Investigating the mechanisms leading to CHK1 inhibitor resistance in cancer therapy

DNA复制应激是由停滞的DNA复制叉引起的，是癌细胞的一个特征，它可以导致基因组不稳定性，从而驱动肿瘤发生。细胞对DNA复制应激反应的关键调节因子是检查点激酶ATR和CHK 1。肿瘤细胞可以对这种途径上瘾，因为它使它们能够在持续的、潜在致命的基因组不稳定性中存活下来。因此，CHK 1抑制剂代表了一类潜在的新型抗癌疗法，目前正在进行临床试验。由于肿瘤经常对靶向激酶的药物产生耐药性，我们一直在更详细地研究导致CHK 1抑制剂耐药性的机制。这包括大量未发表的RNA Seq基因表达谱和蛋白质组学数据，这些数据涉及CHK 1抑制剂耐药性的发展，将用于支持该项目。为了更好地了解这些机制是否在临床中发生，我们还对一组已知影响来自CHK 1抑制剂试验患者循环游离DNA（cfDNA）的ATR/CHK 1途径的基因进行了测序分析，以确定这种治疗引起的突变。因此，我们已经确定了似乎由CHK 1抑制剂治疗引起的基因突变。然而，我们不知道它们是否对这些基因编码的蛋白质具有功能性影响，也不知道它们是否有助于CHK 1抑制剂抗性的过程。该项目的目标是：（1）研究导致CHK 1抑制剂耐药的机制，以提供可用于接受该药物的患者的生物标志物（2）确定CHK 1耐药肿瘤细胞中改变的途径，这些途径可以在耐药肿瘤中靶向我们从临床试验中发现的突变将使用CRISPR/Cas9基因组工程（主要监督）在癌细胞系中重建。将使用各种分子和细胞生物学试验评价这些药物对ATR/CHK 1通路活性和CHK 1抑制剂治疗的影响（主要主管，Neil Perkins教授; http：//www.ncl.ac.uk/camb/staff/profile/neil.perkins; @ndperkins）。这些包括细胞培养、定量PCR分析、细胞活力分析、共聚焦显微镜和蛋白质印迹。由于缺氧（缺氧）后再氧合已知会激活ATR/CHK 1，这是肿瘤的常见特征，因此将确定这些突变对细胞在这些条件下生长能力的影响（第二主管; Prof Sonia罗查; https：//www.rochalab.com/; @srochaliv）。将使用异种移植小鼠模型分析经证明具有体外效应的基因组工程改造的癌细胞系，以研究对肿瘤生长、肿瘤缺氧反应和体内CHK 1抑制剂敏感性的影响（第三主管; Dr Jill Hunter; @JillHunter2185）。该项目为学生提供了一个独特的机会，利用基础科学技术将研究成果转化为极具前景的新型抗癌药物的临床应用。

DNA replication stress results from stalled DNA replication forks and is a feature of cancer cells, where it can lead to the genomic instability driving tumorigenesis. Critical regulators of the cellular response to DNA replication stress are the checkpoint kinases ATR and CHK1. Tumour cells can become addicted to this pathway, since it enables them to survive on-going, potentially lethal, genomic instability. For this reason, inhibitors of CHK1 represent a potential new class of anti-cancer therapies, and are currently in clinical trials. Since tumours frequently develop resistance to drugs targeting kinases, we have been investigating the mechanisms leading to CHK1 inhibitor resistance in more detail. This includes a large amount of unpublished RNA Seq gene expression profiling and proteomics data concerning the development of CHK1 inhibitor resistance that will be used to support this project. To understand better if these mechanisms are occurring in the clinic, we have also performed sequencing analysis of a panel of genes known to affect the ATR/CHK1 pathway from circulating free DNA (cfDNA) from patients enrolled in a CHK1 inhibitor trial, to identify mutations arising from this therapy. Consequently, we have identified genetic mutations that appear to arise from CHK1 inhibitor therapy. However, we do not know if they have a functional effect on the proteins encoded by these genes nor if they contribute to the process of CHK1 inhibitor resistance. The objectives of this project are to:(1) Investigate the mechanisms leading to CHK1 inhibitor resistance to provide biomarkers that can be used in patients receiving this drug(2) Identify the pathways altered in CHK1 resistant tumour cells that could be targeted in resistant tumours The mutations we have identified from the clinical trial will be recreated in cancer cell lines using CRISPR/Cas9 genome engineering (primary supervisor). These will be evaluated for effects on ATR/CHK1 pathway activity and CHK1 inhibitor treatment using a variety of molecular and cell biological assays (primary supervisor, Prof Neil Perkins; http://www.ncl.ac.uk/camb/staff/profile/neil.perkins; @ndperkins). These include cell culture, quantitative PCR analysis, cell viability analysis, confocal microscopy and western blotting. Since hypoxia (lack of oxygen) followed by reoxygenation, is known to activate ATR/CHK1 and this is a common feature of tumours, the effect of these mutations on the ability of the cells to grow under these conditions will be determined (second supervisor; Prof Sonia Rocha; https://www.rochalab.com/; @srochaliv). Genome engineered cancer cell lines with mutations demonstrated to have an effect in vitro will be analysed using xenograft mouse models to investigate effects on tumour growth, the tumour hypoxia response and CHK1 inhibitor sensitivity in vivo (third supervisor; Dr Jill Hunter; @JillHunter2185). This project provides a unique opportunity for a student to use fundamental scientific techniques to translate research findings into the clinical use of highly promising and new anti-cancer drugs.