New C3H KitN824K/WT cancer mouse model develops late-onset malignant mammary tumors with high penetrance.

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 KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

Pharmacological Inhibition of Insulin Growth Factor-1 Receptor (IGF-1R) Alone or in Combination With Ruxolitinib Shows Therapeutic Efficacy in Preclinical Myeloproliferative Neoplasm Models.

Even after development of the JAK1/JAK2 inhibitor ruxolitinib, myeloproliferative neoplasm (MPN) patients require novel therapeutic options. While ruxolitinib can considerably improve quality of life and prolong survival, it does not modify the natural disease course in most patients. Moreover, resistance develops with prolonged use. Therefore, various combination treatments are currently being investigated. Published data provide a compelling rationale for the inhibition of insulin growth factor-1 receptor (IGF-1R) signaling in MPN. Here we report that genetic and pharmacological inhibition of IGF-1R selectively reduced Jak2V617F-driven cytokine-independent proliferation ex vivo. Two different structurally unrelated IGF-1R inhibitors ameliorated disease phenotype in a murine MPN model and significantly prolonged survival. Moreover, in mice, low-dose ruxolitinib synergized with IGF-1R inhibition to increase survival. Our data demonstrate preclinical efficacy of IGF-1R inhibition in a murine MPN model.

The transcription factor NFE2 enhances expression of the hematopoietic master regulators SCL/TAL1 and GATA2.

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.

Enhanced expression of the sphingosine-1-phosphate-receptor-3 causes acute myelogenous leukemia in mice.

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.

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.

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.

Nuclear factor I X deficiency causes brain malformation and severe skeletal defects.

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.

Genomic organization, splice products and mouse chromosomal localization of genes for transcription factor Nuclear Factor One.

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.

Nuclear factor I-B (Nfib) deficient mice have severe lung hypoplasia.

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.