Investigating the mechanisms leading to CHK1 inhibitor resistance in cancer therapy

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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 This includes a large amount of unpublished RNA Seq gene expression profiling and proteomics data concerning the development of CHK1 inhibitor resistance that will be used to support this project.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.