Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein1. One approach to addressing this challenge is to define frequently co-occurring mutations with KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function (LOF) mutations in Kelch-like ECH-associated protein 1 (KEAP1)2-4, a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response5-10. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR/Cas9-based approach in a mouse model of Kras-driven LUAD we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyper-activates Nrf2 and promotes Kras-driven LUAD. Combining CRISPR/Cas9-based genetic screening and metabolomic analyses, we show that Keap1/Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for sub-stratification of human lung cancer patients with KRAS-KEAP1 or -NRF2-mutant tumors as likely to respond to glutaminase inhibition.

Summary Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nrf2/Nfe2l2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine EGFR signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

Protein Persulfide Detection Protocol reveals vital roles for thioredoxin and glutathione systems in maintaining sulfane sulfur homeostasis in cells and in vivo.

The bacteriophage P1 Cre͞loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. Although in vitro studies have shown that Cre can catalyze recombination between cryptic ''pseudo-loxP'' sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals. We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids. All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected. Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating. Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility. Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance. Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected. This study indicates that, following the use of the Cre͞loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.T he bacteriophage P1 recombinase, Cre, is a member of the integrase family of site-specific recombinases that catalyzes recombination between loxP DNA elements as a part of the normal viral life cycle (1, 2). All members of the integrase family share a similar mechanism of action (3). In Cre-mediated recombination, individual loxP sites are bound by Cre homodimers (4). Two Cre-bound loxP sites interact in the recombination reaction. The loxP site is a 34-bp element consisting of two 13-bp inverted repeats separated by a directional 8-bp core (1, 5-7). During cleavage, Cre Tyr 324 becomes covalently linked to the 3Ј-phosphate, forming a DNA-phosphotyrosine intermediate (8, 9). Interaction with a second loxP-bound Cre dimer allows nucleophilic attack of the 3Ј-phosphotyrosine in one loxP site by the free 5Ј-hydroxyl of the other. This ligation reaction induces a structural rearrangement of the tetrameric complex, allowing the same reaction to occur on the second strand of each loxP site, thus completing the strand exchange reaction (8,(10)(11)(12).Cre has proven to be a valuable tool for manipulating the genomes of mammalian cells and of mice in situ (13-19). As an example, Cre-mediated recombination has all but eliminated the problem of ''selection cassette effects'' associated with some targeted mutations (15). Although Cre is most commonly used to generate short deletions in transgenic mice (13), it has also proven useful for recombination between sites up to several centimorgans apart on individual c...

Abstract. Cell sizes can differ vastly between cell types in individual metazoan organisms. In rat liver, spleen, and thymus, differences in average cell size roughly reflect differences in RNA:DNA ratios. For example, hepatocytes were found to have a cytoplasmic:nuclear volume ratio and an RNA:DNA ratio which were 34-and 21-fold higher, respectively, than those in thymocytes. RNA synthesis per DNAequivalent in the hepatocytes was 25-fold greater than that in thymocytes, suggesting that differences in overall transcriptional activity, not differences in overall RNA stability, were primarily responsible for determining cellular RNA:DNA ratios.

Investigation of the role of TrxR1 as a regulator of Nrf2 activation will facilitate further studies of redox control in diverse cells and tissues of mammals, and possibly also in animals of other classes.

BackgroundMetabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins.Principal FindingsHere we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each.ConclusionsChronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response.

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