and Villunger, A. (2013). Loss of − − phenomenon that for unclear reasons is not seen in Raidd / mice, PIDD limits NF-kappaB activation and cytokine production but not cell survival or (Peintner et al., 2015). Moreover, given the role of PIDD1 in TLS transformation after DNA damage. Cell Death Differ. 20, 546-557. Bouchier-Hayes, L. and Green, D. R. (2012). Caspase-2: the orphan caspase. Cell (Logette et al., 2011), it would be highly interesting to see whether Death Differ. 19, 51-57. mice lacking the PIDDosome show a delay in UV-driven skin cancer. Bruning, J. B. and Shamoo, Y. (2004). Structural and thermodynamic analysis of human PCNA with peptides derived from DNA polymerase-delta p66 subunit and flap endonuclease-1. Structure 12, 2209-2219. Conclusions and further directions Cuenin, S., Tinel, A., Janssens, S. and Tschopp, J. (2008). p53-induced protein For many years, PIDD1 and its binding partners were considered to with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB only have a role in the DNA-damage response, which severely and caspase-2 in response to genotoxic stress. Oncogene 27, 387-396. hampered the search for potential pleiotropic, physiological roles of Ditchfield, C., Johnson, V. L., Tighe, A., Ellston, R., Haworth, C., Johnson, T., Mortlock, A., Keen, N. and Taylor, S. S. (2003). Aurora B couples chromosome PIDD1. With the newly established role for PIDD1 as a sensor of alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. aberrant centrosome numbers, one can now also envision a potential J. Cell Biol. 161, 267-280. role of the PIDDosome in cellular differentiation processes and Dodson, H., Bourke, E., Jeffers, L. J., Vagnarelli, P., Sonoda, E., Takeda, S., Earnshaw, W. C., Merdes, A. and Morrison, C. (2004). Centrosome during organogenesis. Our own findings clearly show that the amplification induced by DNA damage occurs during a prolonged G2 phase PIDDosome restricts ploidy in the liver by activating p53 in a and involves ATM. EMBO J. 23, 3864-3873. CASP2-dependent manner. Accordingly, the DNA content of Duan, H. and Dixit, V. M. (1997). RAIDD is a new ‘death’ adaptor molecule. Nature hepatocytes that are deficient of the PIDDosome is substantially 385, 86-89. Fava, L. L., Bock, F. J., Geley, S. and Villunger, A. (2012). Caspase-2 at a glance. increased and, thereby, copies the p53-null mutant phenotype (Fava J. Cell Sci. 125, 5911-5915. et al., 2017; Kurinna et al., 2013). Clearly, PIDDosome-deficient Fava, L. L., Schuler, F., Sladky, V., Haschka, M. D., Soratroi, C., Eiterer, L., animals constitute an interesting model to study the impact of ploidy Demetz, E., Weiss, G., Geley, S., Nigg, E. A. et al. (2017). The PIDDosome activates p53 in response to supernumerary centrosomes. Genes Dev. 31, 34-45. on liver function and regeneration, without facing the adverse effects Flinterman, M., Guelen, L., Ezzati-Nik, S., Killick, R., Melino, G., Tominaga, K., of global p53 deficiency. Of note, a number of cell types become Mymryk, J. S., Gäken, J. and Tavassoli, M. (2005). E1A activates transcription polyploid during organ development or in response to infection: of p73 and Noxa to induce apoptosis. J. Biol. Chem. 280, 5945-5959. Guo, Y., Srinivasula, S. M., Druilhe, A., Fernandes-Alnemri, T. and Alnemri, cardiomyocytes, for example, increase their ploidy during terminal E. S. (2002). Caspase-2 induces apoptosis by releasing proapoptotic proteins differentiation, whereas viral infection can cause cell−cell fusion, and from mitochondria. J. Biol. Chem. 277, 13430-13437. bacterial infection may trigger the formation of macrophage giant Han, J.-W., Flemington, C., Houghton, A. B., Gu, Z., Zambetti, G. P., Lutz, R. J., cells (Aguilar et al., 2013). Under all these conditions, extra Zhu, L. and Chittenden, T. (2001). Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals. Proc. Natl. Acad. Sci. centrosomes arise in cells and it is tempting to speculate that USA 98, 11318-11323. PIDD1 is called into action in some settings, marking PIDD1 also as a Heath, E. M., Chan, S. M., Minden, M. D., Murphy, T., Shlush, L. I. and Schimmer, target for the pharmacological modulation of these processes. A. D. (2017). Biological and clinical consequences of NPM1 mutations in AML. Leukemia 31, 798-807. Heijink, A. M., Krajewska, M. and van Vugt, M. A. T. M. (2013). The DNA damage Acknowledgements response during mitosis. Mutat. Res. 750, 45-55. We are thankful to all members of the Division of Developmental Immunology at MUI Ho, L. H., Read, S. H., Dorstyn, L., Lambrusco, L. and Kumar, S. (2008). for helpful discussion. Caspase-2 is required for cell death induced by cytoskeletal disruption. Oncogene 27, 3393-3404. Competing interests Hodel, A. E., Hodel, M. R., Griffis, E. R., Hennig, K. A., Ratner, G. A., Xu, S. and Powers, M. A. (2002). The three-dimensional structure of the autoproteolytic, The authors declare no competing or financial interests. nuclear pore-targeting domain of the human nucleoporin Nup98. Mol. Cell 10, 347-358. Funding Huang, L., Han, D., Yang, X., Qin, B., Ji, G. and Yu, L. (2011). 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