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 catalyze this process in tumors are unknown. We used RNA sequencing, whol...

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Bibliographic Details
Published in:Molecular pharmacology 2019-06, Vol.95 (6), p.638-651
Main Authors: Hunter, Francis W, Devaux, Jules B L, Meng, Fanying, Hong, Cho Rong, Khan, Aziza, Tsai, Peter, Ketela, Troy W, Sharma, Indumati, Kakadia, Purvi M, Marastoni, Stefano, Shalev, Zvi, Hickey, Anthony J R, Print, Cristin G, Bohlander, Stefan K, Hart, Charles P, Wouters, Bradly G, Wilson, William R
Format: Article
Language:English
Subjects:
Cell Line, Tumor
Cell Proliferation - drug effects
Cell Survival - drug effects
CRISPR-Cas Systems
Electron Transport - drug effects
Gene Expression Regulation - drug effects
Gene Regulatory Networks - drug effects
HCT116 Cells
Humans
Mitochondria - drug effects
Mitochondria - genetics
Neoplasms - drug therapy
Neoplasms - genetics
Nitroimidazoles - pharmacology
Phosphoramide Mustards - pharmacology
Prodrugs
RNA, Small Interfering - pharmacology
Sequence Analysis, RNA - methods
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Summary: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 ( 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 , ( ), and , in addition to the key regulator of mitochondrial function, , 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 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 , , and 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.
ISSN:0026-895X
1521-0111
DOI:10.1124/mol.118.115196