The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

Nuclear factor E2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper (CNC-bZIP) transcription factor that is well established as a master regulator of phase II detoxification and antioxidant gene expression and is strongly expressed in tissues involved in xenobiotic metabolism including liver and kidney. Nrf2 is also abundantly expressed in adipose tissue; however, the exact function of Nrf2 in adipocyte biology is unclear. In the current study we show that targeted knock-out of Nrf2 in mice decreases adipose tissue mass, promotes formation of small adipocytes, and protects against weight gain and obesity otherwise induced by a high fat diet. In mouse embryonic fibroblasts, 3T3-L1 cells, and human subcutaneous preadipocytes, selective deficiency of Nrf2 impairs adipocyte differentiation. Fat cells play an important role in energy storage and metabolism and secrete a variety of factors that influence appetite, insulin sensitivity, inflammation, and many other pathways of biologic and clinical significance (1). Because of the significance of adipocyte biology in the pathogenesis of obesity and related metabolic, cardiovascular, and inflammatory disorders, there has been intense interest in defining the network of transcription factors that controls the expression of genes involved in fat cell development. Peroxisome proliferator-activated receptors (PPARs) 4 are ligand-activated transcription factors that belong to a nuclear hormone receptor family including related molecules that function by forming heterodimers with retinoid X receptors (2). PPAR␥ is expressed abundantly in adipose tissue and is considered to be the dominant transcriptional regulator of adipogenic differentiation (3). Accordingly, much attention has been directed at identifying factors that control PPAR␥ expression during the process of adipogenesis.CCAAT enhancer-binding proteins (C/EBPs) are leucine zipper transcription factors expressed in both white and brown adipose tissue and have been extensively studied for their roles in regulating PPAR␥ activity and adipogenesis (4, 5). C/EBP␤ and C/EBP␦ are transiently expressed at the onset of the adipose differentiation program (6, 7). This phase is then followed by induction of PPAR␥ and C/EBP␣ expression (8 -10). In addition, PPAR␥ and C/EBP␣ form a positive loop by regulating each other's expression (11,12). Although the enforced expression of C/EBP␣ in fibroblasts can trigger adipocytic differentiation, C/EBP␣ is incapable of inducing adipogenesis in the absence of PPAR␥ (13). In contrast, PPAR␥ can induce adipogenic differentiation in C/EBP␣-null cells indicating that PPAR␥ is proximal in effecting adipogenesis (14). Apart from C/EBP␤, -␦, and -␣, relatively few transcription factors have been described that bind to the Ppar␥ promoter and positively regulate Ppar␥ transcription during adipogenesis (15).The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a member of the cap-n-collar basic leucine zipper (CNC-bZIP) family of transcription factors and has been shown to pl...

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. We report here that mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibited steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer. hepatocellular carcinoma ͉ oxidative stress ͉ knockout mouse T ranscription of many cytoprotective genes and phase-2 xenobiotic metabolizing genes is regulated through cis-active sequences known as antioxidant response elements (ARE) (1, 2). Regulation of ARE function is mediated by various basic leucine zipper (bZIP) transcription factors including members of the ''cap n collar'' (CNC)-bZIP and small-Maf family of proteins. Nrf1 and Nrf2 are CNC-bZIP proteins, and they function as obligate heterodimers by complexing with small-Maf and other bZIP proteins (3). An important role for Nrf2 in xenobiotic metabolism and oxidative stress response had been identified through knockout studies in mice (4-10). These and other studies indicate that Nrf2 is an important activator of AREs.In contrast, the function of Nrf1 is not fully understood. Mice deficient in Nrf1 function die during development (11). Analysis of nrf1 and nrf1::nrf2 mutant cells suggests that Nrf1 is also involved in the oxidative stress response (12, 13). However, the importance of Nrf1 in this function in an intact animal is not certain because early lethality precludes analysis of Nrf1-deficient animals beyond embryonic development. Chimeric mice generated with Nrf1-deficient embryonic stem cells showed widespread apoptosis in fetal livers at late gestation, demonstrating a cell autonomous role of Nrf1 in the survival of hepatocytes (14). This finding suggests that Nrf1 is required for normal function of hepatocytes. Based on these findings, we hypothesize that Nrf1 is critical to the oxidative stress response in the adult liver, and that it plays an important role in oxidative stress-induced liver disease. To bypass embryonic lethality, we used a Cre-lox system ...

Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells.Reactive oxygen species (ROS) 1 are generated during aerobic respiration and normal metabolic processes, and they are also byproducts of metabolism of a wide range of environmental agents (1). High levels of ROS are detrimental to the cell, since they react readily with intracellular molecules, causing cell injury and death (1). Cells are equipped with a variety of defense mechanisms that work in parallel or in sequence to minimize ROS levels. These defenses include enzymes that are involved in ROS metabolism and biotransformation of xenobiotics (2). Examples of these enzymes include superoxide dismutases, glutathione peroxidases, thioredoxins, and heme-oxygenases as well as phase 2 enzymes, such as glutathione S-transferases. In addition to enzymatic defenses, cells are also equipped with molecules such as glutathione, metallothioneins, and ferritins that scavenge ROS and metal ions. Basal and inducible expression of a number of these antioxidant defense genes are mediated in part by a cis-acting DNA element known as the antioxidant response element (3-6). Activation through the antioxidant response element (ARE) appears to be driven by conditions that promote intracellular oxidative stress (5,7,8). A number of different transcription factors including basic leucine zipper proteins, AP-1, have been shown to bind the ARE (3, 9, 10).Recent studies implicate the CNC subfamily of bZIP proteins in mediating ARE function (11). Members o...