Overexpression of NF-E2 in vivo: A Murine Model of Myeloproliferative Neoplasms
The transcription factor nuclear factor erythroid-2 (NF-E2) is expressed in hematopoietic stem cells as well as in myeloid, erythroid and megakaryocytic precursors. NF-E2 deficient mice display marked anemia at birth and die perinatally due to thrombopenia, demonstrating an essential role for NF-E2 in both in erythropoiesis and platelet formation. We have previously shown that NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPNs). However, the effect of augmented transcription factor activity has not been studied in vivo. We therefore engineered two independent transgenic mouse lines expressing human NF-E2 under the control of the vav-Promoter, which has previously been shown to direct transgene expression in hematopoietic stem cells as well as in precursor cells of all lineages.
The two founder lines show overlapping but distinct phenotypes. Epo-independent colony formation, a pathognomonic feature of polycythemia vera, is significantly increased in NF-E2 transgenic animals. Bone marrow histopathology shows findings characteristically seen in MPNs, including the presence of increased megakaryopoiesis with cytologically abnormal forms, often in clusters. Both NF-E2 transgenic strains display significantly increased mortality. Upon autopsy, between 15 and 20% of mice in both strains present with major gastrointestinal bleeding in conjunction with splenic atrophy. Histopathological examination of all spleens revealed mild to moderately expanded red pulp with increased numbers of iron containing histiocytes. This observation indicates increased red cell destruction and may explain the fact that neither hematocrit nor hemoglobin are elevated in NF-E2 transgenic animals. At 18 months of age, one mouse developed acute leukemia, which is currently being phenotyped. In summary, in a murine model moderate NF-E2 overexpression causes a phenotype resembling Essential Thrombocythemia. In addition, our preliminary data indicate that NF-E2 overexpression may predispose to the development of acute leukemia.
The role of transcription factor NF-E2 in the pathophysiology of MPNs
The molecular etiology of Polycythemia vera (PV) remains unknown, despite the recent description of a point mutation in the JAK2 kinase (JAK2V617F).
Gene expression profiling has been successfully used to reveal candidate genes involved in disease development.
We thus performed cDNA microarray analysis on 40 patients with PV 50 healthy controls and 12 patients with secondary erythrocytosis (SE) (Goerttler, 2005).
253 genes upregulated and 391 genes downregulated more than 1.5-fold in PV compared to healthy controls (p < 0.01) were identified. Of these, the transcription factor NF-E2, is overexpressed between 2- and 40-fold in PV patients. In PV bone marrow, NF-E2 is overexpressed in megakaryocytes, erythroid and granulocytic precursors. It has been shown that overexpression of NF-E2 leads to the development of Epo-independent erythroid colonies and that ectopic NF-E2 expression can reprogram monocytic cells towards erythroid and megakaryocytic differentiation.
Transcription factor concentration may thus control lineage commitment. We therefore propose that in PV patients elevated concentrations of NF-E2 lead to an overproduction of erythroid and, in select patients, megakaryocytic cells/platelets. In this model the level of NF-E2 overexpression determines both the severity of erythrocytosis and the concurrent presence or absence of thrombocytosis.
The recent description of the Jak2V617F mutation in PV patients raises the question whether this allele exerts its effect on the malignant clone in part through inducing NF-E2 expression.
Both the molecular mechanism leading to NF-E2 overexpression and its effect on human hematopoiesis are not known. Therefore, it is the aim of this project to investigate the cause of NF-E2 overexpression in PV and the effect of NF-E2 overexpression in hematopoietic cells.
Based on the following hypotheses, the specific aims of this project are therefore:
1. Hypothesis: NF-E2 is required for the Epo-independent growth of PV cells Specific Aim: To modulate NF-E2 expression via siRNA knock down and retroviral or lentiviral transduction and examine the consequences on Epo-independent growth in vitro.
2. Hypothesis: NF-E2 and PRV-1 overexpression in PV are mediated by the Jak2V617F allele Specific Aim: To introduce Jak2 wt and V617F alleles in vivo and in vitro and examine the effects on NF-E2 and PRV-1 expression in various models.
Molecular heterogeneity in Essential Thrombycythemia
Essential Thrombocythemia (ET) is one of three diseases termed the chronic myeloproliferative disorders (MPDs). Besides ET this group includes Polycythemia vera (PV) and idiopathic myelofibrosis (IMF). The clinical presentation and course of patients with ET is very heterogeneous. The recent discovery of a point mutation in the JAK2 kinase (JAK2V617F) in only a proportion of ET patients (40 – 60 %) strengthens the hypothesis that several distinct alterations may lead to the development of this clinically defined disorder.
Several molecular markers have been described in ET to date. Besides JAK2V617F, these include clonal hematopoiesis, endogenous erythroid colony (EEC) growth and PRV-1 overexpression. These markers allow the subclassification of ET patients and are potentially useful for the elucidation of distinct molecular disease etiologies. In addition, it has been proposed that hypersensitivity to thrombopoietin (TPO) underlies the development of ET and differentiates it from Polycythemia vera (PV), which is defined by IGF-1 hypersensitivity. However, the presence of molecular markers has not been correlated to growth factor hypersensitivity. Because of these advances, the following questions arise: 1) What alterations underlie the molecular etiology of JAK2wt/PRV-1 normal ET? 2) How does the identical mutation, JAK2V617F, lead to the wide variety of clinical presentations observed in MPD patients? 3) Can molecular markers be used for risk stratification in individual ET patients? Based on the following hypotheses, the Specific Aims of this project are therefore:
Hypothesis: ET is comprised of at least two molecularily distinct entities, which give rise to a similar clinical phenotype. Aim: To perform microarray studies analyzing gene expression in 20 JAK2V617F/PRV-1 positive and 20 JAK2wt/PRV-1 negative ET patients.
Hypothesis: Presence of JAK2V617F and PRV-1 overexpression are correlated with growth factor hypersensitivity. Aim: To determine TPO and IGF-1 hypersensitivities in a cohort of 15 JAK2V617F/PRV-1 positive and 15 JAK2wt/PRV-1 negative ET patients and to correlate the presence of JAK2V617F and PRV-1 overexpresion with this parameter.
Hypothesis: PRV-1 mRNA overexpression leads to an increase in PRV-1 serum protein levels and this is correlated with the risk of developing thrombotic complications. Aim: To determine PRV-1 serum levels by ELISA in a cohort of 100 healthy controls and 140 ET patients and to assess whether PRV-1 protein levels correlate with thrombotic risk.
Epigenetic effects of transcription factor NF-E2 in Myeloproliferative Neoplasms:pathophysiology and therapeutic target
Myeloproliferative neoplasms (MPN) constitute a group of pharmacologically incurable hematologic malignancies. We have previously shown that the expression of transcription factor “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in the vast majority of MPN patients. NF-E2 has been demonstrated to remodel chromatin, to establish histone marks and to recruit both histone acety-lases and the MLL2 methyltransferase complex. Our NF-E2 overexpressing transgenic (tg) mouse model shows a phenotype characteristic of MPNs in humans including thrombocytosis and trans-formation to acute leukemia. In addition, NF-E2 tg mice display alterations in histone modifications. Treatment with the histone deacetylase inhibitor Vorinostat normalized platelet counts in NF-E2 transgenic mice and reversed the altered histone acetylation. Finally, we have identified epigenetic modifiers as novel NF-E2 target genes. Based on these data, we will test the following hypotheses:
1. Hypothesis 1:The pathology of NF-E2 transgenic mice is mediated, at least in part, by epigenetic changes effected by overexpression of the NF-E2 protein.Specific Aim 1:To investigate epigenetic changes (histone marks, chromatin occupancy) in NF-E2 overexpressing mice.
2. Hypothesis 2:The pathologic changes caused by NF-E2 overexpression can be reversed by treatment with inhibitors of chromatin modifying enzymes.Specific Aim 2:To treat NF-E2 transgenic mice with SAHA (Vorinostat) and other inhibitors of histone modifying enzymes and to observe physiological parameters (blood counts, hematopoietic colony formation, bone marrow morphology and spleen size).
3. Hypothesis 3:Novel NF-E2 target genes mediate epigenetic modificationsSpecific Aim 3:To characterizenovel NF-E2 target genes, such as JMJD1C and JMJD2C and their activity in NF-E2 tg mice and MPN patients.
The long-term goals of this project are to demonstrate the pre-clinical efficacy of epigenetic therapy in a MPN model and to translate these results into a novel therapy for patients with MPN.