Abstract 2995: Dissecting renal cell carcinoma vulnerabilities: Using CRISPR/Cas9 to study resistance to glutaminase inhibition

Abstract

Abstract The aim of this study was to identify genes, which when knock out confer resistance to a novel glutaminase inhibitor in Renal Cell Carcinoma (RCC). 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. The most frequent genomic alteration in ccRCC involves loss of 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 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, we have applied a genome-scale CRISPR/Cas9 approach in a cell culture model of ccRCC (786-0 cell line) to identify candidate genes, 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. We conducted the screen in two biological replicates and verified successful genome-scale sgRNA library coverage at baseline in both cases. As a result, we identified candidate genes and pathways that are implicated in tumour metabolism, autophagy and metastasis. Additionally, this analysis identified KD genes, which in combination with CB-839 are synthetic lethal. We have prioritized the top 12 candidate genes, which we are validating in four cell lines (786-0, A498, A704 and Caki-1). After establishing CRISPR/Cas9 single gene knockdown (KD) cell lines, KD is confirmed using RT-qPCR and Western Blot, followed by functional analysis. KD cells were treated with increasing concentrations of the drug in parallel with a cell line containing a scrambled sgRNA to detect growth changes between the two. After in vitro validation, we performed ex vivo validation of the candidate hits. Our data show that the genome-scale CRISPR/Cas9 approach is effective in identifying candidates modulating drug resistance to CB-839. Citation Format: Aleksandra M. Raczka, Paul A. Reynolds. Dissecting renal cell carcinoma vulnerabilities: Using CRISPR/Cas9 to study resistance to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2995.