ag-490 and Polycythemia-Vera

Activating mutations in the tyrosine kinase Janus kinase 2 (JAK2) cause myeloproliferative neoplasms, clonal blood stem cell disorders with a propensity for leukaemic transformation. Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells. Here we show that mouse ES cells carrying the human JAK2V617F mutation were able to self-renew in chemically defined conditions without cytokines or small-molecule inhibitors, independently of JAK signalling through the STAT3 or phosphatidylinositol-3-OH kinase pathways. Phosphorylation of histone H3 tyrosine 41 (H3Y41) by JAK2 was recently shown to interfere with binding of heterochromatin protein 1α (HP1α). Levels of chromatin-bound HP1α were lower in JAK2V617F ES cells but increased following inhibition of JAK2, coincident with a global reduction in histone H3Y41 phosphorylation. JAK2 inhibition reduced levels of the pluripotency regulator Nanog, with a reduction in H3Y41 phosphorylation and concomitant increase in HP1α levels at the Nanog promoter. Furthermore, Nanog was required for factor independence of JAK2V617F ES cells. Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal.

Topics: Adult; Adult Stem Cells; Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chromatin; Chromobox Protein Homolog 5; Embryonic Stem Cells; Female; Histones; Humans; Immunoblotting; Janus Kinase 2; Leukemia Inhibitory Factor; Male; Mice; Mice, 129 Strain; Mice, Transgenic; Mutation; Phosphorylation; Polycythemia Vera; Recombinant Proteins; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

The somatic Jak2 mutation (V617F) was identified in most patients with polycythemia vera (PV). Here, we show that the activating Jak2 V617F mutant completely protected Ba/F3 cells from cytokine withdrawal-induced apoptotic cell death. Interestingly, Ba/F3 cells expressing Jak2 V617F mutant induced rapid tumorigenesis in nude mice, leading to rapid death. Whereas an injection of Ba/F3 cells expressing wild-type Jak2 had no effect, an injection of Ba/F3 cells expressing Jak2 V617F mutant promptly invaded and spread into various distinct organs, such as the liver and spleen. Strikingly, Jak2 inhibitor, AG490 potently inhibited cytokine-independent cell growth induced by the Jak2 V617F mutant. Also, treatment with AG490 effectively delayed Jak2 V617F mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo suggest that Jak2 harboring V617F mutation is a potent oncogene able to promote cell transformation and tumorigenesis.

Topics: Animals; Apoptosis; Cell Line; Cell Transformation, Neoplastic; Genetic Predisposition to Disease; Hematopoietic Stem Cells; Janus Kinase 2; Liver; Mice; Mice, Nude; Mutation; Neoplasm Invasiveness; Neoplasm Transplantation; Polycythemia Vera; Polymorphism, Genetic; Spleen; Transfection; Tyrphostins

JAK2(V617F), a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2(V617F)-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders.

Topics: Amino Acid Substitution; Animals; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Enzyme Activation; Erlotinib Hydrochloride; Growth Inhibitors; Hematopoietic Stem Cells; Humans; Janus Kinase 2; Jurkat Cells; Mice; Phenylalanine; Polycythemia Vera; Protein Kinase Inhibitors; Quinazolines; Valine

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

Topics: ADP-ribosyl Cyclase 1; Amino Acid Substitution; Antigens, CD34; Erythroid Precursor Cells; Hematopoiesis; Hematopoietic Stem Cells; Humans; Janus Kinase 2; Phenylalanine; Point Mutation; Polycythemia Vera; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Thy-1 Antigens; Tyrphostins; Valine