Table 1. Candidates giving CIN-dependent cell death. Killing Cells with Chromosomal Instability Groups Centrosomal JNK pathway DNA damage Wnt signaling pathway Histone kinases Others Candidate symbol Nek2 lok asp tefu bsk bsk pvr slpr Pak3 lok tefu mbt aPKC Wnk CG8878 ball Pink1 lic CG4041 Pask Mammalian homolog NIMA-related kinase 2 (NEK2) checkpoint homolog (Chk2) Abnormal spindle (Aspm) Ataxia telangiectasia mutated (ATM) JUN amino terminal kinase (JNK) JUN amino terminal kinase (JNK) PDGF/VEGF receptor JUN kinase kinase kinase (JNKKK) p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3) checkpoint homolog (Chk2) Ataxia telangiectasia mutated (ATM) Protein kinase C vaccinia-related kinase (VRK)? Nucleosomal histone kinase-1 (Nhk-1) PTEN-induced putative kinase 1 Functional association Cell cycle progression [61] DNA damage [62] Spindle organization [47] DNA damage response [62] JNK signaling pathway [63] JNK signaling pathway [63] JNK activator [64] JNK signaling pathway [65] JNK activator [66] DNA damage [62] DNA damage response [62] Wnt signaling [68] Histone kinase? Histone kinase [70] apoptosis/mitophagy [71] p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) Wnt signaling/cytoskeletal regulation [67] WNK lysine deficient protein kinase 1 (WNK1) Ion regulation, cell cycle progression and adaptation [69] mitogen-activated protein kinase kinase 3 (MAP2K3) MAP kinase-mediated signaling [72] TBC1 domain containing kinase (TBCK)/Rab gtpase? Unknown PAS kinase (PASKIN) cellular energy homeostasis [52,53] Candidates from the viability screening and cell death assay that gave the most CIN-dependent cell death. Some of the candidates are placed in more than one group on the basis of their associations. doi:10.1371/journal.pone.0047447.t001 ly, we examined whether loss of P53 affected the ability of our candidates to induce cell death in a CIN background. Depletion of P53 suppressed the loss of tissue phenotype when Pask and mad2 were both depleted in adult wings (data not shown) and significantly decreased the level of apoptosis in wing discs (Figure 4), suggesting that the cell death in this case was largely p53 dependent. However for asp, depleting P53 had little effect in the double knockdown (asp and mad2) wing discs, showing that, in this case, the apoptosis induced by asp knockdown in CIN cells is largely P53 independent (Figure 4). Other candidates such as bsk (Jun kinase) gave a modest reduction in the level of cell death when P53 was depleted (data not shown), suggesting the involvement of both p53 dependent and independent mechanisms in inducing cell death in this case. Discussion To identify targets that could be depleted to induce apoptosis in cells with chromosomal instability, we carried out RNAi screening of kinases and phosphatases in a CIN model system. We targeted CIN because it is common in cancers and CIN makes these cancers resistant to current therapies [3]. Using Drosophila as a model, we were able to induce CIN in a genetically stable background by depleting ,85% of Mad2, which weakens SAC function and shortens metaphase [25,39]. We found this gives an optimal level of CIN that is enough to screen against, but not so much that cells cannot survive. This approach has some advantages over using vertebrate CIN cell lines [40] which, by definition, have highly diverse and unstable genomes. In particular, we were able to test each candidate on genetically identical control tissues, which allowed us to be confident that any apoptosis was due to the effect of the checkpoint defect, not an artifact of a particular aberrant cell line. The screen identified a significant group of candidates, including Nek2, JNK and Asp, that are directly or indirectly linked to the centrosome (Table 1). The centrosome is a regulatory hub of the major events during mitosis: alterations in centrosomal proteins and numbers result in segregation defects and CIN [41]. Extra centrosomes are common in cancer, and contribute to CIN by forming multi-polar spindles, which produce more merotely and lagging chromosomes and hence whole chromosome aneu- ploidy [42,43]. Our identification of Nek2, which is currently being pursued as therapeutic target for cancer [34], confirms that our screen has the potential to identify clinically significant drug targets for CIN tumors. Members of the JNK pathway, which is known to promote apoptosis, DNA damage response, proliferation, migration and differentiation [44–46] were also found in our screen. Our results suggest a novel role for JNK in preventing cell death in response to mitotic errors. This may potentially explain the anti-apoptotic effect of JNK seen in HCC tumours and leukemia [44]. Abnormal spindle (Asp) binds at the minus end of microtubules, and is required for centrosome attachment [47] and possibly for down-regulating p53 [48]. In this respect it is interesting that we observed p53 independent cell death in asp mad2 double depletions. This suggests that the apoptosis we observed was not simply a result of losing a negative regulator of p53. Further work will be required to determine what triggers cell death in this case. Taken together, these results suggest that CIN cells are highly sensitive to centrosome disruption, responding by apoptosis to treatments that have no effect on normal dividing cells. One PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47447