Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide

Abstract

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on oneelectron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, wholegenome 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 r 0 (r 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 reductasefocused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B r 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.

Impact of Tumour Hypoxia on Evofosfamide Sensitivity in Head and Neck Squamous Cell Carcinoma Patient-Derived Xenograft Models

“…We evaluated MKI67 , POR and SLFN11 expression as other potential candidate predictive biomarkers of evofosfamide sensitivity [49,53,54]. Of these genes, only MKI67 showed evidence of a correlation with the antitumour efficacy of evofosfamide, consistent with our previous finding that evofosfamide is more potent in highly proliferative HNSCC cell lines [49].…”

“…It is epigenetically regulated by methylation, with hypermethylation likely responsible for the decreased expression in PDX tumours, as seen previously in cancer cell lines [74,75]. Since SLFN11 promotes sensitivity to DNA damaging agents that interfere with DNA replication fork progression [60,61,62], possibly including evofosfamide [54], reduced expression of SLFN11 in PDX tumours relative to clinical tumours suggests the PDX tumours could potentially under-predict the clinical activity of evofosfamide in HNSCC.…”

“…Additional markers of sensitivity to evofosfamide have been proposed. These include: POR (NADPH:cytochrome P450 oxidoreductase), which has been reported to catalyse the one-electron reduction of evofosfamide selectively under hypoxia to form Br-IPM [51,52,53]; proliferation markers, since a proliferation metagene can predict evofosfamide sensitivity in HNSCC cell lines in vitro [49] and DNA damage response and mitochondrial electron transport genes (e.g., SLFN11 , YME1L1 ), identified as evofosfamide sensitivity genes through functional genomic screens [54].…”

Single- and Two-Electron Reduction of Nitroaromatic Compounds by Flavoenzymes: Mechanisms and Implications for Cytotoxicity

“…Although the nitroreductase reactions of the latter two enzymes are uncharacterized, one may expect the similarity of their log (rate constant) vs. E 1 7 relationships with those of P-450R and NOS. On the other hand, the contribution of mitochondrial NADH:ubiquinone dehydrogenase and, possibly, other flavoenzymes of mitochondrial respiratory chain to the cytotoxicity of ArNO 2 was recently demonstrated [259]. The comparison of reactivities of P-450R and NOS with those of disulfide reductases (Tables 1 and 2 and Section 3.3) clearly rules out the latter enzymes as the important sources of free radicals of ArNO 2 .…”

The Hypoxia-Activated Prodrug TH-302: Exploiting Hypoxia in Cancer Therapy

“…The reductase involved in this selective activation under hypoxia is not yet fully understood. However, Hunter et al investigated potential modifiers of TH-302 metabolism by RNA sequencing, whole-genome CRISPR knockout, and reductasefocused short hairpin RNA screens, and found that the activation of TH-302 is related to genes involved in mitochondrial electron transfer, DNA damage-response factors and mitochondrial function regulators, such as SLX4IP, C10orf90 (FATS), SLFN11, YME1L1 (Hunter et al, 2019).…”