Projekte

Prof. Dr. Heike L. Pahl

Aktuelle Forschungsprojekte: zurück >


Molekulare Ursachen Myeloproliferativer Erkrankungen:

Mausmodelle Myeloproliferativer Erkrankungen
Die Rolle des Transkriptionsfaktors NF-E2 in der Pathophysiologie der MPNs
Molekulare Heterogenität der Essentiellen Thrombozythämie (ET)
Molekulare Marker Myeloproliferativer Erkrankungen
Molekulare Mechanismen der NF-E2 und PRV-1 Überexpression

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.

Current Lab Members on Project:

Lucas Ganzenmüller, MD student

Monika Gothwal, Ph. D. student

Dr. Albert Gründer, Post doc

Jonas Jutzi, MD student

Kien-Binh Pham, MD student

Alumni:

Tobias Hadlich, (MD pending)

Kai Kaufmann, (MD pending)

Sarah Kayser, (MD pending)

Jan Marx, (MD pending)

Laura Vandré, (MD pending)

Literature

Kaufmann KB, Gründer A, Hadlich T, Wehrle J, Gothwal M, Bogeska R, Seeger TS, Kayer S, Pham KB, Jutzi JS, Ganzenmüller L, Steinemann D, Schlegelberger B, Wagner JM, Jung M, Will B, Steidl U, Aumann K, Werner M, Günther T, Schüle R, Rambaldi A, Pahl HL, (2011) Overexpression of transcription factor NF-E2: a novel murine model of myeloproliferative neoplasms, J. Exp. Med, in press.

Wang W, Schwemmers S, Hexner EO, Pahl HL (2010) AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2. Blood, 116 (2): 254-266

Goerttler PS, Kreutz C, Donauer J, Faller D, Maiwald T, März E, Rumberger B, Sparna T, Schmitt-Gräff A, Wilpert J, Timmer J, Walz G, Pahl HL (2005) Gene Expression Profiling in Polycythemia vera: Overexpression of transcription factor NF-E2., Br. J. Haematol, 129: 138-150.95.

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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 (JAK2^V617F).
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 Jak2^V617F 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 Jak2^V617F 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.

Current Lab Members on Project:

Ruzhica Bogeska, Ph. D. student

Aitomi Essig, MD student

Dr. Thalia Seeger, Post doc

Julius Wehrle, MD student

Alumni:

Christian Arnold, (MD pending)

Martina Buerge, (MD 2011)

Dr. Angela Magin

Manuel Mutschler, (MD 2009)

Roland Roelz, (MD 2010)

Literature

Mutschler M, Magin AS, Buerge M, Roelz R, Schanne DH, Will B, Pilz I, Migliaccio AR, Pahl HL (2009) NF-E2 Overexpression Delays Erythroid Maturation and Increases Erythrocyte Production. , Br. J. Haematol, 146: 203-217.

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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 (JAK2^V617F) 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 JAK2^V617F, 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, JAK2^V617F, 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:

1. 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 JAK2^V617F/PRV-1 positive and 20 JAK2wt/PRV-1 negative ET patients.

2. Hypothesis: Presence of JAK2^V617F and PRV-1 overexpression are correlated with growth factor hypersensitivity. Aim: To determine TPO and IGF-1 hypersensitivities in a cohort of 15 JAK2^V617F/PRV-1 positive and 15 JAK2wt/PRV-1 negative ET patients and to correlate the presence of JAK2^V617F and PRV-1 overexpresion with this parameter.

3. 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.

Current Lab Members on Project:

Dr. Sven Schwemmers, Post doc

Dr. Wei Wang, Post doc

Literature:

Schwemmers S, Will B, Waller CF, Abdulkarim K, Johansson P, Andreasson B, Pahl HL (2007) JAK2V617F-negative ET patients do not display constitutively active JAK/STAT signaling. Exp Hematol, 35 (11):1695 -1703.

Griesshammer M, Klippel S, Strunck E, Temerinac S, Mohr U, Heimpel H, Pahl HL (2004) PRV-1 mRNA expression discriminates two types of Essential Thrombocythemia., Ann. Hematol., 83: 364-370.

(PDF-file) (Internet-reference) Earthklein

Goerttler PS, Steimle C, März E, Johansson PL, Andreasson B, Griesshammer M, Gisslinger H, Heimpel H, Pahl HL (2005) The Jak2^V617F mutation, PRV-1 overexpression and EEC formation define a similar cohort of MPD patients. Blood, 106: 2862-2864.

(PDF-file) (Internet-reference) Earthklein

Goerttler PS, März E, Johansson PL, Andreasson B, Kutti J, Moliterno AR, Marchioli R, Spivak JL, Pahl HL for the MPD Research Consortium (2005) Thrombotic and Bleeding Complications in Four Subpopulations of Patients with ET Defined by c-Mpl Protein Expression and PRV-1 mRNA Levels. Haematologica/The Hematology Journal, 90: 851-853.

(PDF-file) (Internet-reference) Earthklein

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Molecular markers in Myeloproliferative Disorders

The gene for Polycythemia rubra vera-1 (PRV-1) was cloned in our laboratory by virtue of its overexpression in granulocytes from patients with Polcythemia vera (PV) compared to healthy controls (Temerinac, 2000). We and others have subsequently demonstrated that PRV-1 overexpression is specific for PV, since it does not occur in patients with secondary erythrocytosis (SE), secondary thrombocytosis (ST), CML or AML. Based on these data, we have developed a quantitative RT-PCR assay for the determination of PRV-1 mRNA levels, which can be used for the diagnosis of PV in patients with erythrocytosis (Klippel, 2003).

However, in addition to PV patients, a subset of patients with Essential Thrombocythemia (ET), also overexpress PRV-1. In a cohort of 30 patients, we have demonstrated that PRV-1 overexpression correlates tightly with the ability to form EPO independent colonies (so called endogenous erythroid colonies, EECs) (Griesshammer, 2004).

We subsequently determined whether PRV-1 positive and PRV-1 negative ET patients differ in their clinical course. In our cohort, PRV-1 positive ET patients had a statistically significantly higher incidence of thromboembolic or microcirculatory events (p=0.003) compared to PRV-1 negative patients. In addition, 40% of the PRV-1-positive patients develop signs and symptoms of PV during the course of their disease. In contrast, none of the 15 PRV-1-negative patients displayed such symptoms (p=0.017) (Griesshammer, 2004).

PRV-1 is one of five molecular markers described in MPD patients, the others being a point mutation in the JAK2 kinase (JAK2^V617F), the presence of EECs, clonal hematopoesis, the reduced expression of c-Mpl. Each of the markers is present in around 50% of ET patients. We therefore investigated whether these markers are acquired concurrently or separately in individual patients. Our results demonstrate that three of the markers, JAK2^V617F, EEC formation, PRV-1 overexpression and clonality coincide in most patients, whereas reduced c-Mpl expression is acquired independently (Goerttler, 2005a and 2005b).

On the basis of these data, we have postulated the distinction of two subtypes of ET: one that is characterized by the JAK2^V617F mutation, the presence of EECs as well as PRV-1 overepression and the second, in which patients present with wt JAK2, absence of EECs and normal PRV-1 levels. Patients in the former category appear by retrospective analysis to be at higher risk for thromboembolic complications any may therefore profit from more aggressive therapy (Griesshammer 2004).

Current Lab Members on Project:

Sven Schwemmers, Ph. D. student

Alumni:

Dr. med. Snezana Temerinac

Steffen Klippel, graduate student (Ph. D. 2003)

Philipp Goerttler, graduate student (Ph. D. 2005)

Cordula Steimle, graduate student, (Ph. D. 2005)

Literature:

Klippel S, Strunck E , Busse CE, Behringer D, Pahl HL (2002) Biochemical Characterization of PRV-1, a Novel Hematopoietic Cell Surface Receptor, which is Overexpressed in Polycythemia rubra vera., Blood, 100: 2441-2448

(PDF-file)

Temerinac S, Klippel S, Strunck E, Röder S, Lübbert M, Lange W, Azemar M, Meinhardt G, Schaefer HE, Pahl HL (2000) Cloning of PRV-1, a Novel Member of the uPAR Receptor Superfamily, which is Overexpressed in Polycythemia rubra vera. Blood 95:2569

(PDF-file) (Internet-reference) Earthklein

Klippel S, Strunck E, Temerinac S, Meinhardt G, Mohr U, Leichtle R, Griesshammer M, Heimpel H, Pahl HL (2003) Quantification of PRV-1 mRNA distinguishes Polycythemia vera from Secondary Erythrocytosis., Blood, 102: 3569-3574.

(PDF-file) (Internet-reference) Earthklein

Griesshammer M, Klippel S, Strunck E, Temerinac S, Mohr U, Heimpel H, Pahl HL (2004) PRV-1 mRNA expression discriminates two types of Essential Thrombocythemia., Ann. Hematol., 83: 364-370.

(PDF-file) (Internet-reference) Earthklein

Goerttler PS, Steimle C, März E, Johansson PL, Andreasson B, Griesshammer M, Gisslinger H, Heimpel H, Pahl HL (2005) The Jak2^V617Fmutation, PRV-1 overexpression and EEC formation define a similar cohort of MPD patients. Blood, 106: 2862-2864.

(PDF-file) (Internet-reference) Earthklein

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Molecular etiology of NF-E2 and PRV-1 Overexpression in MPU

The transcription factor NF-E2 and the gene encoding PRV-1 are overexpressed in patients with polycythemia vera (PV). The recent description of a 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. NF-E2 is a promising candidate in the pathophysiology of PV for several reasons:

NF-E2 is overexpressed in stem cells as well as in all three cell lineages affected in PV. In murine cells NF-E2 overexpression leads to Epo-independent growth and differentiation. NF-E2 may thus play a pivotal role in causing the erythrocytosis of PV. However, both the molecular mechanism leading to NF-E2 overexpression and its effect on human hematopoiesis are not known. In addition, the cause of PRV-1 overexpression remains unclear. Therefore, it is the aim of this project to investigate the cause of NF-E2 and PRV-1 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 or its overexpression modulates hematopoietic differentiation. Specific Aim: To modulate NF-E2 expression via siRNA knock down and retroviral or lentiviral transduction and examine the consequences on Epo-dependent and 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.

3. Hypothesis: NF-E2 and PRV-1 overexpression result from aberrant transcriptional activation. Specific Aim: To characterize protein/DNA interaction on the NF-E2 and PRV-1 promoters in PV and healthy control cells to determine aberrantly activated transcription factors.

Current Lab Members on Project:

Wei Wang, Ph. D. student

Literature

Wang W, Schwemmers S, Hexner EO, Pahl HL (2010) AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2. Blood, in press

Temerinac S, Klippel S, Röder S, Lübbert M, Lange W, Azemar M, Meinhardt G, Pahl HL (2000) Cloning of PRV-1, a Novel Member of the uPAR Receptor Superfamily, which is Overexpressed in Polycythemia rubra vera. Blood, 95: 2569-2576.

(PDF-file) (Internet-reference) Earthklein

(PDF-file)

(PDF-file) (Internet-reference) Earthklein

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