Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor

Abstract

The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: ATG12, ATG7, GOSR1, IFT172, NRXN2, RAB6A, VPS37A, and the well-known negative regulator of NRF2, KEAP1. Of these, ATG12, ATG7, KEAP1, and VPS37A are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.

Nuclear Factor Erythroid 2-Related Factor 2 in Regulating Cancer Metabolism

“…Other reports contradict the induction of NRF2 in autophagy by p62. A Crispr-based screen focused on finding novel negative regulators of NRF2 signaling ( 133 ) also identified a number of autophagy-related genes in human kidney HK2 cells. However, subsequent functional analyses demonstrated that changes in the phosphorylation of p-p62 do not necessarily induce NRF2 in HK2 cells, indicating that the autophagy-NRF2 loop can proceed in a p62-independent manner.…”

Autophagy and cancer: Modulation of cell death pathways and cancer cell adaptations

“…NRF2 has previously been linked to autophagy, and p62 sequesters and inhibits the NRF2-negative regulator, KEAP1 (Komatsu et al, 2010). Moreover, a recent genome-wide CRISPR screen showed that KO of a large subset of autophagy genes results in up-regulation of the NRF2 signaling pathway (Kerins et al, 2019). As a consequence of NRF2 up-regulation, the autophagy-deficient cells developed an increased sensitivity to pharmacological proteasome inhibitors, a phenotype that was exacerbated with NRF2 knockdown (Towers et al, 2019).…”

Targeting Autophagy to Treat Cancer: Challenges and Opportunities

“…Of note, restoring autophagy promoted spontaneous tumor development, suggesting that prolonged autophagy inhibition may select for additional stress-response pathways which accelerate disease progression when autophagy is restored. Recent studies demonstrate that overactivation of the oxidative stress response by the transcription factor Nrf2 is a dominant consequence of autophagy-related gene deletion in cancer cells ( Kerins et al, 2019 ; Towers et al, 2019 ). The association between autophagy inhibition and Nrf2 was reported even earlier, predominantly due to accumulation of p62 ( Komatsu et al, 2010 ; Inami et al, 2011 ; Ichimura et al, 2013 ).…”

“…Nonetheless, genetic models still comprise the large majority of evidence supporting more specific nodes of autophagy as targets. Beyond canonical members of the pathway, genetic phenotypic screens using engineered reporter cell lines have provided new insights for modulation of autophagic flux ( DeJesus et al, 2016 ; Morita et al, 2018 ; Kerins et al, 2019 ; Shoemaker et al, 2019 ). In addition, pharmacological screens with the same cellular tools have revealed inhibitors with new mechanisms of action.…”