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 catalyze this process in
tumors are unknown. We used
RNA sequencing, whole-genome CRISPR knockout, and
reductase-focused
short hairpin RNA 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 (ρ 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 nonmitochondrial flavoreductases. In UT-SCC-74B cells,
evofosfamide oxidized
cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching
reactive oxygen species 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
cytochrome P450 oxidoreductase is likely to be futile.