Epigenetic regulation of NFE2 overexpression in myeloproliferative neoplasms.

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.

A novel role for nuclear factor-erythroid 2 in erythroid maturation by modulation of mitochondrial autophagy.

We have recently demonstrated that the transcription factor nuclear factor-erythroid 2, which is critical for erythroid maturation and globin gene expression, plays an important role in the pathophysiology of myeloproliferative neoplasms. Myeloproliferative neoplasm patients display elevated levels of nuclear factor-erythroid 2 and transgenic mice overexpressing the transcription factor develop myeloproliferative neoplasm, albeit, surprisingly without erythrocytosis. Nuclear factor-erythroid 2 transgenic mice show both a reticulocytosis and a concomitant increase in iron deposits in the spleen, suggesting both enhanced erythrocyte production and increased red blood cell destruction. We therefore hypothesized that elevated nuclear factor-erythroid 2 levels may lead to increased erythrocyte destruction by interfering with organelle clearance during erythroid maturation. We have previously shown that nuclear factor-erythroid 2 overexpression delays erythroid maturation of human hematopoietic stem cells. Here we report that increased nuclear factor-erythroid 2 levels also impede murine maturation by retarding mitochondrial depolarization and delaying mitochondrial elimination. In addition, ribosome autophagy is delayed in transgenics. We demonstrate that the autophagy genes NIX and ULK1 are direct novel nuclear factor-erythroid 2 target genes, as these loci are bound by nuclear factor-erythroid 2 in chromatin immunoprecipitation assays. Moreover, Nix and Ulk1 expression is increased in transgenic mice and in granulocytes from polycythemia vera patients. This is the first report implying a role for nuclear factor-erythroid 2 in erythroid maturation by affecting autophagy.

The anti-inflammatory effects of E-α-(p-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone are mediated via HO-1 induction.

Inflammation plays a central role in the pathophysiology of many diseases. The inducible enzyme heme oxygenase-1 (HO-1) protects cells against inflammation and can be induced by electrophilic compounds like the chalcones (1,3-diphenylprop-2-enones) from the class of α,ß-unsaturated carbonyl compounds. We hypothesized that the synthetic chalcone E-α-(p-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) exerts anti-inflammatory effects in RAW264.7, Jurkat lymphocytes and HK-2 cells via HO-1 induction. RAW264.7 cells were treated with lipopolysaccharide prior to E-α-p-OMe-C6H4-TMC treatment. Subsequently, HO-1 protein induction and activity were analyzed, as well as expression of pro- and anti-inflammatory mediators, transcription factors and mitogen-activated protein kinases to evaluate the possible molecular mechanism. These results were confirmed in human cell lines (Jurkat T-lymphocytes and HK-2 epithelial cells). We found that the E-α-p-OMe-C6H4-TMC exerts significant anti-inflammatory effects in a dose dependent manner, showing no toxic effects in LPS-treated RAW264.7 macrophages. E-α-p-OMe-C6H4-TMC induced HO-1 and SOD-1 protein expression and HO-1 enzyme activity, reduced the upregulation of COX-2 and iNOS, while inducing the translocation of Nrf2. NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment accompanied by the downregulation of proinflammatory cytokines IL-1ß, IL-6 and MCP-1. Pretreatment with E-α-p-OMe-C6H4-TMC revealed significant changes in phosphorylation of ERK and p38, but not JNK. These anti-inflammatory effects of E-α-p-OMe-C6H4-TMC were approved in Jurkat and HK-2 cells, furthermore revealing a downregulation of IL-8 and IL-10. In conclusion, it is tempting to speculate about E-α-p-OMe-C6H4-TMC as a new and non-toxic agent, inducing HO-1 in cells. This opens up new opportunities regarding the development of therapeutic agents using beneficial effects of HO-1 and its products.

MPN patients harbor recurrent truncating mutations in transcription factor NF-E2.

The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs.

A novel murine model of myeloproliferative disorders generated by overexpression of the transcription factor NF-E2.

The molecular pathophysiology of myeloproliferative neoplasms (MPNs) remains poorly understood. Based on the observation that the transcription factor NF-E2 is often overexpressed in MPN patients, independent of the presence of other molecular aberrations, we generated mice expressing an NF-E2 transgene in hematopoietic cells. These mice exhibit many features of MPNs, including thrombocytosis, leukocytosis, Epo-independent colony formation, characteristic bone marrow histology, expansion of stem and progenitor compartments, and spontaneous transformation to acute myeloid leukemia. The MPN phenotype is transplantable to secondary recipient mice. NF-E2 can alter histone modifications, and NF-E2 transgenic mice show hypoacetylation of histone H3. Treatment of mice with the histone deacetylase inhibitor (HDAC-I) vorinostat restored physiological levels of histone H3 acetylation, decreased NF-E2 expression, and normalized platelet numbers. Similarly, MPN patients treated with an HDAC-I exhibited a decrease in NF-E2 expression. These data establish a role for NF-E2 in the pathophysiology of MPNs and provide a molecular rationale for investigating epigenetic alterations as novel targets for rationally designed MPN therapies.

Nitrate assimilatory enzymes of Spirulina (Arthospira) platensis are more thermotolerant than those of rice.

In order to assess the potential of Spirulina (Arthospira) platensis as a source of abundant, thermostable nitrate assimilatory enzymes, the specific activities and thermal tolerance of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) were compared with those of rice in crude extracts in vitro. The results show that Spirulina enzymes have relatively higher thermotolerance. When the extracts were pre-exposed to 80 °C for 1 hr, Spirulina enzymes retained higher activities by 3.4, 1.7 and 3.7 fold, respectively than corresponding enzymes in rice. This property was not due to salts and other small proteins/molecules, as their removal by gel filtration (G-25) did not affect their thermotolerance.