Wernig G, Gonneville JR, Crowley BJ, Rodrigues MS, Reddy MM, Hudon HE et al.. The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes. Blood 111: 3751-3759

Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Blood (Impact Factor: 10.45). 05/2008; 111(7):3751-9. DOI: 10.1182/blood-2007-07-102186
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The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.

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Available from: James D Griffin, Oct 02, 2015
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    • "EpoR homodimers are pre‐formed prior to their interaction with the ligands [Livnah et al., 1999; Constantinescu et al., 2001]; the latter trigger activation via inducing a conformational change leading to JAK2 activation [Remy et al., 1999]. This dimerization is required even for enhanced signaling mediated by a constitutively active JAK2 V617F oncogene found in patients with diverse myeloproliferative diseases and present in almost all patients with polycythemia vera [Lu et al., 2008]; accordingly, the integrity of the FERM domain in this mutant is essential for its high intrinsic activity [Wernig et al., 2008]. It would be expected that the latter activity should promote receptor elimination. "
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    ABSTRACT: Activation of cytokine receptor-associated Janus kinases (JAKs) mediates most, if not all, of the cellular responses to peptide hormones and cytokines. Consequently, JAKs play a paramount role in homeostasis and immunity. Members of this family of tyrosine kinases control the cytokine/hormone-induced alterations in cell gene expression program. This function is largely mediated through an ability to signal towards activation of the signal transducer and activator of transcription proteins (STAT) as well as towards some other pathways. Importantly, JAKs are also instrumental in tightly controlling the expression of associated cytokine and hormone receptors, and, accordingly, in regulating the cell sensitivity to these cytokines and hormones. This review highlights the enzymatic and non-enzymatic mechanisms of this regulation and discusses the importance of the ambidextrous nature of JAK as a key signaling node that integrates the combining the functions of forward signaling and eliminative signaling. Attention to the latter aspect of JAK function may contribute to emancipating our approaches to the pharmacologic modulation of JAKs. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 01/2014; 115(1). DOI:10.1002/jcb.24647 · 3.26 Impact Factor
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    • "It is thought that the JAK2 V617F mutant gains full transforming activity only when the mutant JAK2 dimerization is promoted by receptor dimerization. This is supported by the need for oncogenic JAK2 V617F activation of an intact FERM (homologous to protein 4.1, ezrin, radixin, moesin)-like domain,12 a domain that normally mediates JAK binding to cytokine receptors. This is the case for EpoR, TpoR or G-CSFR, but other receptors can support JAK2 V617F activation. "
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    ABSTRACT: Janus kinases (JAKs) are non-receptor tyrosine kinases essential for activation of signaling mediated by cytokine receptors that lack catalytic activity, including receptors for erythropoietin, thrombopoietin, most interleukins and interferon. Upon hormone binding, JAKs phosphorylate tyrosine residues in the receptor cytoplasmic domains and in JAKs themselves leading to recruitment and activation of downstream signaling proteins such as signal transducer and activator of transcription (STAT). The JAK-STAT pathway is important for functional hematopoiesis and several activating mutations in JAK proteins have recently been described as underlying cause of blood disorders. One of the best studied examples is the JAK2 V617F mutant which is found in 95% of polycythemia vera patients and 50% of patients suffering from essential thrombocythemia and primary myelofibrosis. Much effort has been made to understand how the JAK2 V617F affects hematopoietic stem cell (HSC) renewal and lineage differentiation, since convincing evidence has been provided to support the notion that the mutation is acquired at the HSC level. We discuss several in vivo models that support contrary conclusions with respect to the advantage given to HSCs by JAK2 V617F. Moreover, we provide the current knowledge about STAT5 activation and its link to HSC expansion as well as amplification of the erythroid compartment. Evidence for both JAK2 V617F mutated HSCs exhibiting skewed differentiation potential and for amplification occurring after erythroid commitment has been provided, and we will discuss whether this evidence is relevant for the disease.
    07/2012; 1(3):184-190. DOI:10.4161/jkst.22071
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    • "Even though the crystal structure of the JAK2 kinase domain has been solved 3, 4, it is not known how exactly the V617F mutation in the pseudokinase domain leads to constitutive activation. The JAK2V617F mutation seems insufficient for its kinase activation and association with a cytokine receptor, such as the erythropoietin receptor (EpoR) appears to be required 5. Lack of a functional FERM domain in JAK2V617F, which mediates interaction with cytokine receptors, results in a loss of its transforming activity 6. It is likely that inhibitory constraints, normally overcome by ligand binding, are targeted by the JAK2V617F mutation, therefore leading to hyperresponsiveness or factor-independent growth. "
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    ABSTRACT: The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2011; 26(4):708-15. DOI:10.1038/leu.2011.255 · 10.43 Impact Factor
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