Regulation of chemosensitivity by the novel daughter strand gap suppressor MRNIP

新型子链间隙抑制子 MRNIP 对化学敏感性的调节

• 批准号: MR/X024040/1
• 负责人: Christopher Staples
• 金额: $ 75.89万
• 依托单位: Bangor University
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX024040%2F1
• 关键词: Regulation; chemosensitivity; novel; daughter; strand

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.

所有细胞 - 甚至癌细胞 - 如果要分裂，则必须通过称为DNA“复制”的过程来复制其DNA，在此过程中，遗传物质很容易破裂，进而导致细胞死亡。癌细胞杀伤是可取的，因此许多化学疗法通过靶向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中的突变，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通过防止化学疗法治疗后DNA中的有毒间隙的形成，并通过帮助修复治疗诱导的DNA断裂来促进癌细胞的存活。因此，患有BRCA突变的患者通常对传统化学疗法（如顺铂和更先进的“精度”药物Olaparib。我们发现MRNIP - 一种与BRCA基因相似的新型细胞因子，我们也发现了Cisplatin和Olaparib癌症中的DNA GAPS。我们已经使用了一种称为CRISPR的技术来删除mRNIP基因，并发现缺乏mRNIP的细胞对这些药物均敏感，并且在治疗后积累了较高水平的DNA间隙和断裂。我们可以通过防止缺乏mRNIP的细胞中DNA间隙的形成来扭转这种破裂和灵敏度。我们的研究还表明，大多数正常和癌细胞含有mRNIP。但是，我们发现一部分卵巢癌细胞没有可检测到的mRNIP。这增加了某些患者患有缺乏mRNIP的癌症并因此对特定治疗做出良好反应的可能性。因此，MRNIP状态可能证明是“精确医学”中的有用工具，其中有关每个患者以及他们受苦的癌症的信息被用来确定最有效的治疗方法。我们的策略是提高我们在多个级别上对mRNIP的了解。我们想了解mRNIP的功能，确定其在卵巢癌中的作用，并找到杀死缺乏mRNIP的癌细胞的新方法。我们将采用一种三管齐下的方法，如下所示。我们通常执行“ DNA纤维测定”，这使我们能够在癌细胞中复制DNA。使用此测试的修改版本，我们将评估新形成的DNA中DNA间隙的流行率，在该细胞中我们使用CRISPR技术去除了mRNIP基因。我们还确定了对MRNIP蛋白的几种修饰，这些修饰对于促进癌细胞耐药性的能力所需的需要，我们将与研究这些修饰的专家合作，以确定它们如何和为什么重要。IAM2：mRNIP在卵巢癌中的作用是什么？我们目前正在使用CRISPR从一个mRNIP阳性癌细胞系中删除mRNIP基因，并正在设计一种基于病毒的方法，以将mRNIP水平恢复到无法检测到的mRNIP的卵巢癌细胞中。这将使我们找出mrnip损失在卵巢癌中的重要性。我们将评估如上所述的DNA间隙的流行率，并采用实验来测试癌细胞对各种化学疗法的敏感性。这项工作与我们与曼彻斯特的肿瘤学家的合作协同作用。IAM3：mRNIP缺陷型癌细胞依赖于什么来生存？ CRISPR技术是一种强大的工具，也可以利用以识别新型药物靶标。我们将采用CRISPR“筛选”，单独删除mRNIP缺陷癌细胞基因组中的每个基因，以识别这些细胞存活所需的基因，而不是用于含有mRNIP的细胞的存活。这些基因的产物可能证明在DNA间隙患病率升高的癌症中使用新型药物靶标。我们的工作将提供对MRNIP功能的机械见解，也可以提供探索最终患者利益的潜力的途径。