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Mol Pharmacol. 2019 Jun;95(6):638-651. doi: 10.1124/mol.118.115196. Epub 2019 Apr 12.

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

Author information

1
Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health Sciences (F.W.H., C.R.H., A.K., I.S., W.R.W.), Maurice Wilkins Centre for Molecular Biodiscovery (F.W.H., A.J.R.H., C.G.P., W.R.W.), School of Biological Sciences, Faculty of Science (J.B.L.D., A.J.R.H.), and Department of Molecular Medicine and Pathology, School of Medical Sciences, Faculty of Medical and Health Sciences (P.T., P.M.K., C.G.P., S.K.B.), University of Auckland, Auckland, New Zealand; Threshold Pharmaceuticals, South San Francisco, California (F.M., C.P.H.); Princess Margaret Genomics Centre (T.W.K.) and Princess Margaret Cancer Centre (S.M., Z.S., B.G.W.), University Health Network, and Departments of Radiation Oncology (B.G.W.) and Medical Biophysics (B.G.W.), University of Toronto, Toronto, Ontario, Canada f.hunter@auckland.ac.nz.
2
Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health Sciences (F.W.H., C.R.H., A.K., I.S., W.R.W.), Maurice Wilkins Centre for Molecular Biodiscovery (F.W.H., A.J.R.H., C.G.P., W.R.W.), School of Biological Sciences, Faculty of Science (J.B.L.D., A.J.R.H.), and Department of Molecular Medicine and Pathology, School of Medical Sciences, Faculty of Medical and Health Sciences (P.T., P.M.K., C.G.P., S.K.B.), University of Auckland, Auckland, New Zealand; Threshold Pharmaceuticals, South San Francisco, California (F.M., C.P.H.); Princess Margaret Genomics Centre (T.W.K.) and Princess Margaret Cancer Centre (S.M., Z.S., B.G.W.), University Health Network, and Departments of Radiation Oncology (B.G.W.) and Medical Biophysics (B.G.W.), University of Toronto, Toronto, Ontario, Canada.

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 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.

PMID:
30979813
DOI:
10.1124/mol.118.115196

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