Regulation of chemosensitivity by the novel daughter strand gap suppressor MRNIP

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All cells - even cancer cells - must copy their DNA if they are to divide, via a process called DNA 'replication' during which the genetic material is vulnerable to breakage, which in turn leads to cell death. Cancer cell killing is desirable, and therefore many chemotherapies work by targeting DNA replication to induce DNA breaks. Some cancers contain mutations in genes that function in DNA break repair, and these mutations render them sensitive to certain therapies. For example, the tumour suppressor genes BRCA1 and BRCA2 promote cancer cell survival by preventing the formation of toxic gaps in the DNA following chemotherapy treatment, and by helping repair therapy-induced DNA breaks. Patients with BRCA mutations often thus respond well to traditional chemotherapies like Cisplatin and the more advanced 'precision' medicine Olaparib.We discovered MRNIP - a novel cellular factor that acts in a similar way to the BRCA genes, and which we have found also suppresses DNA gaps in Cisplatin and Olaparib-treated cancer cells. We have used a technique called CRISPR to delete the MRNIP gene, and discovered that cells lacking MRNIP are sensitive to both these drugs and accumulate higher levels of DNA gaps and breakage following treatment. We can reverse this breakage and sensitivity by preventing the formation of DNA gaps in cells lacking MRNIP. Our studies also indicate that most normal and cancer cells contain MRNIP. However, we find that a subset of ovarian cancer cells have no detectable MRNIP. This raises the possibility that certain patients suffer from cancers that lack MRNIP, and who may therefore respond well to particular treatments. MRNIP status may therefore prove a useful tool in 'precision medicine', in which information about each individual patient and the cancer from which they suffer is used to determine the most effective treatment.Our strategy is to advance our knowledge about MRNIP on several levels. We want to understand how MRNIP functions, to determine its role in ovarian cancer, and to find novel ways to kill MRNIP-deficient cancer cells. We will undertake a three-pronged approach, as follows.Aim 1: How does MRNIP function in cancer cells? We routinely perform 'DNA fibre assays' which allow us to track DNA while it is being replicated in cancer cells. Using a modified version of this test, we will assess the prevalence of DNA gaps in newly-formed DNA in cells from which we have removed the MRNIP gene using CRISPR technology. We have also identified several modifications to the MRNIP protein that are required for its ability to drive cancer cell resistance to therapy, and we will work with an expert who studies these modifications to determine how and why they are important.Aim 2: What is the role of MRNIP in ovarian cancer? We are currently using CRISPR to delete the MRNIP gene from a panel of MRNIP-positive cancer cell lines, and are designing a virus-based method to restore MRNIP levels to ovarian cancer cells in which MRNIP is undetectable. This will let us find out how important MRNIP loss is in ovarian cancers. We will assess the prevalence of DNA gaps as detailed above, and employ experiments to test cancer cell sensitivity to diverse chemotherapies. This work synergises with our collaboration with Manchester-based oncologists.Aim 3: What do MRNIP-deficient cancer cells rely on to survive? CRISPR technology is a powerful tool that can also be harnessed to identify novel drug targets. We will employ CRISPR 'screening', individually deleting every gene in the genome of MRNIP-deficient cancer cells to identify which genes are required specifically for the survival of these cells, but not for survival of cells that contain MRNIP. The products of these genes may prove novel drug targets for use in cancers with an elevated prevalence of DNA gaps. Our work will provide both mechanistic insights into MRNIP function and avenues to explore the potential for eventual patient benefit.