Investigating resistance mechanisms to glutaminase inhibition in renal cell carcinoma using a genome-wide CRISPR/Cas9 approach.


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Aleksandra Raczka1,Aleksandra Raczka1,Paul Reynolds1
1School of Medicine, University of St Andrews

Abstract

Background

Renal cell carcinoma (RCC) represents 90% of kidney cancers and around 3% of all malignancies worldwide. Clear cell RCC (ccRCC) comprises around 70-80% of all RCC. The most frequent genomic alteration in ccRCC involves loss of the 3p chromosomal arm which harbours VHL gene in >90% of ccRCC cases. Large-scale cancer genomics sequencing studies have identified several driver genes beyond VHL, particularly PBRM1 (40%), SETD2 (15%), and BAP1 (10%). 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. Several tumour types, including ccRCC, have been recently shown not only to rely on glutamine for energy generation and maintenance of redox homeostasis, but also use glutamine for 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 as a monotherapy and also as part of a combination therapy, and shows promising results.

Method

Given the significant incidence of resistance to previously approved therapies, we have applied a genome-wide CRISPR/Cas9 approach in a cell culture model of ccRCC to identify candidate genes, which when knocked down will 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.

Results

We conducted the screen with two biological replicates and verified successful genome-scale sgRNA library coverage at baseline in both cases. We identify candidate genes and pathways altered upon drug selection, which are implicated in tumour metabolism, autophagy and metastasis. The screen has also identified candidate genes and pathways predicted to be synthetic lethal with CB-839.

Conclusion

Our data show that the genome-wide CRISPR/Cas9 approach is effective in identifying candidates modulating drug resistance to CB-839.