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

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.National Institute of Environmental Health Sciences [R21ES027920]; National Science Foundation Graduate Research Fellowship Program [DGE-1746060]; [R01CA226920]; [R01DK109555]; [R01ES026845]; [P42ES004940]; [P30ES006694]6 month embargo; accepted manuscript posted online 22 April 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A catalogue of somatic NRF2 gain-of-function mutations in cancer

Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis. Despite its apparent significance, a comprehensive catalogue of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalogue NRF2 mutations in The Cancer Genome Atlas (TCGA) database. 226 unique NRF2-mutant tumors were identified from 10,364 cases. NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a pro-tumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research.National Science Foundation Graduate Research Fellowship Program [DGE-1143953]; University of Arizona College of Pharmacy; University of Arizona Health Sciences; National Institute of Environmental Health Sciences [R21ES027920]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH-/-) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH-/- cell line UOK262. Preferential cell death against UOK262-FH-/- was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH-/- sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH-/-, and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.National Science Foundation [DGE-1143953]; NIEHS [R21ES027920]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2–related factors 1 and 2 (NRF1 and NRF2)

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1(+/+) than in KEAP1(-/-) isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1(-/-) cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.National Institutes of Health [R01 CA154377, R01 DK109555, R01 ES026845]12 month embargo; published online: 18 December 2017This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.National Science Foundation Graduate Research Fellowship Program [DGE-1143953]; University of Arizona College of Pharmacy; University of Arizona Health Sciences grant [214565]6 month embargo; published online 13 March 2017.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The NRF2-p97-NRF2 negative feedback loop

p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients