Killing Cells with Chromosomal Instability Figure 3. DNA damage (anti-P-H2AvD) staining of third instar larval wing discs. (a–c9) Dotted line shows the en-CD8GFP marked test region in which genes were depleted. The other half of each wing disc expressed no transgenes. (a, a9) Negative control (LacZ RNAi) with and without Mad2 (b, b9) PASK depletion with and without Mad2. (c, c9) aPKC depletion with and without Mad2. Significant induction of DNA damage in the depleted area is seen in Pask, mad2 discs but not LacZ, mad2 or aPKC, mad2 discs. doi:10.1371/journal.pone.0047447.g003 the centrosome and events at the kinetochore that renders SAC- deficient cells particularly dependent on centrosomal signals. This dependence may relate to the centrosomal localization of p53 [48,49], DNA damage repair proteins [50] or even Mad2 itself [51]. Several interesting candidates identified in our screen, however, are not localized on centrosomes (e.g. Pask and aPKC). Furthermore, some candidates have no reported connection of any kind with mitosis, indicating that we may have detected novel pathways that sense segregation defects and provide stability to cancer cells against CIN. For example, Pask is a serine/threonine kinase involved in sensing and regulating cellular energy homeostasis [52,53]. Here we show its novel role in preventing DNA damage and p53 dependent apoptosis in CIN cells. Our screen also isolated candidates that are involved in the DNA damage response (tefu, lok, bsk, and pp1a), suggesting a role for the DNA damage response pathway in responding to CIN. This is not surprising given the recent work showing that anaphase errors result in DNA damage [36,54] and the role of Mad2 in delaying anaphase onset to give time for repair [55]. It seems clear that the DNA damage we observed in our candidates in the presence of CIN is not simply part of the apoptotic program: we see strong apoptosis and little DNA damage in aPKC knockdown in CIN cells. Furthermore our highest levels of DNA damage were seen in Pask mad2 double depletions, which gave no more apoptosis than aPKC. It is striking that none of our candidates alone, nor mad2 alone, gives significant levels of DNA damage. Our interpretation of this is that the cellular DNA damage response can keep the level of damage below our detection sensitivity in any of the single depletions, and it is only when multiple checkpoint and repair mechanisms are depleted that we see unrepaired damage and widespread apoptosis. Depletion of Asp or Bsk gives some P53 independent apoptosis in CIN cells, which could make them desirable therapeutic candidates in a clinical setting, where P53 is often absent [56], and indeed JNK inhibitors are currently in clinical trials [57]. Unfortunately, JNK is involved in many processes that make it problematic as a drug target [58]. However, the other candidates that regulate JNK signaling, (Hep: hemipterous/JNKK, Slpr: slipper/ JNKKK, Pak3/Pak2 and Pvr: PDGF/VEGF receptor), may have potential as good CIN-specific targets. In summary, we have used a new model for CIN in Drosophila to demonstrate the principle that it is possible to selectively kill CIN cells. Our RNAi knockdown identified candidates not previously known to have mitotic roles but whose depletion has the potential to kill proliferating CIN cells. Further character- ization of screened candidates and their pathways may help to PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47447