Development of forward genetic screens for anticancer drug sensitivity genes

Abstract

The aim of the project was to explore forward genetic screening approaches for identifying genetic factors whose products influence sensitivity of cells to cytotoxic anticancer drugs. The intention was to then use these approaches to identify potential biomarkers that predict sensitivity to hypoxia activated prodrugs (HAPs). Hypoxia (oxygen deficiency) is a feature of many cancers and contributes to tumour progression as well as resistance to therapy, which makes it a potential therapeutic target. The focus of this study was on drugs that cause DNA lesions through cross-links, either directly or through their reduced metabolites. The study explored two genetic screening approaches. First, a DNA transposon (“jumping gene”) called piggyBac, discovered in insects, was used to generate a genome-wide library in cancer cell lines. A piggyBac plasmid named Slingshot, which is a tamoxifen-inducible gene trap transposon vector, was used for generating a whole genome library of mutations in two different cancer cell lines, HCT116 and SUM159PT. Resistance to the cross-linking agent chlorambucil was not observed, possibly because of low gene trapping efficiency and the lack of phenotype of single-allele mutations in diploid cells. Therefore, a near-haploid human leukaemia cell line KBM7 was validated and tested for its applicability in genome-wide genetic screening. KBM7 could not be transfected with the piggyBac plasmids, leading to exploration of an alternative system for mutagenesis based on lentiviral CRISPR/Cas9 vectors. CRISPR/Cas9 utilises the elements of a microbial defence system to mutate (knock out) essentially all known human genes, using a commercially available custom designed single-guide RNA (sgRNA) library. KBM7 cells expressing Cas9 nuclease (KBM7-Cas9) were generated by lentiviral transduction using the spinoculation method. Whole genome sgRNA mutated library of KBM7-Cas9 cells was generated and screened for resistance to the cytotoxin 6-thioguanine (6-TG), as a proof-of-principle to identify genes which are part of the mismatch repair pathway, whose disruption causes resistance to 6-TG.