Functional CRISPR and shRNA screens identify involvement of mitochondrial electron transport in the activation of evofosfamide

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyse this process in tumours are unknown. We used RNA sequencing, whole-genome CRISPR knockout and reductase-focused shRNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (rho zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage response factors SLX4IP , C10orf90 ( FATS ) and SLFN11 , in addition to the key regulator of mitochondrial function YME1L1 and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes including increased expression of non-mitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidised cytochromes a, b and c and inhibited respiration at complexes I, II and IV without quenching ROS production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as P450 oxidoreductase (POR) is likely to be futile.