Targeting chromosomal instability: screening and characterization of CIN killers.

Abstract

Chromosomal INstability (CIN), a hallmark of cancer cells, refers to a state in which cells have an increased rate of gain or loss of whole chromosomes or large chromosomal fractions. CIN is linked to the progression of tumours with poor clinical outcomes such as drug resistance and metastasis. Chromosomal instability is mainly caused by defective chromosomal segregation during mitosis and normally prevented by cellular checkpoints. As CIN is not found in normal cells, it offers a cancer-specific target for therapy, which may be particularly valuable because CIN is common in advanced tumours that are resistant to conventional therapy. In this study, to identify targets which can specifically induce apoptosis in CIN cells, a CIN model was generated by knocking down the spindle assembly checkpoint in Drosophila. Defects in the checkpoint lead to high rate of chromosomal segregation defects (lagging chromosomes and chromosome bridges). An RNAi screening approach was used and the set of kinases and phosphatases was screened to identify those candidates that induce apoptosis only in CIN cells. Genes identified include those involved in JNK signaling pathways, mitotic cytoskeletal regulation and metabolic pathways. This screen demonstrates that it is feasible to selectively kill cells with CIN induced by spindle checkpoint defects. It has identified candidates that are currently being pursued as cancer therapy targets (e.g. Nek2: NIMA related kinase 2), confirming that the screen is able to identify promising drug targets of clinical significance. Further screening and characterization of the JNK pathway demonstrated that signalling through JNK is required to tolerate CIN which is consistent with the fact that many tumours show high levels of JNK expression. JNK signaling is involved in the DNA damage repair process by maintaining an efficient damage repair and anti-oxidant levels which resolves DNA damage before entry into mitosis. Knockdown of the JNK pathway results in unrepaired DNA damage which leads apoptosis only in CIN cells via the caspase dependent pathway, partly independent of p53. Similarly, it was observed that the G2 length, which is required for DNA damage repair is crucial for the survival of CIN cells. These results suggest that JNK is necessary for the proper regulation of the DNA damage induced delay prior to mitotic entry and crucial for the survival of CIN cells, which are already coping with elevated levels of stress. In addition, CIN can enable tumours to acquire genetic diversity which can provide an advantage in terms of growth and proliferation under stress and also provide resistance against cancer therapies, but this comes at the cost of significant stress to tumour cells. CIN cells evolve their metabolic pathways to increase the ability to tolerate and survive under oxidative and proteotoxic stress, but are still sensitive to these pathways. This study demonstrates the possibility to target both CIN and metabolism for the treatment of highly diverse drug resistant tumours. Further metabolic genes were screened and we demonstrated that CIN cells are particularly sensitive to certain metabolic alterations that do not affect normal cells. These metabolic disruptions lead to high levels of oxidative stress in CIN cells, which are already managing elevated reactive oxygen species (ROS) levels. These potential therapeutic targets are clinically highly desirable because of their potential effects on unstable and highly resistant CIN tumours. In conclusion, a new Drosophila model for CIN was used to demonstrate the principle that it is possible to selectively kill CIN cells. Our RNAi screen identified candidates whose depletion has the potential to kill proliferating CIN cells without affecting their normal counterpart. An efficient DNA damage repair mechanism is required to tolerate CIN and can be used as a target to kill these unstable cells which are already dealing with high levels of DNA damage from ROS. Furthermore, CIN cells are sensitive to metabolic alterations, especially those which are needed to tolerate high levels of proteotoxic and oxidative stress. This study is a significant advance in understanding the target pathways which are involved in CIN tolerance. Further characterization of these pathways may help to identify mechanisms by which cancer cells can tolerate the adverse effects of CIN and aneuploidy which in turn may lead to the identification of novel targets that can specifically kill advanced and drug resistant-CIN tumour cells without harming normal cells.