transforming-growth-factor-beta and Polycythemia-Vera

The BCR-ABL-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), entered the spotlight in 2005 when the unique somatic acquired JAK2 V617F mutation was described in >95% of PV and in 50% of ET and PMF patients. For the very rare PV patients who do not harbor the JAK2 V617F mutation, exon 12 JAK2 mutants were discovered also to result in activated forms of JAK2. A minority of ET and PMF patients harbor mutations that constitutively activate the thrombopoietin receptor (TpoR). In bone marrow reconstitution models based on retroviral transduction, the phenotype induced by JAK2 V617F is less severe and different from the rapid fatal myelofibrosis induced by TpoR W515L. The reasons for these differences are unknown. Exactly by which mechanism(s) one acquired somatic mutation, JAK2 V617F, can promote three different diseases remains a mystery, although gene dosage and host genetic variation might have important functions. We review the recent progress made in deciphering signaling anomalies in PV, ET and PMF, with an emphasis on the relationship between JAK2 V617F and cytokine receptor signaling and on cross-talk with several other signaling pathways.

Topics: Animals; Disease Models, Animal; Hematopoietic Stem Cells; Humans; Janus Kinase 2; Mutation; Phosphorylation; Polycythemia Vera; Primary Myelofibrosis; Receptors, Erythropoietin; Receptors, Granulocyte Colony-Stimulating Factor; Receptors, Thrombopoietin; Signal Transduction; STAT Transcription Factors; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Thrombocythemia, Essential; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF), peripheral blood CD34+ cells isolated from patients with IMF, polycythemia vera (PV), and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is, therefore, likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9, therefore, likely contribute to the development of many pathological epiphenomena associated with IMF.

Topics: Adult; Aged; Antigens, CD34; Apoptosis; bcl-X Protein; Cells, Cultured; Culture Media, Conditioned; Female; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Humans; Hyperplasia; Integrin beta3; Male; Matrix Metalloproteinase 9; Megakaryocytes; Middle Aged; Polycythemia Vera; Primary Myelofibrosis; Thrombopoietin; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

In excess of 90% of patients with polycythaemia vera (PV) express a mutated form of Janus kinase 2 (JAK2), JAK2V617F. Such aberrant proteins offer great potential for the treatment of these diseases; however, inhibitors to JAK2 have had limited success in the clinic in terms of curing the disease. To understand the effects of this oncogene in haematopoietic cells with the aim of improving treatment strategies, we undertook a systematic evaluation of the effects of JAK2V617F expression using proteomics. The effects of JAK2V617F on over 5000 proteins and 2000 nuclear phosphopeptide sites were relatively quantified using either SILAC or eight-channel iTRAQ mass spectrometry. Pathway analysis of the proteins identified as changing indicated disruption to the p53 and MYC signalling pathways. These changes were confirmed using orthogonal approaches. The insight gained from this proteomic analysis led to the formation of hypothesis-driven analysis on inhibitor-mediated effects on primary cells from patients with a JAK2V617F mutation. Simultaneous inhibition of MYC and upregulation of p53 led to the preferential extinction of JAK2V617F-positive CD34+ cells, illustrating a potential therapeutic benefit from combined targeting of p53 and MYC.

Topics: Amino Acid Substitution; Cell Line; Codon; Humans; Janus Kinase 2; Mutation; Polycythemia Vera; Protein Interaction Mapping; Protein Interaction Maps; Proteome; Proteomics; Proto-Oncogene Proteins c-myc; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Topics: Animals; Antigens, CD34; Benzamides; Biomarkers; Cell Cycle; Cells, Cultured; Dioxoles; Disease Models, Animal; Drug Evaluation, Preclinical; Erythroblasts; Fetal Blood; Gene Expression Profiling; Hematopoietic Stem Cells; Humans; Immunophenotyping; Janus Kinase 2; Mice; Molecular Targeted Therapy; Mutation; Phenotype; Polycythemia Vera; Primary Myelofibrosis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor beta-1 (TGF beta-1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGF beta-1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGF beta-1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGF beta-1 expression in IMF and other Ph(-) CMPD, total bone marrow cells as well as laser-microdissected megakaryocytes were quantitatively analysed by real-time RT-PCR. OPG mRNA expression in fibrotic IMF was correlated with TGF beta-1 mRNA expression in a case-specific manner. Both OPG and TGF beta-1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGF beta-1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non-neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGF beta-1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGF beta-1 protein was predominantly expressed by the myeloid lineage in Ph(-) CMPD and non-neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGF beta-1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGF beta-1 expression occurs in megakaryocytes as well as myeloid cells in Ph(-) CMPD. TGF beta-1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non-fibrotic Ph(-) CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGF beta-1 expression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Marrow Cells; Chronic Disease; Diagnosis, Differential; Female; Gene Expression; Glycoproteins; Hematopoiesis; Humans; Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative; Male; Megakaryocytes; Microdissection; Middle Aged; Myeloproliferative Disorders; Osteoprotegerin; Polycythemia Vera; Primary Myelofibrosis; Protein Array Analysis; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombocythemia, Essential; Transforming Growth Factor beta; Transforming Growth Factor beta1

Clonal stem cell proliferation and increased erythrocyte mass are hallmarks of the myeloproliferative disorder polycythemia vera (PV). The molecular basis of PV is unknown.. We carried out a genome-wide screening for loss of heterozygosity (LOH) and analyzed candidate genes within the LOH loci.. Three genomic regions were identified on chromosomes 9p, 10q, and 11q. The presence of these LOHs in both myeloid and lymphoid cells indicated their stem cell origin. The 9pLOH prevalence is approximately 33% and is the most frequent chromosomal lesion described in PV so far. We report that the 9pLOH is due to mitotic recombination and therefore remains undetectable by cytogenetic analysis. Nineteen candidate genes were selected within the 9pLOH region for sequencing and expression analysis. No mutations were found in these genes; however, unexpectedly, increased expression of the transcription factor NFI-B was detected in granulocytes and CD34(+) cells in PV with 9pLOH. Since a member of the NFI gene family (NFI-X) was reported to result in TGF-beta resistance when overexpressed in vitro (TGF-beta is a known inhibitor of hematopoiesis), we transfected the NFI-B gene to the mouse 32D cell line. We found that overexpression of the NFI-B gene confers TGF-beta resistance in vitro.. We characterized a new region on chromosome 9p frequently involved in LOH in PV. Analysis of genes within this 9pLOH region revealed increased expression of the NFI-B gene. Our in vitro studies suggest that TGF-beta resistance may be the physiologic mechanism of clonal stem cell expansion in PV.

Topics: Animals; Antigens, CD34; Carrier Proteins; Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 9; DNA Mutational Analysis; Drug Resistance; Gene Expression; Genes, p16; Hematopoietic Stem Cells; Humans; Loss of Heterozygosity; Membrane Proteins; Mice; Mutation; NFI Transcription Factors; Polycythemia Vera; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transfection; Transforming Growth Factor beta; Uniparental Disomy