Janus Kinase 2 (JAK2) Mutations
Polycythemia Vera

Author: Elisabetta Signorino
Date: 14/09/2011


JAK family of kinases and their role in disease

The four Janus kinases (JAK1, JAK2, JAK3 and non-receptor tyrosine-protein kinase 2 (TYK2)) are differentially activated by different cytokines. Erythropoietin, thrombopoietin, granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL 3), IL 5, growth hormone and prolactin convey their signals through JAK2. Interferon-γ (IFNγ), IL 6, IL 10, IL 11, IL 19, IL 20 and IL 22 signal through both JAK1 and JAK2.
Germline targeting of the Jak1 or Jak2 alleles results in perinatal death and day 12.5 embryonic lethality, respectively. Jak1 deficient mice exhibit defective neural function and defective lymphoid development. Mice lacking JAK2 die as a consequence of a block in definitive erythropoiesis in spite of normal lymphoid development. Deficient JAK3 signalling in humans and mice causes severe combined immunodeficiency (SCID). Tyk2 deficient mice exhibit impaired signalling in response to IFNα/β and IL 12, poor signal transducer and activator of transcription 3 (STAT3) activation and defective clearing of the vaccinia virus. An array of coding and non-coding IL 23 receptor variants has been shown to modulate the susceptibility to Crohn’s disease, ulcerative colitis, psoriasis and seronegative inflammatory diseases such as ankylosing spondylitis.
In view of the involvement of the JAK2V617F mutation in the pathogenesis of myeloproliferative neoplasms, such as Polycythemia Vera, several JAK2 inhibitors (for example, ruxolitinib, TG101348 and CYT387) are currently being investigated in clinical trials for patients with these malignancies.

Janus kinase inhibitors for the treatment of myeloproliferative neoplasias and beyond, 2011

The JAK2 exon 12 mutations: a comprehensive review, 2011.

JAK Family


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Cytokine receptors and JAK2

Proliferation, survival and differentiation of the different haematopoietic lineages are processes tightly regulated by secreted cytokines. Cytokines exert their biological effects through binding to receptors on the cell surface. Type I and type II cytokine receptors, which are classified according to the three-dimensional structure of their ligands, lack intrinsic tyrosine kinase activity and rely on receptor-associated Janus kinases (JAKs) to transmit their signals to the cytoplasm. Type I cytokines have a four α helical bundle structure, whereas the structure of type II cytokines, such as that of interferons (IFNs), is more diverse. The majority of cytokine receptors modulating the activity of the haematopoietic system belong to the class I cytokine receptor family. These receptors commonly share signal transducing components such as gp130 or the common β (βc) or γ (γc) chains. Type II cytokine receptors are mainly bound by interferons. There are four JAKs (JAK1, JAK2, JAK3 and non-receptor tyrosine protein kinase 2 (TYK2)), which are differentially activated by different cytokines. In contrast to JAK1 and JAK2, JAK3 associates only with the γc chain, which is used exclusively by the receptors for a selected group of cytokines that are critical in T cell and natural killer cell development as well as in B cell function.
JAKs have seven homologous domains (JH1–7), including the catalytic domain (JH1) and the catalytically inactive pseudokinase domain (JH2), which putatively downregulates the activity of the kinase domain. The JH3–JH4 domain of JAKs shares homology with Src homology 2 (SH2) domains. The amino terminal domain (JH4–JH7) is known as the FERM (short for 4.1 protein, ezrin, radixin and moesin) domain, which engages with cytokine receptors.

Functional consequences of JAK2 mutations in MPNs

The JAK2V617F mutation maps to the JH2 domain of JAK2, which has significant homology to the tyrosine kinase (JH1) domain but lacks catalytic activity. Current evidence suggests that JH2 is involved in the auto-inhibition of JAK2 activity.
The mutant JAK2V617F is constitutively activated. Expression of JAK2V617F in FDCP (factor-dependent cell progenitor) cells that co-express EPO-R allows these cells to grow independently of EPO and confers hypersensitivity to Epo.

Erythroid colonies can be grown from the peripheral blood of patients carrying JAK2 exon 12 mutations in the absence of exo-genous EPO. However, the erythroid colonies from patients with exon 12 mutations are typically heterozygous for the mutation, in contrast with JAK2V617F carriers in which the mutation is frequently found in the homozygous state. Interestingly, in contrast to the JAK2V617F mutation, exon 12 mutations appear to be restricted to PV, which could be associated with activation of distinct signalling pathways and perhaps subtle clinical phenotypes.

When JAK2V617F is expressed in haematopoietic cells, several signalling pathways that are important for proliferation and survival are activated, including STAT3, STAT5, mitogen-activated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) and the phos¬phoinositide 3 kinase (PI3K)–AKT pathway.

Experimental evidence in mouse models and in patient samples suggests that JAK2V617F causes erythrocytosis and progression to myelofibrosis. Furthermore, JAK2V617F allele burden correlates with white blood cell counts and haemoglobin levels in patients with PV.

Discovery of the JAK2 mutation in MPNs

In 2005, several groups using different approaches discovered a recurrent acquired somatic mutation in the JAK2 gene in a significant proportion of patients with MPNs. This dominant gain-of-function mutation is a guanine-to-thymidine substitution at nucleotide 1,849 of the JAK2 gene that results in a valine-to-phenylalanine substitution at codon 617 of JAK2 (Refs 18–22). It is currently unknown why JAK2V617F is the only mutation described at codon 617 of JAK2 in human disease, given the fact that substitutions for valine at the same codon involving other residues — such as tryptophan, methionine, isoleucine or leucine — have been shown to constitutively activate JAK2 and transform haematopoietic progenitors in vitro.

Recently, a series of novel somatic gain-of-function mutations affecting JAK2 at exon 12 have been described in patients with JAK2V617F-negative PVs. Exon 12 JAK2 mutations and JAK2V617F mutations are mutually exclusive. Exon 12 mutations map just 5′ of the JH2 domain of JAK2, therefore they are likely to cause structural changes that result in JAK2 activation. In one study, the most frequent of all exon 12 mutations was N542 E543del. Importantly, exon 12 mutations are associated with isolated erythrocytosis and suppressed serum erythropoietin production. Therefore, a mutation in the JAK2 gene can be identified in virtually all patients with PV.

JAK2V617F signalling pathways in myeloproliferative neoplasms

The JAK2V617F mutation causes the constitutive activation of the enzyme. Accordingly, STAT3 is constitutively activated in the absence of cytokine stimulation in a subset of patients with polycythaemia vera (PV). STAT5 is also activated in megakaryocytes and CD34+ progenitors from patients with primary myelofibrosis. JAK2V617F also activates the phosphatidylinositol 3 kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR)–forkhead transcription factors (FOXO) signalling proteins as well as the RAS pathway that transmit signals for survival and proliferation, thereby preventing apoptosis of haematopoietic progenitor cells. Furthermore, enforced expression of JAK2V617F in human haematopoietic stem cells and myeloid progenitors steers differentiation towards the erythroid lineage, which is accompanied by decreased expression of the transcription factor PU.1 and enhanced expression and phosphorylation of erythroid transcription factor (GATA1).

Wild-type Janus kinase 2 (JAK2) as well as the constitutively active JAK2V617F mutant have been detected both in the cytoplasm and in the nucleus of various human leukaemic cell lines and primary CD34+ haematopoietic progenitors. In the nucleus, JAK2 phosphorylates histone H3 at tyrosine 41 (H3Y41). The levels of phosphorylated H3Y41 correlate with JAK2 activity in vivo. Notably, JAK2 appears to be the only kinase responsible for H3Y41 phosphorylation, as treatment with JAK2 inhibitors — such as TG101209 or AT9283 — abrogates the phosphorylation of H3Y41 in the nucleus.

Of note, most JAK2 regulated genes do not contain a predicted signal transducer and activator of transcription 5 (STAT5) binding site, suggesting that these genes are regulated by signals other than the JAK2–STAT5 pathway. Of special interest among these genes is lmo2, which is involved in normal haematopoiesis and leukaemogenesis.

JAK2 exon 12 mutations.

Surprisingly, given that the JAK2V617F mutation was thought to result in constitutive kinase activity by perturbing inhibitory interactions between the kinase and pseudokinase domains, the amino acids affected by the exon 12 variants identified are all located immediately adjacent to the pseudo-kinase domain, in a linker between this and the SH2 domain. Although not strictly located within the pseudo-kinase domain, the residues affected by an exon 12 mutation may nonetheless contribute to auto-inhibitory intradomain interactions.

As illustrated using a theoretical model of JAK2 structure , the residues affected by a JAK2 exon 12 mutation (highlighted in yellow) map near to V617 (shown in blue); both regions are located at the interface between the pseudo-kinase and kinase domains.
Molecular dynamics simulations have suggested that the loop defined by residues 539 to 544 is important in supporting the local conformation near V617; a second homology-based
model of JAK2 structure suggests that it is F537 and K539 that mediate interactions with the b4 strand that contains V617. In either scenario, perturbations within this loop structure would disrupt interactions between the pseudokinase and kinase domains, releasing the former from a closed, inactive conformation.

Nevertheless, JAK2V617F and JAK2 exon 12 mutants have a number of similar activities in vitro and in vivo: both stabilize tyrosine-phosphorylated SOCS3 and inhibit DNA damage-mediated apoptosis via the Bcl-xL deamidation pathway. These data collectively suggest that, despite
having similar consequences on cytokine-independent proliferation in vitro, the V617F and exon 12 mutants have overlapping but distinct effects on intracellular signaling
pathways downstream of JAK2. The biological significance of these signaling differences is uncertain but is likely to explain in part the phenotypic differences noted in patients.

Patients with a JAK2 exon 12 mutation present with a hematologic disorder consistent with the diagnosis of PV; this is characterized by erythrocytosis, with a raised hematocrit and hemoglobin level, reduced serum EPO levels, and EPO-hypersensitive erythroid progenitors, but often lacks the proliferation of cells of the granulocytic or megakaryocytic lineages generally observed in patients with classic JAK2V617F-positive PV. Nevertheless, patients in these PV subgroups have comparable incidences of disease complications, such as thrombosis or emorrhage, comparable rates of progression to myelofibrosis and/or acute leukemia, and a similar pattern of secondary acquired genetic events (including mutation of the TET2, ASXL1 or EZH2 genes).


Janus kinase inhibitors for the treatment of myeloproliferative neoplasias and beyond, 2011

The JAK2 exon 12 mutations: a comprehensive review, 2011.

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