RESUMEN
Aims: The nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (NRF2-KEAP1) pathway plays an important role in the cellular response to oxidative stress but may also contribute to metabolic changes and drug resistance in cancer. However, despite its pervasiveness and important role, most of nuclear factor erythroid 2-related factor 2 (NRF2) target genes are defined in context-specific experiments and analysis, making it difficult to translate from one situation to another. Our study investigates whether a core NRF2 gene signature can be derived and used to represent NRF2 activation in various contexts, allowing better reproducibility and understanding of NRF2. Results: We define a core set of 14 upregulated NRF2 target genes from 7 RNA-sequencing datasets that we generated and analyzed. This NRF2 gene signature was validated using analyses of published datasets and gene sets. An NRF2 activity score based on expression of these core target genes correlates with resistance to drugs such as PX-12 and necrosulfonamide but not to paclitaxel or bardoxolone methyl. We validated these findings in our Kelch-like ECH-associated protein 1 (KEAP1) knockout cancer cell lines. Finally, our NRF2 score is prognostic for cancer survival and validated in additional independent cohorts for lung adenocarcinoma and also novel cancer types not associated with NRF2-KEAP1 mutations such as clear cell renal carcinoma, hepatocellular carcinoma, and acute myeloid leukemia. Innovation and Conclusions: These analyses define a core NRF2 gene signature that is robust, versatile, and useful for evaluating NRF2 activity and for predicting drug resistance and cancer prognosis. Using this gene signature, we uncovered novel selective drug resistance and cancer prognosis associated with NRF2 activation.
RESUMEN
Synthetic oleanane triterpenoids (SOTs) are small molecules with broad anticancer properties. A recently developed SOT, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-2-yl)-1H-imidazole (CDDO-2P-Im or '2P-Im'), exhibits enhanced activity and improved pharmacokinetics over CDDO-Im, a previous generation SOT. However, the mechanisms leading to these properties are not defined. Here, we show the synergy of 2P-Im and the proteasome inhibitor ixazomib in human multiple myeloma (MM) cells and 2P-Im activity in a murine model of plasmacytoma. RNA sequencing and quantitative reverse transcription PCR revealed the upregulation of the unfolded protein response (UPR) in MM cells upon 2P-lm treatment, implicating the activation of the UPR as a key step in 2P-Im-induced apoptosis. Supporting this hypothesis, the deletion of genes encoding either protein kinase R-like endoplasmic reticulum kinase (PERK) or DNA damage-inducible transcript 3 protein (DDIT3; also known as CHOP) impaired the MM response to 2P-Im, as did treatment with ISRIB, integrated stress response inhibitor, which inhibits UPR signaling downstream of PERK. Finally, both drug affinity responsive target stability and thermal shift assays demonstrated direct binding of 2P-Im to endoplasmic reticulum chaperone BiP (GRP78/BiP), a stress-inducible key signaling molecule of the UPR. These data reveal GRP78/BiP as a novel target of SOTs, and specifically of 2P-Im, and suggest the potential broader utility of this class of small molecules as modulators of the UPR.