Mercher T, Wernig G, Moore SA, Levine RL, Gu TL, Frohling S et al.. JAK2T875N is a novel activating mutation that results in myeloproliferative disease with features of megakaryoblastic leukemia in a murine bone marrow transplantation model. Blood 108: 2770-2779
Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However, there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL, based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation, and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy, we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model, JAK2T875N induced a myeloproliferative disease characterized by features of AMKL, including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL.
"In 3% of PV patients where the V617F mutation is not present, four somatic gain-of-function mutations in exon 12 of JAK2 have been reported, out of which JAK2 K539L, located in the linker region between the SH2 and JH2 domains, was the most common . Another point mutation first identified in a megakaryoblastic cell line derived from an acute megakaryoblastic leukemia (AMKL) patient, and subsequently detected in a screen of human AMKL cell lines for STAT5 activation, presented a T875N substitution in the kinase domain of JAK2 . The JAK2 R683G mutation located in the hinge region between N-terminal and C-terminal lobes of the pseudokinase domain, was identified in a screen of pediatric acute lymphoblastic leukemia (ALL) patient samples . "
[Show abstract][Hide abstract] ABSTRACT: The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.
PLoS ONE 06/2010; 5(6):e11157. DOI:10.1371/journal.pone.0011157 · 3.23 Impact Factor
"In PV, activation of signal transducer and activator of transcription (STAT) 5 and STAT3 was described (Roder et al., 2001; Garcon et al., 2006). The constitutively active Jak2 mutants have been shown to lead to cytokine receptor-dependent constitutive activation of various signaling proteins, such as STATs, mitogen-activated protein kinases and phosphatidylino- sitol-3-kinase/AKT (James et al., 2005; Lu et al., 2005; Mercher et al., 2006; Scott et al., 2007). SOCS proteins negatively regulate signal transduction in a classical feedback loop (Yoshimura et al., 1995; Endo et al., 1997; Naka et al., 1997; Starr et al., 1997). "
[Show abstract][Hide abstract] ABSTRACT: Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia.
[Show abstract][Hide abstract] ABSTRACT: The acquired Janus kinase 2 (JAK2) V617F mutation shows a high frequency in diverse BCR/ABL-negative chronic myeloproliferative disorders (CMPD), and it is typically associated with polycythemia vera (PV). The frequency of JAK2 V617F mutation is about 90% in patients with PV, 50-60% in patients with essential thrombocythemia (ET), primary myelofibrosis (PMF), and less in patients with other myeloid neoplasms, while extremely rare in lymphoid malignancies. About 20 kinds of novel mutations of JAK2 other than V617F have been reported recently in the literature. Among these mutations, only one case of JAK2 V617F/C618R has been reported in a 67-year-old patient with PV. Here, we report a rare case of JAK2 V617F/C618R in a 41-year-old Korean male patient with review of the relevant literature on JAK2 mutations other than V617F. Although the frequency of JAK2 mutations other than the V617F is very low, this study emphasizes the need for assiduous analysis of the JAK2 gene to characterize new mutations, to determine their frequency, and to improve understanding of the clinical phenotypes as well as prognostic and biologic features associated with these mutations.
Cancer genetics and cytogenetics 03/2009; 189(1):43-7. DOI:10.1016/j.cancergencyto.2008.09.010 · 1.93 Impact Factor
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