Regulation of chemosensitivity by the novel daughter strand gap suppressor MRNIP

所有细胞--甚至癌细胞--如果要分裂，就必须复制它们的DNA，这一过程被称为DNA复制，在这个过程中，遗传物质很容易被破坏，这反过来又会导致细胞死亡。杀死癌细胞是可取的，因此许多化疗药物通过靶向DNA复制来诱导DNA断裂。一些癌症包含在DNA断裂修复中起作用的基因突变，这些突变使它们对某些疗法敏感。例如，肿瘤抑制基因BRCA1和BRCA2通过防止化疗后DNA中形成有毒缺口，以及帮助修复治疗诱导的DNA断裂来促进癌细胞存活。因此，BRCA突变的患者通常对传统化疗方案反应良好，如顺铂和更先进的精准药物奥拉帕米。我们发现了MRNIP-一种新的细胞因子，其作用方式类似于BRCA基因，我们发现它还抑制顺铂和奥拉帕利治疗的癌细胞中的DNA缺口。我们使用了一种名为CRISPR的技术来删除MRNIP基因，并发现缺乏MRNIP的细胞对这两种药物都很敏感，并在治疗后积累了更高水平的DNA缺口和断裂。我们可以通过阻止缺乏MRNIP的细胞中DNA缺口的形成来逆转这种破坏和敏感性。我们的研究还表明，大多数正常细胞和癌细胞都含有MRNIP。然而，我们发现卵巢癌细胞的一个子集没有检测到MRNIP。这增加了某些患者患有缺乏MRNIP的癌症的可能性，因此他们可能对特定的治疗反应良好。因此，MRNIP状态可能会被证明是‘精确医学’中的一个有用工具，即使用关于每个患者及其所患癌症的信息来确定最有效的治疗。我们的战略是在几个层面上促进我们对MRNIP的了解。我们想要了解MRNIP是如何发挥作用的，确定它在卵巢癌中的作用，并寻找新的方法来杀死MRNIP缺陷的癌细胞。我们将采取三管齐下的方法，如下。目标1：MRNIP如何在癌细胞中发挥作用？我们经常进行‘DNA纤维分析’，这使我们能够在DNA在癌细胞中复制时追踪它。使用这项测试的改进版本，我们将使用CRISPR技术评估新形成的DNA中使用CRISPR技术移除MRNIP基因的细胞中DNA缺口的发生率。我们还确定了MRNIP蛋白的几个修饰，这些修饰对于它驱动癌细胞抵抗治疗的能力是必要的，我们将与研究这些修饰的专家合作，以确定它们如何以及为什么它们是重要的。目标2：MRNIP在卵巢癌中的作用是什么？我们目前正在使用CRISPR从一组MRNIP阳性的癌细胞系中删除MRNIP基因，并正在设计一种基于病毒的方法，以恢复MRNIP无法检测到的卵巢癌细胞的MRNIP水平。这将让我们发现MRNIP缺失在卵巢癌中有多重要。我们将如上所述评估DNA缺口的流行程度，并利用实验测试癌细胞对不同化疗药物的敏感性。这项工作与我们与曼彻斯特的肿瘤学家的合作协同进行。目标3：MRNIP缺陷的癌细胞依赖什么生存？CRISPR技术是一个强大的工具，也可以用来识别新的药物靶点。我们将使用CRISPR‘筛选’，单独删除MRNIP缺陷癌细胞基因组中的每一个基因，以确定哪些基因是这些细胞生存所特需的，而不是含有MRNIP的细胞生存所必需的。这些基因的产物可能被证明是用于DNA缺口患病率上升的癌症的新药物靶点。我们的工作将提供对MRNIP功能的机械性见解和探索最终患者受益的可能性的途径。

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.