The JAK2 gene, located on chromosome 9p24 in humans, includes 25 exons encoding a protein of about 1132 amino acids. This protein is named JAK2 and it is a member of the Janus kinase (JAK) family. Four members of this family have already been identified: JAK1, JAK2, JAK3 and Tyk2.20

In 2005, the discovery of the JAK2 mutation (JAK2 V617F) expanded the knowledge about the pathogenesis of BCR-ABL-negative neoplasms. A missense mutation at position g.1849 with a guanine-thymine transversion (g.1849 G>T)5,6 was identified in exon 14 of the JAK2 gene. This mutation results in the substitution of valine for phenylalanine at amino acid position p.617,5 affecting the pseudokinase domain (JH2) of the protein. The JAK2 V617F mutation is present in most patients with PV and in a subgroup of patients with ET and PMF (∼95% in PV, 50–60% in ET and 50–60% in PMF).5–9,19

It has been shown that the JAK2 V617F mutation causes genetic instability in gene expression by inducing changes in the chromatin structure and by reducing the apoptotic response to DNA damage. These are mechanisms that can increase the accumulation of genetic lesions leading to malignant transformations.12 Besides the g.1849 G>T (p.V617F) mutation,5,6 other mutations were found in exon 12 in 2007 namely: p.N542-543del, p.E543-D544del, p.K539L, p.F537-K539delinsL, p.H538-p.K539delinsL, p.H538QK539L, p.V536-1546dup11, p.F537-1546dup10+F547L, p.R541 E543delinsK-and-p.I540 E543delinsMK.21–23 These were found to show low frequencies of about 3–4% in PV patients. There are valid techniques that detect these mutations and their active search is justified not only due to the absence of the exon 14 mutation, but also in patients that have erythrocytosis and in those with low levels of erythropoietin.24

Members of the JAK2 protein family have seven homologous domains (JH) that are numbered from 1 to 7. The JH1 domain acts as a kinase domain, which contains the adenosine triphosphate (ATP)-binding and signaling pathway activation regions. The JH2 domain is a pseudokinase that is homologous to the JH1 domain. It lacks catalytic activity due to a substitution of an aspartate residue in the catalytic loop (His–Arg–Asp – HRD motif). JH2 has an inhibitory function that regulates both the basal activity of JAK and the cytokine-induced activation by interacting with the active site, JH1, blocking ATP and/or substrates binding to the catalytic site.25–27

Mutations in exon 12 of JAK2 are found in the binding region of the Src homology 2 (SH2) and JH2 domains. Although not directly located in the JH2 domain, these mutations may modify the structure of the pseudokinase domain, inducing erythropoietin hypersensitivity similar to that caused by the JAK2 p.V617F mutation.5,9,28,29

As our knowledge about the JAK/STAT pathway has increased, important mechanisms for down-regulation of this pathway have been elucidated. These include removal of phosphates from cytokine receptors and activated STATs by tyrosine phosphatases, as well as by protein inhibitors of activated STAT (PIAS). PIAS inhibit transcriptional activation by binding and blocking access to target DNA. The next section will discuss three PIAS: SOCS, CBL and Lyme neuroborreliosis (LNB) proteins.

SOCS proteins are a family of cytokine signaling suppressors of the JAK/STAT pathway, characterized by the presence of an amino-terminal domain that is variable in size. It includes an inhibitory domain, a SH2 domain and a carboxy-terminal domain called SOCS box. Currently, there are eight members in this family, namely: SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7 and CIS. Under normal conditions, they are expressed at low levels in unstimulated cells while their expression increases when STATs are activated.30,31,38

SOCS can inhibit the JAK/STAT signaling through two mechanisms: (1) inhibition of JAK2 kinase activity by competing with the STATs SH2 domains for binding sites in the cytoplasmic domain of the receptor or (2) proteasomal degradation of signaling proteins by binding to the JH1 domain of JAK2, inhibiting the activation of all other JAK/STAT-associated pathways.39

Some mutations in different SOCS have been identified in MPNs, but are rarely found. Hypermethylation of CpG islands in SOCS1 and SOCS3 is associated with a decrease in the expression of these regulators in p.V617F-positive PV and ET patients.5,10

Other important components of this negative regulatory complex include multifunctional proteins with ubiquitin ligase activity, such as CBL. CBL is usually involved in the negative feedback of tyrosine kinase receptors by competitive blocking signaling. It induces proteasomal degradation of tyrosine kinases activated by ubiquitination. All CBL have single domain proteins that recognizes phosphorylated tyrosine residues that are present on activated tyrosine kinases.10,40

LNK, included in the SH2B protein family, negatively regulates the activity of JAK2 by binding to SH2-phosphorylated tyrosine. It also regulates the signaling of TPO-R and EPO-R, and it is detected in both JAK2 V617F-positive and -negative patients.10 Increased LNK deficiency increases the oncogenic ability of JAK2 to expand myeloid progenitors in vitro and in vivo.41

In addition to activating the JAK/STAT pathway, the p.V617F mutation5 also constitutively activates the rat sarcoma (RAS)/Mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, aka Akt) pathways, which results in an increased expression of mitotic proteins, regulatory proteins from the cellular cycle [cyclins D1, CDC25A (cell division cycle)] and anti-apoptotic genes [B-cell lymphoma-extra-large (Bcl-XL) and Bcl2]. These abnormalities involving the JAK/STAT and related pathways result in proliferation of affected cells (Figure 1).10

Figure 1.

Signaling pathways activated by cytokine receptor binding. PI3K, Akt and STAT are activated after the phosphorylation of JAK2. The active negative feedback regulatory proteins complete the activation pathways. Such control is ineffective in the presence of a mutation in the JH2 domain of JAK2. PI3K: phosphoinositide 3-kinase; Akt: protein kinase B; STAT: signal transducers and activators of transcription; JAK2: Janus kinase 2; EPO-R: erythropoietin receptor; SOCS: suppressors of cytokine signaling; Bcl-XL: B-cell lymphoma-extra-large; Myc: myelocytomatosis oncogene.

(0,27MB).

In mutated cells, down-regulation becomes inadequate and/or inefficient. SH2 domains, normally present in PIAS proteins, cannot dephosphorylate JAK2. In addition, the anti-apoptotic mechanisms are expressed at much higher levels than normal. An acquired mutation in one of these regulatory proteins can trigger an exacerbated activation of STAT, even in the absence of specific mutations in JAK2.42

Additional molecular events appear to interfere with the activity of JAK2, such as cytogenetics, which increase the kinase activity of the protein, the regulatory ability of phosphorylases and SOCS proteins, polymorphisms, and mutations in cytokine receptors.43

A further intriguing question for researchers regarding MPNs is how the same mutation can trigger three phenotypically different diseases. There is still no clear understanding of the molecular mechanisms involved in the process, just hypotheses. One of them is the dose–response gene hypothesis, which is associated with allele load. Another is that there may be epigenetic changes that predispose the patient's organism to develop certain MPNs as well as changes in genes specific in the development of malignancies, as well as mutations in other signaling pathways that might affect signaling in regulatory proteins.16

For Kralovics et al., Plo et al., Smith & Fan, Cross and Pagliarini,26,44–47 the phenotype of the disease is related to the concept of allelic load. Thus, low levels of mutant alleles would result in ET phenotype, while higher levels of mutated alleles would lead to a phenotype similar to PV. These higher levels of mutated alleles in hematopoietic stem cells could be associated with the patient's homozygous condition, caused by loss of heterozygosity, as explained by the theory of uniparental disomy.48

Presumably, homozygosity confers a proliferative advantage over other cells, which present only one mutated allele, increasing the activity level of the tyrosine kinase generated by the mutated JAK2. Patients with ET and PMF are mostly heterozygous and seldom show homozygosity.48

Besides the association between phenotype and homozygosity/heterozygosity, there are other hypotheses that have attempted to correlate the different phenotypes observed related to the JAK2 V617F mutation.

In their study, Plo et al.45 discuss the gene-dose hypothesis, which postulates that the JAK2 mutation can be a starting point for the three pathologies. They also discuss the existence of other genetic events that can modify the JAK2 kinase activity, which may explain the heterogeneity present in classic MPNs. Several mechanisms have been proposed to aid the understanding of loss of heterozygosity and/or the presence of new genetic abnormalities, including the double-stranded break repair mechanisms in DNA, homologous recombination and non-homologous end joining. All these lead to genomic instability, which highlights how essential the precise regulation of these mechanisms is for the maintenance of genome stability and diversity.49–51

According to Mascarenhas et al.52 biological events that lead to the initiation and progression of MPNs are linked not only to acquisition of genetic mutations such as JAK2 V617F. They are also due to epigenetic changes that do not affect the primary DNA sequence, but the gene expression responsible for chromatin remodeling. Deregulation of modulation processes between nucleosomes may result in silencing of tumor suppressor as well as differentiation genes, promoting cell survival by blocking apoptosis and senescence, contributing to malignant transformation. The researchers also highlighted two categories of epigenetic alterations in MPNs: (1) changes in the genes that encode proteins involved in remodeling of the chromatin structure, such as the TET2, ASXL1, JAK2 genes, among others, and (2) changes in the promoter site of genes essential for cell survival, differentiation and proliferation. Besides its role in signaling, the JAK2 V617F mutation also affects chromatin structure by blocking the recruitment of repressor proteins.9