Development of whole-genome CRISPR-Cas9 functional screens for the discovery of TH-302 sensitivity genes

Abstract

Hypoxia, a central pathophysiological feature of many tumours, has long been considered as a potential target for solid tumour-selective therapy. In addition to solid tumours, recent evidence indicates the role of hypoxic bone marrow in advancing leukaemia and thus suggesting that hypoxic bone marrow niche may also be considered for targeted therapies in leukaemic patients. Hypoxia-activated prodrugs (HAPs) contain a bioreductive cytotoxic moiety which is selectively activated in hypoxic tumour cells. The clinical development of the HAP TH-302 has been challenging. This thesis aimed to develop the methodologies for harnessing the recent, RNA guided CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 nuclease (CRISPR-Cas9) for performing high-throughput genome-scale knockout (GeCKO) to identify the genetic sensitivity determinants of TH-302. Production of high titer lentiviruses (LV) using sucrose gradient centrifugation improved the transduction efficiency of Cas9 expressing pseudohaploid chronic myeloid leukaemia cell line KBM7 (KBM7-Cas9). Using high titer LV, a GeCKO library targeting 19,050 genes with unique 122,411 guide RNA sequences was produced at an MOI of ~0.25 in KBM7-Cas9 cells. The ensuing KBM7-Cas9-GeCKO library was screened with TH-302 and its active metabolite Br-IPM, to identify genetic sensitivity determinants. Drug screening methodology was optimised where low concentrations yet repeated cycles of drug exposure prevented the surviving fraction from undergoing tremendous genetic bottleneck, thus retaining a significant proportion of the library with adequate representation. To determine the functioning of the current bioinformatics pipeline, MAGeCK -VISPR analysis of the IIlumina HiSeq data from previously performed pilot-study with 6-thioguanine (6-TG) in a small KBM7-Cas9-GeCKO library, was carried out. The high-ranking hits identified several genes with known roles in 6-thioguanine sensitivity (NUDT5, MLH1, MSH6, PMS2, HPRT1). The end-to-end pipeline of CRISPR-Cas9 functional screens at ACSRC was established to answer the important long term objective of the project, i.e., to identify the determinants of sensitivity to TH-302.