Abstract
Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = −0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7–8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.
Highlights
Nuclear factor-like 2 (NFE2L2), or nuclear factor erythroid 2-related factor 2 (NRF2), is a transcription factor that maintains cellular redox homeostasis by regulating the expression of antioxidant genes, including glutathione peroxidase, thioredoxin 1, thioredoxin reductase, peroxiredoxin 1, and sulfiredoxin 1 [1]
NAD(P)H quinone oxidoreductase-1 (NQO1) is a detoxification gene that is regulated by the NRF2 pathway, and NQO1 gene expression or promoter activity has been used as a reporter for NRF2 activity [2, 35]
On the basis of these data, we propose the following as the mechanism of action model for NRF2 synthetic lethality compound-01 (NSLC01) (Fig. 6): NSLC01 selectively activates eEF2K by inhibiting its phosphorylation at S366; activated eEF2K phosphorylates the downstream eEF2 at T56, inhibiting its translation elongation function; this inhibition of translation reduces the availability of enzymes (ASNS, GHPDH, phosphohydroxythreonine aminotransferase 1 (PSAT1), and PSPH) that drive the synthesis of asparagine, and likely, serine, necessary for cancer cell survival and proliferation
Summary
Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), or nuclear factor erythroid 2-related factor 2 (NRF2), is a transcription factor that maintains cellular redox homeostasis by regulating the expression of antioxidant genes, including glutathione peroxidase, thioredoxin 1, thioredoxin reductase, peroxiredoxin 1, and sulfiredoxin 1 [1]. NRF2 is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1), which binds to NRF2 and the E3 ubiquitin ligase Cullin-3 (CUL3) protein to drive proteasome degradation of NRF2 [2,3,4,5]. Other oncogenic signaling pathways, including AKT, MAPK, PKC, and c-Myc, regulate KEAP1/NRF2 pathways through direct phosphorylation of NRF2 or through transcriptional or post-transcriptional regulation of NRF2 expression [7,8,9]. Gene mutations in the KEAP1/ NRF2 pathway are rare; NRF2 expression levels are high in over 93% of pancreatic adenocarcinomas [14]. The overexpression is believed to be a consequence of the near-universal presence of oncogenic KRAS gene mutations and downstream activation of the MAPK pathway and high levels of c-Myc [14, 15]. The oncogenic pathway-induced NRF2 expression in pancreatic cancer enhances ROS detoxification and modulates protein translation to promote tumorigenesis and drug resistance [14,15,16]
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