Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis

Dongxin Zhao; Mehmet G Badur; Jens Luebeck; Jose H Magaña; Amanda Birmingham; Roman Sasik; Christopher S Ahn; Trey Ideker; Christian M Metallo; Prashant Mali

doi:10.1016/j.molcel.2018.01.017

Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis

Mol Cell. 2018 Feb 15;69(4):699-708.e7. doi: 10.1016/j.molcel.2018.01.017.

Authors

Affiliations

¹ Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
² Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, CA, USA.
³ Center for Computational Biology and Bioinformatics, University of California, San Diego, La Jolla, CA, USA.
⁴ Department of Medicine, Division of Genetics, University of California, San Diego, La Jolla, CA, USA; Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA.
⁵ Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA; Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA. Electronic address: cmetallo@ucsd.edu.
⁶ Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA. Electronic address: pmali@ucsd.edu.

Abstract

The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using ¹³C and ²H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.

Keywords: KEAP1; NADPH; NRF2; combinatorial CRISPR screening; genetic interactions; glycolysis; metabolic enzyme compensation; metabolic enzyme essentiality; metabolic flux analysis; redox metabolism.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Cas Systems*
Glycolysis
HeLa Cells
Homeostasis
Humans
Kelch-Like ECH-Associated Protein 1 / antagonists & inhibitors
Kelch-Like ECH-Associated Protein 1 / genetics
Kelch-Like ECH-Associated Protein 1 / metabolism*
Metabolic Networks and Pathways*
NF-E2-Related Factor 2 / antagonists & inhibitors
NF-E2-Related Factor 2 / genetics
NF-E2-Related Factor 2 / metabolism*
Oxidation-Reduction
Pentose Phosphate Pathway
Signal Transduction
Transcriptome*

Substances

KEAP1 protein, human
Kelch-Like ECH-Associated Protein 1
NF-E2-Related Factor 2
NFE2L2 protein, human

Abstract

Publication types

MeSH terms

Substances

Grants and funding