NRF2: KEAPing Tumors Protected

Abstract

The Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway plays a physiologic protective role against xenobiotics and reactive oxygen species. However, activation of NRF2 provides a powerful selective advantage for tumors by rewiring metabolism to enhance proliferation, suppress various forms of stress, and promote immune evasion. Genetic, epigenetic, and posttranslational alterations that activate the KEAP1/NRF2 pathway are found in multiple solid tumors. Emerging clinical data highlight that alterations in this pathway result in resistance to multiple therapies. Here, we provide an overview of how dysregulation of the KEAP1/NRF2 pathway in cancer contributes to several hallmarks of cancer that promote tumorigenesis and lead to treatment resistance.

Significance: Alterations in the KEAP1/NRF2 pathway are found in multiple cancer types. Activation of NRF2 leads to metabolic rewiring of tumors that promote tumor initiation and progression. Here we present the known alterations that lead to NRF2 activation in cancer, the mechanisms in which NRF2 activation promotes tumors, and the therapeutic implications of NRF2 activation.

Figure 1:. Physiological Activation and Regulation of NRF2

Under basal conditions, NRF2 is bound by KEAP1 via the DLG and ETGE motifs in the Neh2 domain of NRF2 in cytosol and leads to binding of CUL3, poly-ubiquitination and proteasomal degradation. NRF2 is also regulated by KEAP1-indepent mechanisms via phosphorylation of the Neh6 domain by GSK-3 and proteasomal degradation by β-TrCP. Reactive oxygen species (ROS), drugs, and toxins react with cysteine residues on KEAP1 resulting in structural changes and the accumulation of NRF2 to translocate to the nucleus and function as a transcriptional factor. In the nucleus, NRF2 heterodimerizes with small MAF proteins and binds to antioxidant response elements to induce a series of target genes for detoxification of ROS, toxins, and drugs. KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; β-TrCP, beta-transducin repeat containing protein; GSK-3, glycogen synthase kinase 3; Ub, ubiquitin; ARE, antioxidant response element; MAF, musculoaponeurotic fibrosarcoma; TXN, thioredoxin; TXNRD1, thioredoxin reductase 1; PRDX1, peroxiredoxin-1; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; NQO1, NADPH-quinone Dehydrogenase 1; ABC, ATP-binding cassette

Figure 2:. Mutation Spectrum in the KEAP1/NRF2 Pathway

A) Frequency of mutations in NRF2 and KEAP1 in solid tumors generating using cBioportal TCGA data. B) Map of KEAP1 with mutations based on cBioportal TCGA datasets. KEAP1 is divided up into the following domains: NTR, BTB, IVR, 6 Kelch domains, and CTR. Key cysteine residues for sensing ROS and toxins are indicated. C) Map of NRF2 with 7 Neh domains and mutations labelled. The KEAP1 binding motifs, DLG and ETGE, are indicated in the Neh2 domain, while the loci of phosphorylation by β-TrCP is located in the Neh6 domain. Somatic mutations in NRF2 are highly concentrated in DLG and ETGE motifs. SCC, squamous cell carcinoma; pRCC, papillary renal cell carcinoma; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; NTR, N terminal region; BTB, Bric-a-brac; IVR, intervening region; DGR, double glycine repeat; CTR, C terminal region; Maf, musculoaponeurotic fibrosarcoma; ARE, antioxidant response element

Figure 3:. Metabolic Rewiring by NRF2

Activation of NRF2 dramatically enhances generation of glutathione by increasing synthesis of glutathione from intracellular glutamate, cysteine, and glycine. Intracellular glutamate is derived from glutamine through GLS-1. Cystine is imported by the NRF2 target SLC7A11. Serine and glycine are synthesized via NRF2 dependent processes. NADPH is synthesized to support redox metabolism by the pentose phosphate pathway. GLS-1 and SLC7A11 function can be impaired by CB-839 and erastin respectively. PHGD, phosphoglycerate dehydrogenase; PSAT1, phosphoserine aminotransferase; PSPH, phosphoserine phosphatase; SHMT, serine hydroxymethyltransferase; G6PD, glucose-6-phopshate dehydrogenase; PGLS, 6-phosphogluconolactonase; 6PGD, 6-phosphogluctonate dehydrogenase; TKT, transketolase; TAL, transaldolase; GPX4, glutathione peroxidase 4; GR, glutathione reductase; GLS1, glutaminase 1; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GSS, glutathione synthetase; TR, thioredoxin; NAD(P)H, nicotinic adenine dinucleotide (phosphate); PUFA, polyunsaturated fatty acid; THF, tetrahydrofolate

Figure 4:. NRF2 Dependent Drug Detoxification

Aquated form of cisplatin targets both mitochondrial and genomic DNA by anchoring to nucleotides at guanine-guanine or guanine-adenine bonds, which cause severe DNA damage and generates ROS. Hyperactivated NRF2 maintains high glutathione and thioredoxin levels, which scavenge ROS. Toxic aquated forms of cisplatin are conjugated with glutathione by a NRF2 induced transferase, GST, and then excreted by multi-drug resistant pumps, which are also transcriptional targets of NRF2. ROS, reactive oxygen species, GSH, glutathione; GSSG, glutathione disulfide; TXN, thioredoxin; glutathione synthetase; GR, glutathione reductase; TXNRD1, thioredoxin reductase 1; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GST, glutathione s-transferase; MRP, multi-drug resistance protein

Figure 5:. Impact of Keap1/Nrf2 Mutation on Tumor Microenvironment and Anti-tumor Immune Responses

Keap1 LOF/ Nrf2 GOF mutation harboring tumors display increased uptake of non-essential amino acids such as glycine, serine, and glutamine. Cystine is imported through NRF2 regulated transporter xCT. Overall, in the microenvironment of Keap1/Nrf2 mutant tumors, glutamate is increased while cystine, glycine, glutamine, serine is depleted These metabolic changes can inhibit effector T cell function (expansion, production of IFN-γ) and induce apoptosis. Besides amino acids, Keap1/Nrf2 mutations result in increased glycolysis and thus increased glucose consumption and lactate secretion which can be deleterious for T cell function. NRF2 is a master regulator of antioxidants that decrease ROS and lipid peroxides which can impact dendritic cells, T cell effector cells and T regulatory cells. Hyperactivation of NRF2 results in altered heme metabolism and the byproducts of these pathways can impact neutrophils, macrophages, T regulatory cells and cytotoxic T lymphocytes.