Investigating the mechanisms leading to CHK1 inhibitor resistance in cancer therapy

DNA复制压力源于DNA复制分叉的停滞，是癌细胞的一个特征，它可以导致基因组的不稳定，从而驱动肿瘤的发生。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CHK1。肿瘤细胞可能会对这种途径上瘾，因为它使它们能够在持续的、可能致命的基因组不稳定中存活下来。因此，CHK1抑制剂代表了一种潜在的新型抗癌疗法，目前正在临床试验中。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CHK1抑制剂耐药的机制。这包括大量未发表的RNA Seq基因表达谱和蛋白质组学数据，这些数据与CHK1抑制剂耐药性的发展有关，将用于支持本项目。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ATR/CHK1途径的基因进行了测序分析，这些基因来自参加CHK1抑制剂试验的患者的循环游离DNA (cfDNA)，以确定这种治疗引起的突变。因此，我们已经确定了似乎由CHK1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有功能影响，也不知道它们是否有助于CHK1抑制剂耐药性的过程。该项目的目标是：(1)研究导致CHK1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CHK1耐药肿瘤细胞中可能被靶向的通路。我们从临床试验中发现的突变将使用CRISPR/Cas9基因组工程（主要主管）在癌细胞系中重建。这些将通过各种分子和细胞生物学测定来评估对ATR/CHK1通路活性和CHK1抑制剂治疗的影响（主要导师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western blotting。由于缺氧（缺氧）之后再氧化，已知会激活ATR/CHK1，这是肿瘤的共同特征，这些突变对细胞在这些条件下生长能力的影响将被确定（第二导师；Sonia Rocha教授；https://www.rochalab.com/; @srochaliv）。研究人员将使用异种移植小鼠模型分析基因工程癌细胞系对肿瘤生长、肿瘤缺氧反应和体内CHK1抑制剂敏感性的影响（第三导师；JillHunter博士；@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具有很高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 This includes a large amount of unpublished RNA Seq gene expression profiling and proteomics data concerning the development of CHK1 inhibitor resistance that will be used to support this project.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.