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

Francis W Hunter1, Jules B L Devaux2, Fanying Meng2

  • 1Auckland 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.

Molecular Pharmacology
|April 14, 2019
PubMed

Insights

Evofosfamide activation in cancer relies on mitochondrial electron transport, not just traditional reductases. This finding impacts how we predict patient response to this hypoxia-activated prodrug.

Area of Science:

  • Molecular Biology
  • Cancer Therapeutics
  • Cellular Respiration

Background:

  • Evofosfamide (TH-302) is a hypoxia-activated prodrug for cancer therapy.
  • Its activation requires one-electron reduction, but tumor reductases remain unidentified.

Purpose of the Study:

  • To identify the reductases responsible for evofosfamide activation in tumors.
  • To investigate the role of mitochondrial electron transport in evofosfamide's mechanism of action.

Main Methods:

  • RNA sequencing, CRISPR knockout, and shRNA screens were used to identify modifiers of evofosfamide activation.
  • Experiments utilized cancer cell lines, including mitochondrial DNA-deficient cells (143B ρ0) and biochemical assays.

Main Results:

  • Evofosfamide potency correlated with mitochondrial electron transfer gene expression.
  • CRISPR and shRNA screens identified mitochondrial factors (e.g., YME1L1, Complex I) and DNA damage factors (SLX4IP, FATS, SLFN11) as modifiers.
  • Mitochondrial DNA-deficient cells showed enhanced activation, suggesting non-canonical pathways.

Conclusions:

  • The mitochondrial electron transport chain significantly contributes to evofosfamide activation.
  • Predicting evofosfamide sensitivity based on canonical bioreductive enzymes like cytochrome P450 oxidoreductase may be unreliable.

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