Investigating resistance mechanisms to CB-839 in renal cell carcinoma using a whole-genome CRISPR/Cas9 approach

Abstract

Renal cell carcinoma (RCC) represents around 3% of all malignancies worldwide, with its most common type, clear cell RCC (ccRCC), making up around 70-80% of all RCC. RCC is characterised by significant tumour heterogeneity and inherent (in 25-30% cases) or acquired resistance to available chemotherapeutics. Glutamine addiction is a potential new therapeutic target for RCC as it has been recently shown not only to rely on glutamine for energy generation and maintenance of redox homeostasis, but also de novo pyrimidine synthesis. CB-839 is a small, orally administered reversible inhibitor of human kidney-type glutaminase (GLS). CB-839 is currently in early phase clinical trials and shows promising results.

Given the significant incidence of resistance to previously approved therapies, this thesis describes a genome-scale CRISPR/Cas9 screen approach in a cell culture model of ccRCC (786-0 cell line) to identify candidate genes (hits), which when knocked down confer resistance to CB-839. Next generation sequencing data analysis of drug-selected sgRNA library representation from two timepoints was performed using the MAGeCKFlute bioinformatics workflow. To validate generated hits, single-gene knockout cell lines were created using several independent sgRNAs per gene of interest.

Knockout of FOXC1, which appeared as a strong hit, could not be achieved using multiple approaches. Additionally, knockout of RPLP2, gene, a hit in both timepoints, could not be confirmed, likely due to it being an essential gene. FOXO3 and ALDH18A1 knockout pools were confirmed using Western blotting, however, their resistance to CB-839 compared to wild type 786-0 was not significantly different. Upon further inspection of expression databases, the hits selected for validation did not show significant difference in expression or impact on survival between normal and kidney cancer tissues. However, a CB-839 resistant cell line (10-fold more resistant) was created from 786-0 cells, suggesting that resistance factors do potentially exist.

While the screen aligned with metrics usually associated with good quality control, no candidate genes were validated when subsequently assessed individually. This study highlights the challenges in performing and validating CRISPR/Cas9 screens and describes the issues surrounding false-positive candidates.