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Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H Kit

Acute myeloid leukemia (AML) carries a 10–100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P3) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P3 signaling to leukemogenesis that warrants the exploration of S1P3 antagonists in preclinical AML models.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders.

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 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.

Mutations in the transcription factor NF-E2 in patients with myeloproliferative neoplasms result in a truncated protein that enhances the function of wild-type NF-E2 and causes erythrocytosis and throbocytosis in a mouse model., 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.

Mice expressing a transgene encoding the transcription factor NF-E2 in hematopoietic cells exhibit features of myeloproliferative neoplasms, including thrombocytosis, Epo-independent colony formation, stem and progenitor cell overabundance, leukocytosis, and progression to acute myeloid leukemia., 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.

The transcription factor family of nuclear factor I (NFI) proteins is encoded by four closely related genes: Nfia, Nfib, Nfic, and Nfix. A potential role for NFI proteins in regulating developmental processes has been implicated by their specific expression pattern during embryonic development and by analysis of NFI-deficient mice. It was shown that loss of NFIA results in hydrocephalus and agenesis of the corpus callosum and that NFIB deficiency leads to neurological defects and to severe lung hypoplasia, whereas Nfic knockout mice exhibit specific tooth defects. Here we report the knockout analysis of the fourth and last member of this gene family, Nfix. Loss of NFIX is postnatally lethal and leads to hydrocephalus and to a partial agenesis of the corpus callosum. Furthermore, NFIX-deficient mice develop a deformation of the spine, which is due to a delay in ossification of vertebral bodies and a progressive degeneration of intervertebral disks. Impaired endochondral ossification and decreased mineralization were also observed in femoral sections of Nfix−/− mice. Consistent with the defects in bone ossification we could show that the expression level of tetranectin, a plasminogen-binding protein involved in mineralization, is specifically downregulated in bones of NFIX-deficient mice.

Transcription factor Nuclear Factor One (NFI) proteins are derived from a small family of four vertebrate genes (NFIA, B, C and X), all of which produce a fair number of protein variants by alternative splicing. In order to ultimately locate RNA signal sequences around exon/intron borders for the production of regulated splice variants, we have determined the exon structure of the chicken NFIB gene as the last of the four vertebrate genes for which the gene structure was not yet elucidated. This made it possible to compile nine newly isolated and sequenced mouse NFI cDNA sequences together with all previously available ones and to deduce corresponding splicing patterns for the orthologous vertebrate genes of all four paralogous gene types. Results from the analysis of alternative splicing and of NFI gene mapping in the genome of human and mouse argue for a phylogenetic route in which the four vertebrate NFI genes result from a single duplication of a genomic segment containing two NFI intermediate genes rather than from two independent duplications of two separated single ancestor genes.

Binding sites for transcription factor nuclear factor one (NFI) proteins, encoded by four genes in the mouse, have been characterized from many tissue-specific genes. NFI genes are expressed in unique but overlapping patterns in embryonic and in adult tissues. Nfib is highly expressed in the embryonic lung. Here we show that Nfib null mutants die early postnatally and display severe lung hypoplasia. Heterozygotes do survive, but exhibit delayed pulmonary differentiation. Expression of transforming growth factor beta 1 (TGF-beta1) and sonic hedgehog (Shh) is not down-regulated in mutant lung epithelium at late stages of morphogenesis, which may result in incomplete lung maturation. Our study demonstrates that Nfib is essential for normal lung development, and suggests that it could be involved in the pathogenesis of respiratory distress syndromes in humans.

Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have recently shown that NF-E2 overexpression in a murine model causes a phenotype similar to MPN in humans. This includes thrombocytosis, leukocytosis, expansion of the stem- and progenitor cell compartments as well as spontaneous transformation to acute leukemia. However, both the downstream pathways by which this transcription factor exerts its effects as well as the mechanisms leading to its overexpression in MPN patients remain incompletely characterized. We show here that the histone demethylases JMJD1C and JMJD2C constitute novel NF-E2 target genes. Using chromatin immunoprecipitation (ChIP) we demonstrate NF-E2 binding to the JMJD1C and JMJD2C loci. Both JMJD1C and JMJD2C protein levels are statistically significantly elevated in patients with polycythemia vera (PV) compared to healthy controls. JMJD1C and 2C proteins mediate the demethylation of histone H3K9, converting H3K9me2 via H3K9me to unmethylated H3K9. Consistent with increased demethylase activity, we observed statistically significantly decreased levels of global H3K9me and H3K9me2 in PV patients compared to healthy controls. We subsequently investigated whether the NF-E2 gene is regulated by epigenetic mechanisms and whether these are altered in PV patients. Indeed, using ChIP we demonstrate presence of the repressive H3K9me2 mark at three sites within the NF-E2 locus in healthy control granulocytes, where NF-E2 is expressed at low levels. These repressive histone marks are significantly reduced or completely absent in PV patients, which overexpress NF-E2. At the same time, binding of the repressive heterochromatin protein 1-alpha (HP1a) to the NF-E2 gene, while present in healthy controls, is significantly reduced in PV patients. HP1a chromatin binding is dependent on the presence of the H3K9me2 modification. Decreased levels of H3K9me2 and decreased HP1a binding are therefore both consistent with elevated NF-E2 expression in PV. We hypothesized that increased levels of the H3K9me2 demethylase JMJD1C in PV patients are responsible for the loss of this histone mark in the NF-E2 gene. Consistent with this model, we demonstrate significantly increased binding of JMJD1C to the NF-E2 locus in purified primary PV granulocytes. Our data demonstrate that the NF-E2 gene is regulated by epigenetic mechanisms and that these histone modifications are perturbed in PV patients. Moreover, NF-E2 participates in an auto-regulatory loop, by directly regulating transcription of the histone demethylase JMJD1C. Increased levels of NF-E2 thus cause increased JMJD1C expression. The JMJD1C protein, in turn, demethylates H3K9me2 residues in the NF-E2 locus, thereby further augmenting NF-E2 expression. Besides sustaining this novel autoregulatory loop, increased JMJD1C activity may contribute to MPN pathology by altering gene expression at additional loci. We sought to determine whether elevated NF-E2 levels could be normalized by pharmacological intervention. Treatment with the nucleoside analogue Decitabine reduced NF-E2 expression, and restored both the H3K9me2 marks and HP1a binding to the NF-E2 locus. These data provide a molecular rational for pre-clinical investigation of the effects of histone demethylase inhibitors and Decitabine on MPN cell biology and, subsequently, for a phase I trial investigating a combination of Decitabine and histone demethylase inhibitors in MPN patients. Disclosures: No relevant conflicts of interest to declare.

Recently, we described insertion and deletion mutations in transcription factor NF-E2 in a subset of patients with Myeloproliferative Neoplasms (MPN), most frequently patients with primary or secondary myelofibrosis (PMF or sMF, respectively) as well as patients with polycythemia vera (PV). These mutations lead to the formation of truncated NF-E2 proteins that lack DNA binding activity. Nonetheless, mutant NF-E2 proteins augment wild-type NF-E2 activity both in vitro and in vivo in a murine bone marrow transplant model. Mice expressing mutant NF-E2 develop erythrocytosis and thrombocytosis. Here, we demonstrate that, in contrast to mice expressing wild-type NF-E2, treatment of mice expressing mutant NF-E2 with interferon-alpha does not lower red blood cell numbers (RBC), white blood cell counts (WBC) or platelet numbers. While these parameters are significantly decreased by interferon-alpha (IFN) in mice expressing wild-type NF-E2, mice expressing mutant NF-E2 showed no response to interferon treatment in these counts over a four month period. Two PV patients carrying truncating mutations in NF-E2 received IFN treatment. Blood samples were available both from before treatment as well as from 3 and 4 years after the beginning of therapy, respectively. Purified granulocytes from these samples were analyzed for mutant NF-E2 allele burden by GeneScan analysis. In both patients, the mutant NF-E2 allele burden rose during IFN treatment, increasing from 1% to 30% in one patient and from 24% to 100% in the second. We have demonstrated that acquisition of an NF-E2 mutation confers a proliferative advantage on cells already carrying the JAK2V617F mutation. Using colony assays from primary MPN cells, we showed that cells carrying both mutations outcompete cells carrying only the JAK2V617F mutation in vivo. Cells expressing both mutant NF-E2 and JAK2V617F are statistically significantly more frequently in the S-phase of the cell cycle and express significantly higher levels of several proteins that promote G1/S transition, cyclin D3, CDK4 and CDK6. The data presented here suggest that IFN treatment is insufficient to counteract the proliferative drive conferred by mutant NF-E2. Disclosures: No relevant conflicts of interest to declare.