DDR SYNVIA · Cellular models for human disease: alleviation, mechanisms and potential therapies

Cellular models for human disease: alleviation, mechanisms and potential therapies

The key objective of this Marie Curie research project has been to explore synthetic viability principles applied to DNA damage response (DDR) mechanisms. In this way, we intended to improve our knowledge of DDR processes and, in the longer term, help derive new therapies and/or biomarkers for personalized treatment of cancers as well as hereditary genetic diseases affecting the DDR. As cancer is a major cause of mortality and morbidity in Europe, this project has directly addressed clinical issues of emerging importance, such as the development of resistance against PARP inhibitors and other DDR-targeting small molecules through synthetic viability mechanisms. The host laboratory has made several groundbreaking discoveries in the DDR/cancer field, including insights into the molecular mechanism of interaction between BRCA1 and 53BP1, which represents a prime example of synthetic viability in cancer cells. As such, the host lab has been the ideal place for me to carry out and bring to completion the proposed milestones as they were stated in the original proposal. Given that tumours commonly harbour DDR defects, our results will help to exploit knowledge of the DDR to optimize and further personalize cancer treatments. They may also suggest new treatment regimens for human DDR deficiency syndromes, and might provide a screening platform that can be applied to many inherited human diseases. To achieve the above goal I have carried out synthetic viability screens for genes whose mutation/loss may suppress cellular hypersensitivity to DNA damage caused by loss of key DDR proteins or sensitivity to inhibitors currently in clinical trials as anticancer drugs that selectively target DDR proteins. To do this, I have grown either wild-type or specific DDR-deficient cells and then subjected them to a procedure that induces gene inactivation through the Cas9 nuclease, which creates targeted DNA double-strand breaks in sequence-specific genomic regions matching the sequence of 20bp-long clustered interspaced short palindromic repeats (CRISPRs). The CRISPR/Cas9 technology is a revolution in the field of genome editing and we have used it to generate heterogeneous populations of mutant cells, each with a single-gene deletion, with the aim of identifying suppressor mutations. These cell populations, either defective for DNA repair or wild-type cells treated with a DDR inhibitor, were then grown under DNA-damaging conditions designed to normally kill all cells. Cells that survived this treatment were then analysed to identify the suppressing mutations. Through the implementation of this project I have been able to establish a robust pipeline to perform CRISPR-based genetic screens in human cancer cells, uncovering new genetic interactions among components of the DDR. This methodology has allowed us to identify the first set of validated gene candidates that, when mutated, confer resistance to a selective small molecule inhibitor (currently in clinical trials) of the Ataxia-telangiectasia and Rad3-related kinase (ATR). This protein plays an essential role in regulating DNA replication and the response to DNA damage, and is predicted to be essential in cells undergoing replication stress such as oncogene-driven B-cell and T-cell lymphoma and chronic myeloid leukemia cells. We are currently completing additional studies to establish whether these observations are cell-type specific or can be extended to other cancer cells both in vitro and in mouse models, and we are exploring the function of these genes to determine how their inactivation alleviates the sensitivity to ATR inhibition. Similar CRISPR-based genetic screens were also performed in interstand crosslink repair-deficient and nucleotide excision repair-deficient cell lines modelling two genetic diseases characterized by patients’ hypersensitivity to DNA damage: Fanconi anemia and Xeroderma pigmentosum. We have been able to isolate CRISPR-targeted clones of both cell lines that became resistant to DNA damage and are currently analysing their genetic profiles to identify the suppressing mutations. As with the gene targets identified above, we will carry out studies to determine whether such suppressors could be exploited as potential targets whose therapeutic inhibition could alleviate the clinical symptoms of above-mentioned genetic diseases. Given the power of the CRISPR screening technology to uncover new genetic interactions and to gain insight into the basic biology of the DDR, we plan to start several research projects using these CRISPR libraries in the near future. All the screens we have performed to date have been positive screens whereby we looked for suppressors of sensitivity to DNA damage, but we now plan to perform more challenging negative screens, with the aim of identifying genes that, when ablated, confer hypersensitivity to DNA damage with selective DDR inhibitors, or are synthetically lethal with cancer-prevalent mutations in other DDR genes. For this purpose, we have designed focused lentiviral CRISPR libraries targeting all known DDR genes and other gene families in collaboration with Prof. Allan Bradley’s group at the Wellcome Trust Sanger Institute. These focused CRISPR libraries have the advantage of being of lower complexity compared to the whole-genome libraries we have used so far, hence facilitating the identification of relevant drop out targets in negative screens. Moreover, they target only sets of genes involved in cellular processes that the host laboratory has an in depth knowledge of and is experienced in the techniques necessary to gain insight into their molecular mechanism, if any new DDR-related genetic interactions are discovered. Our expertise in the DDR and in performing CRISPR screens has come to the attention of other academic and commercial groups involved in drug discovery and target identification in relevant cancer models, and we plan, in the near future, to establish collaborations that hold great promise of mutual benefit. The aim of these collaborations will be to validate the gene candidates we have identified in our CRISPR screens as biomarkers of resistance to ATR inhibitors in cancer models that more realistically reproduce the drug response observed in clinical patients. The findings we obtain will have the potential to translate faster into clinical applications faster and lead to improved cancer therapies.

Data: CORDIS, © European Union